UNIVERSIDAD*DE*CANTABRIA*
FACULTAD*DE*MEDICINA*
DEPARTAMENTO*DE*ANATOMÍA*Y*BIOLOGÍA*CELULAR*
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TESIS*DOCTORAL*
“Papel*de*los*factores*de*transcripción*Sp6*y*Sp8*
en*el*desarrollo*de*la*extremidad”*
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Endika*Haro*Gabicagogeascoa*
Santander*2013*
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UNIVERSIDAD*DE*CANTABRIA*
FACULTAD*DE*MEDICINA*
DEPARTAMENTO*DE*ANATOMÍA*Y*BIOLOGÍA*CELULAR*
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“Role*of*Sp*transcription*factors*Sp6*and*
Sp8*in*limb*development”*
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Tesis*Doctoral*
presentada*por*
ENDIKA*HARO*GABICAGOGEASCOA*
Para*optar*al*Grado*de*Doctor*
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Dra.*María*Ángeles*Ros*profesora!de!investigación!y!
Dra.!Federica*Bertocchini,!contratada!Ramón!y!Cajal!
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del!Instituto!de!Biomedicina!y!Biotecnología!de!Cantabria!(CSIC?UC?SODERCAN),!
como! directoras! de! la! Tesis! Doctoral! titulada! “Papel! de! los! factores! de!
transcripción!Sp6!y!Sp8!en!el!desarrollo!de!la!extremidad”!“Role!of!Sp6!and!Sp8!
transcription!factors!in!limb!development”!
CERTIFICAN**
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que*dicho!trabajo!ha!sido!realizado!por!Don!Endika*Haro*Gabicagogeascoa*y!
consideran!que!se!encuentra!terminado!y*reúne!los!requisitos!para!su!
presentación!como!memoria!de!doctorado!por!el!interesado,!al!objeto!de!poder!
optar!al!grado!de!Doctor!por!la!Universidad!de!Cantabria.!
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María%A.%Ros%Lasierra%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%Federica%Bertocchini%
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Santander!a!1!de!octubre!de!2013!
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CONTENTS*
ABBREVIATIONS*.............................................................................................................................*9*
RESUMEN*........................................................................................................................................*13*
1.**INTRODUCTION*.......................................................................................................................*19*
1.1*****Limb*development*......................................................................................................*22*
1.2*****Limb*initiation*..............................................................................................................*25*
1.3*****Signalling*centers*........................................................................................................*32*
1.3.1**Proximo[Distal*axis*and**the*AER**..............................................................*32*
1.3.1.1*Fgf*signalling**.......................................................................................*35*
1.3.1.2*Proximo[Distal*patterning**.............................................................*36*
1.3.2**Anterior[Posterior*axis*and*the*ZPA**........................................................*39*
1.3.2.1*Shh*signalling**.....................................................................................*40*
1.3.2.2*Digit*patterning**.................................................................................*41*
1.3.3**Dorso[Ventral*axis*and*the*non[AER*ectoderm*.....................................*43*
1.3.3.1**Polarizing*the*limb*ectoderm*.......................................................*44*
1.3.3.2**The*non[AER*ectoderm**..................................................................*45*
1.3.3.3**Dorso[Ventral*boundaries*in*the*limb*ectoderm*...................*47*
1.4*****AER*development*........................................................................................................*49*
1.4.1**AER*induction*...................................................................................................*50*
1.4.2**AER*maturation*................................................................................................*50*
1.4.3**AER*regression*.................................................................................................*51*
1.4.4**AER*maintenance*.............................................................................................*52*
1.4.5**Split*hand/foot*malformation*.....................................................................*53*
1.4.6**AER*induction*and*DV*pattern*establishment*.......................................*55*
1.4.6.1**The*role*of*Bmp*signalling*pathway*...........................................*59*
1.4.6.2**The*role*of*Wnt*signalling*pathway*............................................*66*
1.4.6.3**Epistatic*relationship*between*Wnt*and*Bmp**
*signalling*pathways*..........................................................................*70*
1.5*****The*family*of*the*Specificity*Protein*of*transcription*factors*......................*72*
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2.*AIM*OF*THE*PRESENT*THESIS*.............................................................................................*79*
3.*MATERIALS*AND*METHODS*.................................................................................................*83*
3.1*****Mouse*strains**......................................................................................................*83*
3.2*****Mouse*mating*strategies*...................................................................................*84*
3.3*****Mouse*embryos*....................................................................................................*86*
3.4*****DNA*extraction*.....................................................................................................*86*
3.4.1**DNA*extraction*from*tail*biopsies*......................................................*86*
3.4.2**Yolk*sac*DNA*extraction*........................................................................*87*
3.5*****Mice*Genotyping*by*the*PCR*reaction*..........................................................*87*
3.6*****Skeletal*preparations*........................................................................................*88*
3.7*****RNA*probe*synthesis*..........................................................................................*89*
3.7.1**Transformation*........................................................................................*89*
3.7.2**Mini[prep*....................................................................................................*89*
3.7.3**Plasmid*linearization*.............................................................................*90*
3.7.4**In*vitro*transcription*.............................................................................*90*
3.8*****Whole*mount*In*Situ*Hybridization*..............................................................*90*
3.9*****Paraffin*embedding*and*histological*sections*..........................................*92*
3.10**Araldite*embedding*and*semithin*sections*................................................*92*
3.11**In*Situ*Hybridization*in*sections*....................................................................*93*
3.12**LacZ*staining*.........................................................................................................*94*
3.13**Tamoxifen*administration*...............................................................................*95*
3.14**mRNA*extraction*.................................................................................................*95*
3.15**Retro*Transcription*............................................................................................*96*
3.16**Quantitative*PCR*..................................................................................................*96*
3.17**Generation*of*antisense*probes*for*ISH*by*PCR**.......................................*97*
3.18**Isolation*and*cloning*of*the*Sp8*coding*sequence*...................................*98*
3.19**TUNEL*assay*.......................................................................................................*100*
3.20**Immunohistochemistry*..................................................................................*100*
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4.*RESULTS*...................................................................................................................................*105*
4.1*****Analysis*of*the*expression*of*Sp6$in*the*absence*of*Sp8$*....................*106*
4.2*****Limb*phenotype*of*Sp6;Sp8$compound*mutants*...................................*107*
4.3*****Relative*expression*levels*of*Sp6$and*Sp8*in*E10.5*mouse*limbs*....*113*
4.4*****Selection*of*a*limb*ectoderm*specific*Cre*line*and*monitorization**
of*its*activity*......................................................................................................*114*
4.5*****Conditional*inactivation*of*Sp8$with*the*Ap2
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null*background:*Morphological*characterization*of*the**
compound*mutants*by*skeletal*staining*..................................................*116*
4.6*****Conditional*inactivation*of*Sp8$with*the*Msx2)Cre$line*in*an*Sp6**
null*background:*Morphological*characterization*of*the**
compound*mutants*by*skeletal*staining.*.................................................*118*
4.7*****Molecular*and*morphological*characterization*of*Sp6;Sp8$$
double*mutant*limb*buds*..............................................................................*121*
4.7.1**Molecular*characterization*of*Sp6)/);Sp8CreERT2/CreERT2$
$double*mutant*AER*..............................................................................*123*
4.7.2**Cell*death*in*the*developing*limb*bud*in*absence**
of*Sp6$and*Sp8*........................................................................................*127*
4.7.3**Morphological*characterization*of*Sp6)/);Sp8CreERT2/CreERT2$
$double*mutant*AER*..............................................................................*128*
4.7.4**DV*pattern*establishment*in*Sp6)/);Sp8CreERT2/CreERT2$
$double*mutant*limb*buds*..................................................................*130*
4.8*****Molecular*characterization*of*Sp6)/);Sp8+/CreERT2$mutant**
limb*buds**...........................................................................................................*133$
4.8.1**Gene*expression*analysis*of*the*Sp6)/);Sp8+/CreERT2$$
mutant*AER*.............................................................................................*133*
4.8.2*Sp6*and*Sp8*and*the*Tp63*network*leading*to*SHFM*................*136*
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4.8.3**Dorso[Ventral*pattern*establishment*in*Sp6)/);Sp8+/CreERT2$
mutant*limb*buds*..................................................................................*137*
.* 4.9*****Temporally*controlled*deletion*of*genes*specifically*expressed**
in*the*limb*bud*ectoderm*with*the*Sp8CreERT2*line*.................................*139*
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β
)catenin*Gain*of*function*with*the*Sp8CreERT2*line*.................................*140*
5.**DISCUSSION*...........................................................................................................................*147*
5.1*****Sp6*and*Sp8*are*conjointly*required*in*a*redundant**
and*dose*dependent*manner*for*limb*development*...........................*147*
5.2*****Sp6*and*Sp8*are*required*for*Fgf8*induction*in*the*AER*....................*150*
5.3*****Role*of*Sp6*and*Sp8*in*Dorso[Ventral*pattern*establishment.*..........*153*
5.4****Sp6/Sp8*and*Split*hand/foot*malformation*............................................*156*
6.**CONCLUSIONS*.......................................................................................................................*161*
7.**CONCLUSIONES*.....................................................................................................................*165*
8.**REFERENCES*..........................................................................................................................*169*
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ABBREVIATIONS*
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AER Apical Ectodermal Ridge
AP Antero-Posterior
BMP Bone Morphogenetic Protein
DV Dorso-Ventral
Dlx Distal-less homeobox
E Embryonic day
En1 Engrailed-1
FGF Fibroblast Growth Factor
h Hour
GOF Gain of function
HH Hamburger and Hamilton
IHC Immunohistochemistry
ISH In situ hybridization
KO Knock Out
LPM Lateral Plate Mesoderm
Lmx1b LIM homeobox transcription factor 1 beta
LOF Loss of function
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Msx msh Homeobox
min Minute
ON Over night
PBS Phosphate Buffered Saline
PD Proximo-Distal
PCR Polymerase chain reaction
PFA Paraformaldehyde
RA Retinoic Acid
RE Restriction enzyme
RT Room temperature
SEM Scanning Electron Microscopy
SHFM Split hand/foot malformation
Shh Sonic hedgehog
Sp Specificity Protein
Tam Tamoxifen
Tp63 Tumor Protein 63
TUNEL Terminal deoxynucleotidyl transferase–mediated dUTP nick end
labeling
ZPA Zone of Polarizing Activity
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RESUMEN*
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RESUMEN*
Durante!el!desarrollo,!la!formación!de!un!nuevo!organismo!resulta!de!la!
combinación! coordinada! de! múltiples! procesos! como! son! especificación,!
crecimiento!y!muerte!celular.!Entre!los!principales!modelos!para!el!estudio!de!
esos!procesos!se!encuentra!la!extremidad,!puesto!que!no!constituye!un!órgano!
vital!y!puede!ser!genética!o!experimentalmente!manipulada!sin!comprometer!la!
supervivencia!del!embrión.!!
Las! extremidades! se! forman! a! partir! de! pequeños! abultamientos! que!
aparecen! en! la! pared! lateral! del! cuerpo! embrionario! y! están! formadas! por!
mesodermo! recubierto! por! una! capa! de! ectodermo.! Estos! abultamientos,!
llamados!esbozos!de!la!extremidad,!bajo!la!influencia!de!los!llamados!“centros!
señalizadores”!forman!una!extremidad!completa!y!con!un!patrón!perfecto.!!
Uno!de!los!centros!señalizadores!mas!importantes!en!el!desarrollo!de!la!
extremidad!es!la!cresta!ectodérmica!apical!(AER),!que!dirige!el!crecimiento!a!lo!
largo!del!eje!próximo?distal.!Se!trata!de!un!epitelio!situado!en!el!extremo!mas!
distal! de! la! extremidad! en! desarrollo.! El! hecho! de! que! el! desarrollo! de! la!
extremidad! quede! truncado! cuando! la! AER! es! dañada! o! extirpada!
quirúrgicamente,! demuestra! claramente! su!necesidad! para! el! correcto!
desarrollo!de!la!extremidad.!La!función!principal!de!este!centro!señalizador!es!la!
de!regular!la!correcta!expresión!génica,!supervivencia!celular!y!proliferación!en!
el!mesodermo!subyacente!a!ella.!Estas!funciones!son!llevadas!a!cabo!mediante!la!
producción! de! diversos! miembros! de! la! familia! de! Factores! de! Crecimiento!
Fibroblástico!(Fgfs).!
La! formación! de! la! AER! comienza! con! la! inducción! de! las! células!
precursoras!de!la!misma.!En!ratón,!estas!células!están!inicialmente!localizadas!
en! el! ectodermo! ventral! del! esbozo! de! extremidad! y! mediante! movimientos!
morfogenéticos! quedan! compactadas! en! el! límite! dorso?ventral,! dando! lugar! a!
un! epitelio! poliestratificado! denominado! AER! madura.! Se! trata! de!un! proceso!
muy!complejo,!estrechamente!ligado!al!inicio!del!esbozo!de!la!extremidad!y!al!
establecimiento!del!eje!dorso?ventral.!Dicho!proceso!está!dirigido!por!complejas!
interacciones!entre!diferentes!vías!de!señalización,!entre!las!que!cabe!destacar!
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las!vías!de!señalización!por!Bmp,!Wnt/β?catenina!y!Fgf,!que!actúan!tanto!en!el!
ectodermo!como!entre!los!componentes!del!ectodermo!y!del!mesodermo.!
Actualmente!se!acepta!que!una!actividad!Wnt/β?catenina,!en!ectodermo,!
es!necesaria!tanto!para!la!inducción!de!la!AER!como!para!su!mantenimiento.!Por!
otra!parte,!la!señalización!por!Bmp!también!es!esencial!para!la!inducción!de!la!
AER,! actuando! probablemente! “upstream”!de! la! señalización! por! Wnt/β?
catenina.!Paradójicamente,!una!vez!que!la!AER!ha!sido!inducida,!la!señalización!
por! Bmp! se! vuelve!perjudicial! para! el! mantenimiento! de! ésta! y! termina! por!
tomar!parte!en!su!regresión.!
A! pesar! del! gran! número! de! estudios! que! hacen! referencia! a! los!
mecanismos! de! inducción! y! mantenimiento! de! la! AER,! pocos! factores! de!
transcripción! han! sido! descritos! como! mediadores! en! estos! procesos.! En!
particular,! dos! miembros! de! la! familia! de! factores! de! transcripción! Specificity!
Proteins! (SP),! Sp6! y! Sp8,! conocidos! también! como! Epiprofin! (Epfn)!y!
Buttonhead! (Btd)! respectivamente,! han! sido! descritos! como! imprescindibles!
para!el!mantenimiento!y!la!maduración!de!la!AER.!
Ambos! factores! de! transcripción! constan! de! un! patrón! de! expresión!
similar!en!el!ectodermo!del!esbozo!de!extremidad,!particularmente!en!la!AER.!En!
lo!que!concierne!a!la!formación!del!eje!próximo?distal,!diversos!estudios!sitúan!a!
estos! dos! factores! de! transcripción! “upstream”! de! Fgf8!y! “downstream”! de! la!
señalización!por!Wnt/β?catenina!en!el!ectodermo!de!la!extremidad,!indicando!su!
implicación!en!el!proceso!de!mantenimiento!y!maduración!de!la!AER.!
Las!extremidades!que!se!forman!en!ausencia!de!Sp6!se!caracterizan!por!la!
presencia! de! una!sindactilia! mesoaxial! en! las! extremidades! superiores! y! una!
sinostosis!en!las!inferiores,!además!de!una!dorsalización!parcial!en!la!punta!de!
los! dedos.! En! lo! referente! a! la! AER,! ésta! se! desarrolla! con! defectos! de!
maduración.! En! el! caso! de! los! mutantes! para! Sp8,! en! el! mejor! de! los! casos! el!
individuo! llega! a! nacer! pero! muere! en! el! periodo! perinatal! debido! a! defectos!
neurológicos,!siendo!así!letal!la!deleción!de!este!gen.!Las!extremidades!aparecen!
con!diferentes!grados!de!truncamiento!en!relación!a!una!regresión!prematura!de!
la!AER.!
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Debido!a!la!estrecha!relación!entre!los!genes!Sp6%y!Sp8!y!su!similar!patrón!
de!expresión,!am!factores!podrían!tener!una!función!redundante!en!el!desarrollo!
de!la!extremidad.!También!cabe!destacar!que!en!el!“Knock%out”!(KO)!de!Sp6!se!
mantiene!la!expresión!de!Sp8.!Debido!a!esta!posible!redundancia!en!la!función!
de!estos!dos!factores!de!transcripción,!es!necesaria!la!generación!del!doble!KO%
Sp6;Sp8!para!poder!esclarecer!su!en!el!desarrollo!de!la!extremidad.!
Por!todo!ello,!el!objetivo!de!la!presente!Tesis!Doctoral!es!el!análisis!de!la!
función! de! los! factores! de! transcripción! Sp6! y! Sp8,! así! como! su! posible!
redundancia!en!el!desarrollo!de!la!extremidad,!particularmente!en!la!inducción!
de!la!AER!y!en!el!establecimiento!del!patrón!antero?posterior.!Para!ello!hemos!
utilizado!el!ratón!como!animal!modelo,!para!la!generación!y!la!caracterización!
del! doble! mutante%Sp6;Sp8,! así! como! mutantes! condicionales! de! Sp8!en! un!
“background”!nulo!para!Sp6!con!el!uso!de!dos!líneas,!la!Ap2
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FCre!y!la!Msx2FCre.!
El!análisis!fenotípico!de!los!mutantes!y!su!caracterización!molecular!nos!
ha! permitido! determinar! el! requerimiento! de! Sp6!y! Sp8,! así! como! su!
redundancia,!en!el!desarrollo!de!la!extremidad.!En!ausencia!de!ambos!genes!o!
incluso!cuando!sólo!un!alelo!funcional!de!Sp6!está!presente!(en!ausencia!de!Sp8),!
las!extremidades!no!se!forman,!dando!lugar!a!fenotipos!amélicos.!La!pérdida!de!
un!alelo! de! Sp8!en!ausencia! de! Sp6,!da! lugar! a!un! fenotipo! reminiscente!de! la!
malformación! en! humanos! conocida! con! el! nombre! de! mano! hendida/pie!
hendido.! El! análisis! de! los! mutantes! demostró! que! ambos! factores! de!
transcripción! son! necesarios! de! una! forma! dosis! dependiente! aunque,!
probablemente,! por! su! mayor! nivel! de! expresión! Sp8! realiza! una! mayor!
contribución!que!Sp6!al!desarrollo!de!la!extremidad.!
Finalmente,! los! análisis! moleculares! mostraron! que! ambos! factores! de!
transcripción! son! necesarios! para! la! inducción! de! la! AER,! actuando! de! forma!
redundante!en!la!inducción!de!Fgf8!mediada!por!Wnt/β?catenina.!Además,!estos!
factores! podrían! estar! cooperando! con! la! vía! de! señalización! de! Bmp! en! la!
inducción!de!Engrailed1!y,!por!tanto,!actuando!en!el!establecimiento!del!patrón!
dorso?ventral!de!la!extremidad!
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1.*INTRODUCTION*
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Introduction!
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1.*Introduction*
Developmental! Biology! is! the! field! focused! in! the! understanding! of! the!
transformation! process! that! a! fertilized! egg! suffers! to! generate! a! remarkable!
ordered!cellular!diversity!before!becoming!an!adult!organism!able!to!ensure!the!
continuity!of!life!in!successive!generations.!Living!organisms!are!under!a!constant!
process!of!progressive!changes!termed!development,!process!that!never!ends!due!
to!the!constant!renewal!of!cells! that!constitute!the!adult!organism.!Although!the!
process!of!generating!an!adult!organism!from!a!single!cell!differs!between!different!
species,! several! aspects! can! be! gathered! under! the! same! generalized! features.!
Through!the!coordination!of!multiple!processes!such!as!specification,!growth!and!
programmed!cell!death,!development!leads!to!the!generation!of!new!organisms.!!
Embryology!is!the!discipline!inside!developmental!biology!that!studies!the!
development! of! the! organism! from! fertilization! to! the! fetus! stage.! During! this!
period! of! time! the! organism! is! called! embryo.! In! the! human,! the! term! embryo!
refers!to!the!developing!organism!from!the!moment!the!zygote!is!implanted!until!
the! eight! week! after! conception! (tenth! week! of! pregnancy),! from! this! point!
onwards!it! is! termed! fetus.! Embryology! is! focused! on! the! description! of! the!
processes! that! the! embryo! suffers! during! development,! while! developmental!
biology! centers! its! attention! trying! to! understand! the! causality! of! the!
developmental!processes!within!an!organism.!!
The! generation! of! a! new! organism! starts! with! the! fecundation.! In! this!
process,! the! fusion! of! the! genetic! material! provided! by! two! gametes! (the! sperm!
and!the!egg)!leads!to!the!formation!of!a!fertilized!egg!or!zygote.!Once!the!fertilized!
egg! is! formed,! a! phase! known! as! cleavage! occurs.! During! cleavage,! the! zygote!
undergoes!several!mitotic!divisions!and!develops!into!a!morula,!a!compact!sphere!
composed!of!blastomers.!This!phase!finishes!with!the!generation!of!the!blastula,!a!
fluid! filled! sphere! known! as! blastocoel! surrounded! by! a! cell! layer! termed! the!
trophoblast!that!nourishes!the!blastocyst!and!will!develop!the!embryonary!part!of!
Introduction!
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20!
the! placenta.! A! group! of! blastomers! concentrated! in! one! of! the! poles! of! the!
blastocyst!(the!animal!pole)!forms! the! inner! cell!mass!that!will! develop!into!the!
fetus.!
The! cleavage! is! followed! by! gastrulation.! The! gastrulation! is! the! phase!
where! the! gastrula! is! generated! and! drastic! cell! rearrangements! occur.! These!
cellular!movements!give!rise!to!the!formation!of!the!three!germ!layers!typical!of!
triploblastic! embryos:! ectoderm,! endoderm! and! mesoderm.! After! gastrulation,!
cellular! interactions! lead!to! the! generation! of! the! organs.! During! this! process!
termed!organogenesis!the!ectoderm,!the!outer!layer!of!the!gastrula,!is!responsible!
for!the!development! of! the! epidermis!and!the! nervous! system,! the!endoderm!or!
inner!layer!gives!rise!to!the!digestive!system!and!associated!organs,!such!as!liver,!
pancreas! and! lungs! and! the! mesoderm,! located! between! the! ectoderm! and! the!
endoderm,!produces!the!heart,!the!gonads!and!the!kidney!but!also!blood!cells!and!
vessels,!bones,!tendons!and!muscles.!!
During!development!a!single!cell,!the!zygote,!will!give!rise!to!more!than!200!
different!types!of!cells!that!will!form!part!of!the!more!than!30!different!organs!that!
constitute!about!more!than!10!systems!or!apparatus!present!in!an!organism.!The!
influence! of! different! signalling! pathways! orchestrates! the! generation! of! the!
cellular! diversity! and! pushes! cells! to! acquire! morphological! and! functional!
characteristics!according!to!their!different!fates.!All!these!interactions!enable!the!
generation! of! the! different! tissues! and! organs,! through! a! process! known! as!
differentiation.!
Previous!to!differentiation!cells!acquire!a!commitment!to!a!certain!fate.!This!
commitment! can! be! divided! into! two! states,! specification! and! determination.!
Specification!is!the!reversible!state!of!the!cells!that!precedes!determination.!In!this!
state! the! information! to! acquire! morphological! and! functional! characteristics!
according! to! a! specific! commitment! is! established.! However,! the! exposure! to!
different!signals!could!break!the!commitment!and!change!their!fate.!Determination!
Introduction!
!
21!
instead,!is!an!irreversible!state!and!is!often!closely!followed!by!differentiation.!A!
cell!is!determined!when!the!commitment!to!its!fate!cannot!be!broken,!the!cell!will!
continue! towards! the! specified! fate! even! when! transplanted! into! a! different!
environment.!
Despite! the! bewildering! number! of! cell! types! and! patterns! found! during!
development,! few! signalling! pathways! have! been! shown! to! be! responsible! for!
generating! them.! The! vast! majority! of! the! signalling! molecules,! ligand,! receptor!
and!signal!transducer!belong!to!the!same!five!families:!WNT,!Notch,!Transforming!
Growth! Factor?ß! (TGF?ß),! Fibroblast! Growth! Factor! (FGF)! and! Hedgehog! (Hh)!
families.! Hence,! developmental! biology! tries! to! understand! the! role! of! these!
signalling! pathways! during! development,! as! well! as,! the! interactions! amongst!
them.!
Over!many!decades,!animal!models!have!been!used!as!tools!to!figure!out!the!
regulatory! mechanisms! behind! developmental! processes.! Based! on! the! high!
homology!between!different!animal!models!and!humans,!the!use!of!these!models!
has! become! an! incredibly! helpful! tool! to! gain! insights! into! several! aspects! of!
human!biology!and!diseases.!
Since! vertebrate! limb! is! an! easily! accessible! organ! and! it’s! experimental!
manipulation!does!not!affect!embryo!survival,!it!has!become!a!popular!system!for!
the! study! of! the! mechanisms! orchestrating! the! organogenesis.! Grafting! and!
recombination!experiments!in!chicks!as!well!as!gene?targeted!manipulation!assays!
in! mice! have! provided! incredibly! helpful! insight! concerning! the! intricate!
interactions! between! the! different! signalling! pathways! that! pattern! the! limb.!
Moreover,! the! possibility! to! extrapolate! the! relation! amongst! the! different!
signalling! pathways! during! limb! development! to! other! developmental! process!
makes!the!limb!a!very!useful!tool.!!
*
Introduction!
!
!
22!
1.*1*Limb*development*
The!first!morphological!evidence!of!limb!development!is!appreciable!when!
differences! in! proliferation! rates! between! cells! of! the! flank! and! those! of! the!
prospective!limb!fields!occur.!By!the!time!proliferation!rates!are!maintained!in!the!
prospective!limb!area!but!decrease!in!the!flank!of!the!embryo,!cells!from!the!lateral!
plate! mesoderm! (LPM)! start! to! accumulate! under! the! ectoderm,! leading! to! the!
appearance!of!slight!bulges!in!the!lateral!body!wall!of!the!embryo!(Fig.!1)(Searls!
and!Janners,!1971).!
Figure*1.*The*limb*is*formed*by*cells*from*the*lateral*plate*mesoderm*(LPM;*
limb* skeletal* precursor)* and* from* the* somitic* mesoderm* (limb* muscle*
precursors).*Representation!of!a!16HH!chick!embryo!(left)!and!scanning!electron!
microscopy! (SEM)! section! (right).! The! SEM! photograph! corresponds! to! a!
transversal!section!at!the!level!of!the!wing!(indicated!with!a!red!arrow).!The!limb!
develops!from!the!LPM!and!the!muscular!precursors!cells!colonize!the!limb!form!
the! somitic! mesoderm.! The! intermediate! mesoderm! lies! between! them.! From!
Johnson!and!Tabin,!1997.!
!
Vertebrate! limb! development! is! a! complex! process! governed! by! several!
interactions!mainly!between!the!ectoderm!and!the!mesoderm!but!also!within!the!
ectoderm! and! within! the! mesoderm.! Interactions! amongst! multiple! signalling!
pathways! establish!pattern! along! the! three! main! axes! of! the! limb;! the! proximo?
distal! (PD)! axis! (from! the! shoulder/hip! to! the! tip! of! the! digits),! the! anterior?
posterior!(AP)!axis!(from!the!thumb!to!the!little!finger)!and!the!dorso?ventral!(DV)!
axis!(from!knuckle!to!palm)(Fig.!2A).!!
Introduction!
!
23!
Outgrowth! and! patterning! along! the! three! axes! is! controlled!by! three!
signalling! centers! that! arise! in! the! limb! bud! as! it! emerges! (Mariani! and! Martin,!
2003;! Niswander,! 2003;! Tickle,! 2003).! These! signalling! centers! are! the! Apical!
Ectodermal! Ridge! (AER),! the! Zone! of! Polarizing! Activity! (ZPA)! and! the! non?AER!
ectoderm.! These! signalling! centers! control! development! and! patterning! in! each!
one! of! the! three! axes! through! the! production! of! specific! signalling! molecules!
responsible!for!the!developmental!behavior!of!neighbouring!cells.!!
*
*
*
*
Figure*2.*Illustration*of*limb*axes*and*signalling*centers.*A)!Illustration!of!
a! mouse! embryo! depicting! the! three! spatial! limb! axes! Proximo?distal! (PD),!
anterio?posterior! (AP)! and! dorso?ventral! (DV).! B)! Schematic! representation!
of!a!mouse!limb!bud!showing!the!signalling!centers!AER!(yellow),!ZPA!(red)!
and!non?ectoderm!AER!(Green).*
!
Members!of!the!Fgf!family!emanating!from!the!AER!located!at!the!distal!tip!
of! the! limb,! promotes! outgrowth! and! patterning! of! the! limb! along! the! PD! axis!
(Saunders,!1948;!Sun!et%al.,!2002;!Boulet!et%al.,!2004;!Mariani!et%al.,%2008).!The!ZPA,!
a!signalling!center!formed!by!a!group!of!mesodermal!cells!located!at!the!posterior!
limb!border!drives!patterning!along!the!AP!axis!through!the!production!of!Sonic!
Hedgehog! (Shh)! (Saunders! and! Gasseling,! 1968;! Riddle! et% al.,! 1993;! Yang! et% al.,!
1997;!Gritli?Linde!et%al.,!2001;!Zeng!et%al.,!2001).!Finally,!the!non?AER!ectoderm!is!
responsible! for! DV! asymmetries! of! the! limb! through! the! specific! expression! of!
Wnt7a! in! the! dorsal! limb! ectoderm! and! Engrailed1! (En1)! in! the! ventral! limb!
ectoderm! (Fig.! 2B)(Parr! and! McMahon,! 1995;! Loomis! et% al.,! 1996;!Cygan% et% al.,!
1997).!Importantly,! coordination! and! interaction! amongst! these! centers! is!
Introduction!
!
!
24!
indispensable! for! proper! pattern! establishment! and! development! of! the! skeletal!
elements!that!form!the!limb!(Laufer!et%al.,!1994;!Niswander!et%al.,!1994;!Parr!and!
McMahon,!1995;!Yang!and!Niswander,!1995).!
The!limb!bud!mesoderm!is!mainly!composed!of!cells!from!the!LPM!that!will!
give! rise! to! the! connective! tissue! and! the! skeletal! precursors! (Michaud! et% al.,!
1997).! However,! from! a! very! early! stage! it! is! quite!heterogeneous! due! to! the!
colonization! of! endothelial! and! muscular! precursor! cells! from! the! somitic!
mesoderm!that!will!develop!into!muscles!and!tendons,!whereas!hair,!nails,!feathers!
and!eccrine!glands!are!ectodermal!derivatives!(Chevallier!et%al.,!1977;!Christ!et%al.,!
1977).!!
During! limb! development! mesenchymal! cells! condense! in! a! proximal! to!
distal! fashion! to! form! a! continuous! branching.! Later! on,! this! branching!
differentiates! into! cartilage! and! subsequent! joint! formation! leads! to! the!
segregation!of!individual!elements!(Cohn!and!Bright,!1999;!Shubin,!2002).!Finally,!
due! to! an! ossification! process,! cartilage! is! replaced! by! bone! and! the! basic!
organization!of!the!vertebrate!limb!is!acquired.!
Although! forelimbs! and! hindlimbs! differ! in! morphology! and! acquire! their!
own! and! unique! identity! during! development,! they! are! formed! by! homolog!
elements.! A! well?developed! vertebrate! limb! consists! of! three! main! skeletal!
segments!termed!in!a!proximal!to!distal!sequence,!the!stylopod,!the!zeugopod!and!
the!autopod.!The!stylopod!is!the!most!proximal!segment!and!contains!one!skeletal!
element,!the!humerus!in!the!forelimb!and!the!femur!in!the!hindlimd.!The!zeugopod!
corresponds!to!the!intermediate!skeletal!segment!localized!between!the!stylopod!
and!the!autopod.!It!contains!two!skeletal!elements,!radius!and!ulna!in!the!forelimb,!
and!tibia!and!fibula!in!the!hindlimb.!Finally!the!third!segment!corresponds!to!the!
autopod.!The!autopod!is!the!most!distal!element!of!the!limb!and!contains!several!
elements! as! carpals/tarsals,! metacarpals/metatarsals! and! phalanges! (from!
proximal! to! distal)(Fig.! 3).! While! the! stylopod! and! the! zeugopod! are! highly!
Introduction!
!
25!
conserved! across! tetrapods,! the! autopod! displays! a! more! variable! organization!
amongst!different!species.!!
*
Figure* 3.*Skeletal* elements* of* the* mouse* limb.* Alizarin! red! (bone)! and!
alcian!blue!(cartilage)!skeletal!staining!showing!the!forelimb!skeletal!pattern!
of!a!newborn!mouse.!The!stylopod!containing!a!unique!element!the!humerus,!
the! zeugopod! containing!the! ulna!(u)!and!the! radius!(ra)!and! the! autopod!
containing!carpals,! metacarpals! and! phalanges.! The! numbers! indicate!the!
identity!of!each!digit.!!
!
1.*2*Limb*initiation*
Although!limb?forming!capacity!before!getting!restricted!to!the!prospective!
limb!mesoderm!is!broadly!distributed!along!the!LPM,!limbs!arise!from!very!precise!
locations!along!the!body!axes!(Burke!et%al.,!1995;!Cohn!et%al.,!1997).!Interestingly,!
only! four! limbs! per! embryo! are! specified! in! all! vertebrates! and! they! are! always!
located!opposite!to!each!other!in!respect!to!the!midline.!!
In!the!chick,!the!limb!buds!are!first!appreciable!by!stage!16!of!Hamburger!
and! Hamilton! (HH;! Hamburger! and! Hamilton,! 1951)! after! 51?56! hours! of!
incubation,!while!in!the!mouse!they!are!first!evident!around!embryonic!day!E9.5!of!
gestation.! In! most! tetrapods,! forelimb! always! lies! at! the!cervical! to! thoracic!
transition!and!the!hindlimb!at!the!lumbosacral!transition!(Burke!et%al.,!1995).!The!
Introduction!
!
!
26!
protuberances!are!present!in!the!embryo!trunk!extending!opposite!to!somites!15?
20!in!chick!and!7?12!in!mouse!for!the!upper!limbs,!while!lower!limbs!developed!
opposite!to!somites!26?32!in!chick!and!23?28!in!mouse!(Fernandez?Teran!and!Ros,!
2008).!
!Prospective!limb!field!positioning!along!the!embryo!body!axes!is!under!a!
rigorous! specification! process! in! which! intricate!interactions! between! different!
molecules! establish!their! location.! Therefore,! limb! fields! and! thus! limbs,! do! not!
form! anywhere! along! the! body! axis.! Several! reports! have! proposed! different!
possible!candidates!for!the!specification!of!prospective!limb!fields,!although!this!is!
not!fully!clear.!!
Retinoic!acid!(RA)!was!proposed!as!the!most!upstream!molecule!involved!in!
limb! field! specification,! but! its! role! remains! quite! controversial.! Supernumerary!
limb!developed!after!administration!of!RA!in!mice!at!early!stages,!suggested!a!role!
for! RA! in! limb! induction! (Niederreither! et% al.,! 1996;! Rutledge! et% al.,! 1994).!
However,!it!was!later!demonstrated!that!RA!treatments!were!responsible!for!tail!
bud!duplication!rather!than!supernumerary!limb!formation.!The!absence!of!upper!
limbs!in!both,! mice! lacking!Retinaldehyde%dehydrogenase! (Raldh2;! gene!encoding!
the!enzyme!necessary!for!the!synthesis!of!RA)!(Niederreither!et%al.,!2002,!Zhao!et%
al.,!2009)!and!experiments!in!chick!where!RA!inhibitors!were!applied,!reinforced!
the!possible!role!of!RA!in!limb!induction!(Stratford!et%al.,!1996;!Helms!et%al.,!1996).!
Very!recently,!RA!has!been!proposed!to!be!required!prior!to!limb!bud!induction!to!
antagonize!Fgf8!signal!emanating!from!the!heart!and!the!caudal!proliferative!zone!
to!enable!limb!bud!induction!(Cunningham!et%al.,!2013).!!
Finally,! it! was! shown! that! RA! administration! led!to! homeotic! vertebral!
transformation,! due! to!the!alteration! of! the! expression! boundaries! of! Homeobox!
(Hox)!genes!in!the!paraxial!mesoderm.!This,!together!with!its!ability!to!induce!the!
expression!of!the!Hox%transcription!factors!“in%vitro”,!supports!that!RA!would!act!
Introduction!
!
27!
upstream! of! Hox!genes! in! the! initiation! of! limb! development! (Kessel! and! Gruss,!
1990;!Deschamps,!2007).!
Hox!genes!were!first!identified!in!Drosophila%melanogaster.!They!can!appear!
dispersed! in! the! genome! or! as! a! single! Hox! complex! or! cluster! composed! of! 8!
transcription! factors! arranged! in! a! split! tandem! as! in! D.% melanogaster.! Tetrapod!
vertebrates!can!have!up!to!13!Hox!transcription!factors!arranged!in!tandem!and!
organized!in!4!different!clusters!(HoxA?D)!(Duboule!and!Dolle,!1989;!Tarchini!and!
Duboule,!2006)(Fig.!4).!!
!
Figure* 4.* Hox* genes* in* Drosophila*and*their* phylogenetic*
counterparts* in* mouse. In Drosophila!Hox! cluster! is! composed! of! 8!
genes!arranged!in!tandem!in!a!split?cluster.!In!the!mouse!there!are!39!Hox!
genes!organized!in!four!clusters!A,!B,!C!and!D!in!four!chromosomes!(6,!11,!
15!and!2! indicated!under! each!cluster).!Three! sets!of! paralogs!and! their!
corresponding! ancestral! genes! are! designated! in! color! boxes.! Spatial!
colinearity! with! the! embryonic! craneo?caudal! axis! is! illustrated! (color!
match!between!genes!and!their!expression!domains!along!the!embryonic!
axis).!From!Pang!et%al.,!2010.!!
!
Within! a! single!cluster,! a! 3´! located! Hox!gene! is! expressed! first! during!
development!and!in!a!more!anterior!position!along!the!AP!axis!of!the!embryo!than!
a!5´!located!one!(Kessel!and!Gruss,!1990;!Duboule!and!Dolle,!1989;!Gaunt,!1988;!
Krumlauf,! 1992).! This! intrinsic! property! of! spatial! and! temporal! collinearity!
produces!nested! overlapping!expression! domains! of! the! Hox!genes! along!the! AP!
axis!of!the!vertebrate!embryos!in!correlation!with!their!genomic!arrangement.!The!
staggered! boundaries! in! the! expression! of! Hox!genes! have! been! shown! to! play!
Introduction!
!
!
28!
determinant!roles!in!the!specification!of!positional!identity!along!the!body!axis!of!
insect! segments! and! chordate! neural! tube! and! axial! skeleton,! because!
modifications! in! the! expression! patterns! of! Hox!genes! resulted! in! homeotic!
transformations!(Akam!et%al.,%1994;!Krumlauf,!1994).!
The! analysis! of! Hoxb9,! Hoxc9% and! Hoxd9!expression%in! the! LPM!after!
inducing! ectopic! limbs! in! chick,! suggested! the! involvement! of!the! staggered! and!
dynamic!expression!of!these!Hox9!paralogs!in!the!specification!of!the!prospective!
wing,!flank!and!leg!territories!(Cohn!et%al.,!1997).!In! addition,! Hoxb5,!Hoxc6!and!
Hoxc8! anterior! expression! boundaries! located! at! the! level! of! the! upper! limbs!
(Oliver! et% al.,! 1990;!Rancourt! et% al.,! 1995;!Nelson! et% al.,! 1996),! together! with!an!
anterior! shift! of! the! forelimbs! observed! in! mice! lacking% Hoxb5!supported! the!
implication! of! the! expression! boundaries! of! Hox! genes! in! limb! positioning!
(Rancourt! et% al.,! 1995).! Moreover,! lack! of! morphological! evidence! of! forelimb!
development!in!snakes!was!correlated!with!an!extended!expression!domain!of!Hox!
genes,!in!both!the!paraxial!and!the!LPM!(Cohn!&!Tickle,!1999).!!
Although!the!proposal!for!the!implication!of!Hox!genes!in!limb!positioning,!
gene!targeting!analysis!carried!out!in!mouse!disrupting!different!combinations!of!
Hox! genes! did! not! verify! the! involvement! of! any! member! of! this! family! in! the!
specification!of!limb!positioning.!The!only!case!regarding!the!role!of!Hox!genes!in!
this!aspect! is! the! mentioned! deletion! of! Hoxb5! in! mouse! (Rancourt! et% al.,! 1995).!
However,! a! cervico?thoracic! homeotic! transformation! could! account! for! the!
anterior!shift!of! the! forelimb.!In!spite! of! this!controversy,!Hox!genes!in! the! LPM!
have! been! shown! to! be! required! upstream! of! the! T?box!transcription! factors!
(Minguillon!et%al.,!2005).!!
The! members! of! the! T?box! family! of! transcription! factors! were!shown! to!
play!multiple!roles!during!limb!development!(Rallis!et%al.,!2005;!Papaioannou!and!
Silver!1998).!Expression!of!several!members!of!this!family!in!the!developing!limb!
was! reported! to! be!spatially! and! temporally! consistent! with! a! role! in! the! limb!
Introduction!
!
29!
specification!process!(Gibson?Brown!et%al.,!1996;!Gibson?Brown!et%al.,!1998;!Isaac!
et% al.,! 1998;! Logan! et% al.,!1998;! Takeuchi! et% al.,! 2003).! Further! analysis! in! mice,!
demonstrated!that!expression!of!Tbx5!in!the!forelimb!LPM!was!directly!regulated!
by! Hox!genes!supported! by! the! presence! of! Hox! binding! motifs! in! the% Tbx5!
promoter!(Minguillon!et%al.,!2012).!!
Tbx5!and! Tbx4% are%expressed! in! the! prospective! forelimb! and! hindlimb!
mesoderm! respectively! and! have! been! shown! to! be! required! for! forelimb! and!
hindlimb! induction! (Agarwal! et% al.,! 2003;! Rallis! et% al.,! 2003;! Naiche! and!
Papaioannou,! 2007).! Disruption! of! Tbx5!leading! to! absence! of! pectoral! fin!
induction! in! zebrafish! (Ahn! et% al.,! 2002;! Ng.% et% al.,! 2002;! Garrity! et% al.,!2002),!
together!with!missexpression!experiments!in!chick!and!lack!of!forelimbs!in!mice!in!
which!Tbx5!has!been!disrupted,!demonstrated!that!Tbx5!was!absolutely!required,!
and!sufficient,!for!forelimb!and!pectoral!fin!induction!(Ng%et%al.,!2002;!Takeuchi!et%
al.,!2003;!Rallis!et%al.,!2003).!!
It!has!been!assumed!that!Tbx4%in!the!hindlimb!function!similar!to!Tbx5!in!
the!forelimb.!However,!while!disruption!of!Tbx5!abolished!forelimb!development,!
in!the!absence!of!Tbx4!the!hindlimb!still!formed!(Hasson!et%al.,!2007;!Minguillon!et%
al.,!2005;!Minguillon! et% al.,!2009;!Naiche! and! Papaioannou,! 2007).! Thus,! some!
additional!factor!must!be!cooperating!with!Tbx4!for!hindlimb!induction!(Agarwal!
et%al.,!2003,!Rallis!et!al.,!2003,!Minguillon!et%al.,!2005).!In!chick,!hindlimb!restricted!
expression!of!the!paired!homeodomain!factor,!Pitx1,!was!shown!to!precede!that!of!
Tbx4.! Further! analysis! in! mice! demonstrated! that! it! was! responsible! for! Tbx4!
expression! and! that! both! were! conjointly! required! for! proper! hindlimb!
development! (Logan! and! Tabin,! 1999;! Kioussi!et% al.,! 1999;! Lanctot! et% al.,!1999;!
Szeto!et%al.,!1999;!Duboc!and!Logan,!2011).!
In!the!current!model!limb!induction!is!a!tightly!regulated!process!that!relies!
on!intricate!ephitelial?mesenchymal!interactions!in!which!a!Wnt/ß?catenin!activity!
restricts! the! limb! forming! ability! to! the! prospective! limb! mesoderm! where! a!
Introduction!
!
!
30!
regulatory! loop! between! Fgf!signalling! from! the! mesoderm! and! the! ectoderm!
components!of!the!limb!is!established!(reviewed!by!Tabin!1995).!The!ability!of!the!
members! of! the! Fgf! family! to! develop! ectopic! limbs! when! applied! to!the! flank!
(Cohn! et% al.,!1995;!Crossley! et% al.,! 1996;! Vogel! et% al.,! 1996)! together! with! the!
dynamic!expression!pattern!of!Fgf10!in!the!LPM!during!chick!development!focused!
the! attention! on! it! as! a! possible! candidate! responsible! for! the! induction! of! Fgf!
signalling! in! the! limb! ectoderm! (Ohuchi! et% al.,! 1997).! At! early! stages! of! chick!
development! prior! to! the! appearance! of! the! bud,! Fgf10!is! expressed! in! a! broad!
domain! of! the! LPM,! as! well! as,! in! the! segmental! plate! and! the! intermediate!
mesoderm.!However,!by!14HH!in!chick,!just!prior!to!limb!bud!appearance!and!Fgf8!
expression! in! the! limb! ectoderm,! Fgf10! gets! restricted! to! the! prospective! limb!
mesoderm!(Ohuchi!et%al.,!1997).!
Expression!of!Tbx5!and!Tbx4!was!shown!to!be!required!upstream!of!Fgf10!
in!the!prospective!limb!forelimb!and!hindlimb!mesoderm,!respectively!(Agarwal!et%
al.,!2003;!Rallis!et%al.,!2003;!Takeuchi!et%al.,!2003).!In!chick,!Tbx5!has!been!shown!
to! be! necessary! for! Wnt2b!expression! in! the! prospective! forelimb! mesoderm,!
whereas! Wnt8c%requires! previous! Tbx4! expression! in! the! hindlimb! prospective!
mesoderm! (Takeuchi! et% al.,! 2003).!In! chick,! Wnt2b!is! required!to! restrict! or!
maintain! Fgf10!expression! in! a!Wnt/ß?catenin! dependent! manner! at! the!
appropriate!level!of!the!LPM!where!forelimb!buds!developed,!while!Wnt8c!acts!in!
the!same!manner!in!the!hindlimb!(Kawakami!et%al.,!2001).!In!mouse,!mesodermal!
requirement!of!Wnt/β?catenin!activity!previous!to!restriction!of!Fgf10!expression!
to!the!LPM!has!been!also!suggested!(Kawakami!et%al.,!2011).!However,!the!epistatic!
relation!between!Tbx!and!Wnt/β?catenin!signalling!in!the!LPM!upstream!of!Fgf10!
is!not!clear.!Studies!in!mice!and!chick!support!the!requirement!of!the!Tbx!genes!
upstream! of! Wnt/β?catenin!signalling,! whereas! gene! inactivation! studies! in!
zebrafish!positioned! Wnt2b! upstream! of!Tbx5!(Ng.!et%al.,! 2002).!In! addition,!Hox!
genes! have! been! recently! involved! in! the! direct! regulation! of! Fgf10!in! the! LPM!
(Sheth!et%al.,!2013).!
Introduction!
!
31!
The! limbless! phenotype! of! mice! lacking! Fgf10! demonstrated! its!
requirement!for!limb!outgrowth!and!development,!consistent!with!a!failure!in!Fgf8!
induction! in! the! limb! ectoderm! (Min! et% al.,! 1998;! Sekine! et% al.,! 1999).! However,!
although!mice!lacking!Fgf10%have!no!limbs,!an!initial!bulge!is!formed!implying!the!
irrelevance!of!Fgf10%for!the!initial!accumulation!of!LPM!cells!under!the!ectoderm!
(Sekine!et%al.,!1999).!!
Once!Fgf10!expression!gets!restricted!to!the!prospective!limb!mesoderm,!it!
induces! Fgf8! expression! in! a! set! of! specific! cells! of! the! overlying! ectoderm,! in! a!
Wnt/β?catenin! dependent! manner.! The! induction! of! Fgf8! in! the! ectoderm!
establishes!a!positive!regulatory!feedback!loop!between!Fgf8!and!Fgf10!signalling!
(Ohuchi!et%al.,!1997;!Kawakami!et%al.,!2001).!Mesodermal!Fgf10!signalling!to!the!
ectoderm!through!its!receptor!Fgfr2b%(Xu!et%al.,%1998;!Arman!et%al.,!1999;!Yu!et%al.,!
2008;!Lu!et%al.,!2008)!induces!the!expression!of!Wnt3a!in!the!chick!limb!ectoderm!
(Kengaku!et%al.,!1998).!In!mouse,!gene?targeting!analysis!demonstrated!that!Wnt3!
fulfills!the!role!of!Wnt3a!in!chick!(Barrow!et%al.,!2003).!Next,!these!Wnt!signalling!
molecules! in! the! limb! ectoderm! activate! Fgf8! expression! in! certain! limb!
ectodermal! cells,! in! a! Wnt/β?catenin! dependent! manner! (Kengaku!et% al.,! 1998;!
Soshnikova!et%al.,!2003;!Barrow!et%al.,!2003;!Yu!et%al.,!2008;!Lu!et%al.,!2008).!As!the!
limb!grows!ectodermal!cells!expressing!Fgf8!developed!into!a!mature!AER.!Once!
Fgf8! is! induced! in! the! limb! ectoderm! it! maintains! Fgf10!expression! in! the!
underlying!mesoderm!(Ohuchi!et%al.,%1997),!establishing!a!positive!regulatory!loop!
between!Fgf8!in!the!ectoderm!and!Fgf10!in!the!mesoderm,!absolutely!required!for!
limb!bud!outgrowth!(Ohuchi!et%al.,!1997;!Xu!et%al.,!1998)(Fig.!5).!!
The! current! model! predicts! that! disruption! of! any! component! of! this!
regulatory!Wnt/Fgf!loop!will!result!in!limbless!phenotypes!due!to!a!failure!in!Fgf!
expression!in! the! limb! ectoderm,! as! occurs! in! mice! lacking! Tbx5! (Agarwal! et%al.,!
2003;!Rallis!et%al.,!2003),!Fgf10%(Min!et%al.,!1998;!Sekine!et%al.,!1999),!Fgfr2%(Xu!et%
al.,% 1998;! Arman! et% al.,! 1999;!Yu! et% al.,! 2008;! Lu! et% al.,! 2008),!Wnt/ß?catenin!
(Barrow!et%al.,!2003;!Soshnikova!et%al.,!2003)!or!Wnt3!(Barrow!et%al.,!2003).!!
Introduction!
!
!
32!
Figure* 5.* Illustration* of*
the* regulatory* cascade*
involved*in*limb*initiation.*
A*Hox!code! in! the! lateral!
plate!mesoderm!and!Pitx1!in!
the! hindlimb! territory!
acting! upstream! of!the! Tbx!
transcription! factors! and! a!
Wnt! activity! in! the! lateral!
plate!mesoderm,!Wnt2b!and!
Wnt8c!in! the! forelimb! and!
hindlimb! respectively,! are!
required! for!the! restriction!
of! Fgf10!expression! in! the!
prospective!limb!mesoderm.!
Fgf10!signalling!from! the!
mesoderm! induces! the!
expression!of!Wnt3!in!chick!
or! Wnt3a!in! mouse! limb!
ectoderm! that! in! turns!
activates!the!expression!of!Fgf8!in!the!limb!ectoderm!in!a!Wnt/β?catenin!dependent!manner.!Then,!
Fgf8!signalling!to!the!mesoderm!maintains!Fgf10!expression!and!a!regulatory!loop!is!established.*
So,! Somite;! IM,! Intermediate! mesoderm;! LPM,! Lateral! plate! mesoderm;! Ec,! Ectoderm.! Based! on!
Kawakami!et%al.,!2001.!
!
1.3*Signalling*centers**
1.3.1*Proximo[Distal*axis*and*the*AER**
The!AER!is!the!thickened!epithelium!running!along!the!DV!boundary!of!the!
limb!bud!(Fig.!6A),!responsible!for!the!PD!elongation!and!patterning!of!the!limb!
through! the! maintenance! of! the! underlying! mesenchyme! in! a! proliferative! and!
undifferentiated!state!(Saunders!et%al.,%1948;!Dudley!et%al.,!2002;!Sun!et%al.,!2002;!
Niswander!et%al.,%2003;!Tickle,%2003;!Boulet!et%al.,!2004;!Mariani!et%al.,%2008).!It!is!
Introduction!
!
33!
an! embryonic! transitory! and! very! dynamic! structure! subject! to! morphogenetic!
changes! during! development! (Guo! et% al.,! 2003).! A! mature! AER! consists! of!a!
thickened! pseudostratified! columnar! epithelium! in! birds!and! polystratified! in!
mammals!(Fig.!6B,C).!
!
!
*
*
*
*
*
Figure*6.* AER* Morphology.* 26HH! chick! limb! scanning!
microscopy!photograph!showing!a!distal!view!of!the!AER!
prominence!indicated!by!arrows!(A).!Semithin!sections!of!
20HH!chick!wing!where!the!pseudostrtaified!organization!
is!appreciable!(B)!and!E10.5!mouse!where!a!polystratified!
organization! is! visible!(B).! From! Fernandez?Teran! and!
Ros,!2008.!
!
Surgical!AER!removal!experiments!in!chick!demonstrated!the!importance!of!
this! structure! during! limb! development.! While! surgical! removal! during! early!
stages! of! limb! development! led! to! the! formation! of! severely! truncated! limbs,!
progressively! removal! at! later! stages! resulted! in! the! formation! of! more! distal!
elements! in! a! progressive! fashion.! These! experiments! unveiled! the! relevance! of!
this! signalling! center! during! limb! outgrowth! along! the! PD! axis! (Saunders,! 1948;!
Rowe!et%al.,!1982;!Cohn!et%al.,!1995).!Since!limb!PD!outgrowth!and!patterning!was!
shown! to! be! an! AER! dependent! developmental! process,! understanding! the!
Introduction!
!
!
34!
processes!concerning!its!formation!and!maintenance,!as!well!as,!the!identification!
of!the!mechanism!involved!in!the!function!that!exerts!in!the!underlying!mesoderm!
has!became!one!of!the!major!objectives!within!the!limb!field.!
!Interestingly,! members! of! the! Fgf! family! were! shown! to! be! expressed!
within! the! AER! and! the! finding! that! beads! soaked! in! Fgf! induced! ectopic! limb!
formation! when! applied! into! the! flank! and! also! that! development! of! truncated!
limbs!after!AER!removal!was!rescued!by!either!application!of!Fgf!soaked!beads!or!
Fgf! expressing! cells,! demonstrated! that! AER! activity! was! mediated! by! Fgf!
signalling!(Fallon!et%al.,!1994;!Niswander!et%al.,!1993).!
Several! members! of! the! Fgf! family! Fgf4,! Fgf8,! Fgf9!and! Fgf17!exhibits! a!
restricted!pattern!of!expression!in!the!AER!and!are!termed!the!AER?Fgfs.!However,!
while!Fgf8!expression!spatially!and!temporally!accompanies!the!whole!existence!of!
the! AER,! the! rest! of! AER?Fgfs! showed! a! lower! level! of! expression! and! a! more!
posteriorly!restricted!pattern!of!expression!in!time!and!space!(Heikinheimo!et%al.,!
1994;!Ohuchi!et%al.,!1994;!Mahmood!et%al.,!1995;!Crossley!et%al.,!1995).!!
Combinatorial! deletions! of! the! different! AER?Fgfs! demonstrated! that! they!
are!functionally!redundant!(Fig.!7)!(Sun!et%al.,!2000;!Mariani!et%al.,!2008).!Fgf8!is!
the!major!contributor!to!the!Fgf!dose!provided!by!the!AER,!followed!by!Fgf4,!Fgf9!
and!Fgf17!and!it!is!considered!the!principal!mediator!of!AER!function,!because!it!is!
sufficient! for! proper! limb! development! (Mariani! et% al.,! 2008).! Amongst!the! Fgfs!
expressed! in! the! AER,! Fgf8! is! the! only! one! that! leads! to! limb! defects! when!
disrupted! (Moon!and! Capecchi,! 2000;! Lewandoski! et% al.,! 2000;! Sun! et% al.,! 2002;!
Mariani!et%al.,!2008).!!
In! absence! of! Fgf8,! the! limb! bud! is! reduced! in! size! and! exhibited!aplasia! or!
hypoplasia!of!specific!skeletal!elements!(Moon!and!Capecchi,!2000;!Lewandoski!et%
al.,!2000).!Remarkably,!additional!removal!of!Fgf4!resulted!in!amelic!phenotypes!
implying!that!the!AER!exerts!its!function!through!the!production!of!the!members!
of! Fgf! family! (Sun! et% al.,!2002;! Boulet! et% al.,!2004).! In! addition,! the! mild! limb!
Introduction!
!
35!
phenotype!observed!after!Fgf8!removal!can!be!explained!due!to!the!upregulation!
of!Fgf4!that!was!expressed!earlier!and!in!a!more!anterior!fashion!than!in!normal!
circumstances!(Moon!and!Capecchi,!2000).!
!
Figure* 8.* Effects* of* AER[Fgf*
inactivation* in*mouse*limb*
development.!A)! Forelimb! (a)!
and! hindlimb! (b)! skeleton!
phenotype! of!Fgf4;Fgf9;Fgf17!
triple! KO!mutants.! B)!
Combinations!of! different! AER?
Fgf! inactivation! leading! to! limb!
phenotype.! a´!corresponds! to!
control,!b´!to!Fgf8;Fgf17!DKO,!c´!
to!Fgf8;Fgf9!DKO!(Asterisks!in!b´!
and!c´!indicates!that!the!mutant!
autopod!has!only!four!digits),!d´!
Fgf8;Fgf4!DKO,! e,! and! f´!
illustrates! the! two! different!
phenotypes!obtained!for!Fgf8;Fgf4%DKO;Fgf9F/+!the!more!and!the!less!common!respectively!(the!
inset!is!a!higher!magnification!of!the!distal!elements!and!the!bracket!in!f!indicates!a!gap!between!
the!distal!humerus!and!the!digit?like!element),!g´!Fgf8;Fgf4;Fgf9F/F!TKO.!From!Mariani!et%al,!2008.!
*
1.3.1.1*Fgf*signalling*
Fgf!is!a!large!family!composed!at!least!of!23!known!different!growth!factors!
in! which!alternative! splicing! produces! further! diversity! (Martin,! 1998;! Ornitz,!
2000;!Ornitz!and!Marie,!2002).!Signalling!by!Fgf!members!is!mediated!through!4!
different!fibroblast!growth!factor!receptor!(Fgfr).!Each!of!Fgfr!encodes!a!tyrosine!
kynase! receptor!which!is! further! diversified! by! the! production! of! alternative!
spliced!forms!containing!2!to!3!inmunoglobulin!(Ig)!extracellular!domains!(Hou!et%
al.,!1991;!Werner!et%al.,!1992;!Orr?Urtreger!et%al.,!1993;!Ornitz!et%al.,!1996).!The!
Introduction!
!
!
36!
alternative!splicing!isoforms!have!different!binding!affinities!for!the!different!Fgfs.!
Fgfr!are!selectively!distributed!in! the! limb! bud!(Orr?Urtreger!et% al.,! 1993),!Fgfr1!
expression! is! restricted! to! the! limb! mesoderm! (Partanen! et% al.,! 1998),! different!
isoforms!of!the!Fgfr2!are!expressed!in!both!the!mesoderm!and!the!AER!(Xu!et%al.,!
1998)!and!Fgfr3!is!expressed!in!the!growth!plate!of!long!bones,!whereas!Fgfr4!is!
not!expressed! in! the!limb! bud!(Weinstein! et%al.,! 1998).! The!distribution! and! the!
high!variability!of!both,!the!ligands!and!the!receptors,!together!with!the!different!
affinities!between!them!increase!the!complexity!of!the!Fgf!signallig!pathway.!
Intracellular! signal! transduction! downstream! of! Fgf! signalling!is! highly!
variable!and! leads! to! the! phosphorylation! of! several! target! signal!transducers.!
Heparin! Sulfate! Proteoglycans! (HSPG)! promotes! ligand! binding! and! receptor!
dimerization! (Ornitz,! 2000;! Lin,! 2004).! Dimerization! and! subsequent!
autophosphorylation! of! the! receptor! activates!the!Growth! factor! receptor?bound!
protein!(Grb2)!or!the!Fgfr!stimulated!Grb2!binding!protein!(Frs2)!and!leads!to!the!
formation!of!membrane!associated!complexes!responsible!for!the!recruitment!of!
either!Ras!or!phosphatydil?inositol!3´?OH!kynase!(PI3K).!This!recruitments!leads!
to! the! activation! of! the! mitogen! activated! protein! kynase! pathway! (MAPK)! that!
includes!ERK,!JNK!or!p38!signal!transducers!(Roux!and!Blenis,!2004)!or!the!AKT!
pathway,!respectively!(Datta!et%al.,!1999).!In!addition,!Fgfr!signal!transduction!can!
act!through!JAK/STAT,!as!well!as,!PLC?γ1!(Mohammadi!et%al.,!1991).!
*
1.3.1.2*Proximo[Distal*patterning*
Despite! intensive! studies! have! given! important! clues! regarding! different!
signalling! molecules! and! transcription! factors! involved! in! limb! PD! pattern!
establishment,!the!mechanisms!behind!are!still!unknown!and!several!models!have!
been! proposed.! Interestingly,! Meis1,! Hoxa11!and! Hoxa13!exhibit!a! restricted!
pattern!of!expression!in!the!stylopod,!the!zeugopod!and!autopod!respectively,!and!
Introduction!
!
37!
are!considered!the!best!markers!of!each!of!the!limb!segments,!although!they!are!
not!responsible!for!their!specification!(Tabin!and!Wolpert,!2007).!
Experiments! in! chick,! in! which! the! AER! was! progressively! removed! at!
different!stages!resulting!in!progressively!more!distal!truncations!corresponding!
to!later!AER!removals,!led!to!the!proposal!of!the!first!model!to!explain!the!manner!
in!which!the!limb!is!specified!along!its!PD!axis,!the!Progress*zone*model*(PZM)!
(Summerbell!et%al.,!1973).!According!to!this!model,!mesodermal!cells!located!under!
the!AER!form!the!progress!zone!(PZ).!Fgf!emanating!from!the!AER!maintains!the!
cells! of! the! PZ! in! a! continuous! proliferative! an! undifferentiated! state.! While!the!
limb!grows,!the!size!of!the!PZ!remains!constant!and!cells!from!PZ!are!progressively!
released! from! the! influence! of! the! AER! and! start! to! differentiate.! This! model!
predicts!that!the!longer!the!cells!stay!in!the!PZ!under!the!influence!of!the!AER,!the!
more!distal!elements!they!are!specified!into.!It!also!assumes!that!distal!elements!
are!specified!after!the!specification!of!the!proximal!ones!and!also!that!the!number!
of!divisions!should!be!the!way!by!which!cells!from!the!PZ!realize!the!time!spent!in!
the!PZ.!
Transplantation! experiments! where! the! PZ! from! an! old! chick! wing! was!
transplanted!into!a!young!chick!wing!and!vice%versa!led!to!loss!and!duplication!of!
limb! elements! respectively! (Summerbell! and! Lewis,! 1975),! reinforcing! the!
prevalence! of! the! PZM.! After! 30! years,! conditional! disruption! of! Fgf8!and! Fgf4!
resulted!in! a! phenotype! incompatible!with!the!PZM!and!led!to!the!proposal!of!a!
new!model,!the!Early*specification*model.!Conditional!removal!of!Fgf8!and!Fgf4!
was!performed!with!the!use!of!the!Msx2FCre!line.!The!kinetics!of!this!line!enables!
the! removal! of! both! Fgfs! prior! to! their! induction! in! the! hindlimb,! while! allowed!
early! and! transient! expression! of! both! Fgfs! in! the! forelimb.! While! amelic!
phenotype! resulted! from! removal! of! both! Fgfs! in! the! hindlimb,! the! forelimb!
developed!the!three!segments!of!the!limb!with!proximal!elements!being!severely!
hypoplastic! and! one! or! two! perfectly! developed! digits!(Sun! et% al.,! 2002).! This!
phenotype! was! difficult! to! explain! with! the! PZM.! The! ESM! postulates! that! from!
Introduction!
!
!
38!
very!early!stages!the!three!segments!of!the!limb!are!already!specified!and!as!the!
limb!develops!they!expand.!This!model!proposed!that!AER!removal!experiments!
that! support! the! PZM! could! be! explained!through! the! increase! in!cell! death!
observed!in!the!distal!mesenchyme!after!AER!removal!(Rowe!et%al.,!1982;!Dudley!
et% al.,!2002).! The! lack! of! early! markers! for! the! progenitor! cells! of! the! specified!
segments!did!not!make!of!this!model!a!very!popular!model.!
The!Differentiation*front*model!was!proposed!with!the!aim!to!reconcile!
mouse!genetics!and!manipulation!experiments!in!chick!(Tabin!and!Wolpert,!2007).!
This!model!was!merely!based!in!the!main!characteristics!of!the!two?signal!model!
with!the!difference!that!it!postulates!that!the!state!of!the!early!limb!is!proximal!by!
default.!Specification!of!distal!elements!is!then!achieved!through!the!modification!
of!the!default!state!by!the!action!of!the!AER?Fgfs!as!indicated!by!the!induction!of!
zeugopod!and!autopod!markers,!Hoxa11!and!Hoxa13%respectively.!While!proximal!
cells! are! progressively! determined! distal! cells! are! maintained!in! an!
undifferentiated! state! through! the! influence! of! the! AER?Fgfs! until! they! are!
specified.! The!boundary! between! both!states! corresponds! to! the! differentiation!
front,!boundary!that!becomes!progressively!shifted!distally!as!the!limb!grows.!!
The!phenotype!resulting!from!the!triple!conditional!deletion!of!Fgf8;4;9+/F%
lacking! the! zeugopod! in! the! forelimb! while! reduced! stylopod! and! autopod! are!
developed! was! incompatible! with! the! previous! models! proposed! and! led! to! the!
formulation! of! the! Two[signal* model!(Mariani! et% al.,! 2008).! According! to! this!
model,! it! was! proposed! that! the! limb! bud! develops! under! the! influence! of!
instructive! opposing! diffusible! signals! emanating! from! the! trunk! and! the! AER,!
possibly!RA!and!Fgf!signalling!respectively.!RA!was!proposed!responsible!for!the!
specification! of! proximal! elements! and! Fgf! for! distal! elements.! Finally,! cellular!
interactions! between! proximal! and! distal! domains! might! be! responsible! for! the!
specification!of!the!zeugopod.!A!modified!version!of!this!model!has!been!proposed!
in!which!as!the!limb!bud!grows!the!distal!part!of!the!limb!get!rid!of!the!influence!of!
Introduction!
!
39!
the! proximalizing! factor! and! it! is! subdivided! into! zeugopod! and! autopod!
compartments!(Rosello?Diez!et%al.,!2011;!Cooper!et%al.,!2011).!
Interestingly,!regarding!RA!role!in!PD!establishment,!recent!analysis!carried!
out! in! zebrafish! and! mouse! where! RA! emanating! from! the! trunk! has! been!
abolished,! implies! that! RA! is! not! required! as! an! instructive! signal! for! proximal!
specification!of!limb!segments!as!assessed!by!Meis1/2!expression!in!the!proximal!
mesoderm.! Thereby,! argues! against! an! opposing! diffusible! gradient! between! RA!
and!Fgf,!where!RA!specifies!proximal!fates!(Cunningham!et%al.,!2013).!
!
1.3.2*Anterio[Posterior*axis*and*the*ZPA*
Limb! AP! axis! is! established! very! early! in! development! prior! to! the!
appearance! of! the! limb! bud! (Hamburger,! 1938).! Classical! experiment! in! chick!
demonstrated! that! the! asymmetry! of! the! vertebrate! limbs! along! its! AP! axis! was!
controlled! by! the! ZPA.! This! signalling! center! is! composed! of! mesodermal! cells!
located! at! the! posterior! limb! border.! The! ability! to! perform! mirror! image! digit!
duplications!when!transplanted!into!the!anterior!limb!mesenchyme,!demonstrated!
that!the!ZPA!was!the!signalling!center!responsible!for!the!establishment!of!the!limb!
polarity!along!the!AP!axis!(Saunders!and!Gasseling,!1968).!
The!first!model!to!explain!how!digit!identity!is!specified!was!the!classical!
Morphogen* gradient* model.! This! model! assumes! that!an! unknown! diffusible!
molecule!emanating!from!the!ZPA!was!responsible!for!the!AP!polarity!through!the!
establishment! of! a! concentration! gradient! along! the! AP! axis.! In! addition,! it!
proposed!that!digit!identity!corresponded!to!the!positional!value!acquired!by!the!
cells!according! to! their!position!within!the! gradient.! The! model!determined!that!
posterior!digits!required!the!highest!concentrations!of!the!morphogen!(Yang!et%al.,!
1997).!
!
Introduction!
!
!
40!
Mirror! image! digit! duplication! after! RA! administration! led! to! the!
assumption! that! RA! was! the! mediator! of! the! ZPA! responsible! for! AP! pattern!
establishment!(Tickle!et% al.,! 1982;!Tickle!et% al.,!1985;! Wanek!et%al.,!1991).! Later,!
Shh,!a!signalling!molecule!emanating! from!the!ZPA!establishing!a!gradient!along!
the!limb!AP!axis,!was!shown!to!be!the!morphogen!responsible!for!AP!limb!polarity!
(Riddle!et%al.,!1993;!Lopez?Martinez!et%al.,!1995;!Gritli?Linde!et%al.,!2001).!!
Further!analysis!demonstrated!that!Shh!elicited!the!same!effect!of!the!ZPA!
and!that!AP!duplications!resulting!from!RA!treatments!were!due!to!the!induction!
of!Shh!in! the! anterior!limb! mesenchyme! (Riddle! et%al.,! 1993;! Helms! et%al.,! 1996;!
Lopez?Martinez!et%al.,!1995;!Yang!et%al.,!1997).!!
Gene?targeting! analysis! in! mice! supported! the! role! of! Shh!as! the! critical!
morphogen!essential!for!AP!pattern!establishment!of!the!autopod!regulating!both!
digit!number!and!identity,!while!AP!patterning!of!the!stylopod!was!independent!of!
Shh.!In!the!absence!of!Shh!the!stylopod!is!developed!in!both!the!forelimb!and!the!
hindlimb.! However,! the! zeugopod! lacks! one! element! in! the! forelimb! and! the!
autopod!is!not!developed,!whereas!in!the!hindlimb!both!elements!of!the!zeugopod!
are!partially!present!but!fused!and!only!digit!1!is!developed!(Chiang!et%al.,!2001;!
Chiang!et%al.,!1996;!Kraus!et%al.,!2001).!!
!
1.3.2.1*Shh*Signalling**
Shh!signalling! transduction! is! mediated! by! its! receptor! Patched! (Ptc).! In!
absence!of!Shh,!Ptc%inhibits!the!trans?membrane!protein!Smoothened!(Smo)!and!
avoids! the! activation! of! target! genes.! Shh!binding! to! Ptc,!enables!Shh!signalling!
transduction! through! the! release!of! Smo.! In! vertebrates,! Gli1,! Gli2!and! Gli3%
transcription!factors%members! of! the! Glioma! associated! oncogenes! family,! were!
shown!to!mediate!Shh!signalling!transduction!downstream!of!Smo.!Since!the!limb!
was!correctly!developed!in!absence!of!Gli1!and!disruption!of!Gli2!lead!to!a!delay!in!
Introduction!
!
41!
ossification!(Mo!et%al.,!1997),!whereas!mice!lacking!Gli3!developed!polydactilous!
limbs! (Schimmang! et% al.,% 1992;! Hui!and! Joyner,! 1993),! Gli3!is!the! principal!
mediator!of!Shh!signalling!during!limb!development.!!
Gli3!expression!is!independent!of!Shh!signalling!and!is!uniformly!expressed!
along! the! AP! axis! of! the! limb,! except! in! the! ZPA! where! it! is! not! expressed.! In!
absence! of! Shh!signalling!cleavage! of! the! Gli3!full! length! (Gli3FL)!protein! to! a!
truncated! transcriptional! repressor! form! occurs! (Gli3R)! (Wang! et% al.,% 2000),!
whereas! Shh!signalling!inhibits! the! cleavage! of! the! Gli3FL! generating! a! Gli3R!
gradient!along!the!AP!axis!with!higher!concentrations!in!the!anterior!mesenchyme,!
translating!the!extracellular!gradient!of!Shh!into!an!opposite!intracellular!gradient!
of!Gli3R.!%Shh!dependent!gradient!of!Gli3R!was!shown!to!be!responsible!for!digit!
patterning!of!the!autopod!(Wang!et%al.,%2000).!Moreover,!the!analysis!of!Gli3!and!
the! double! Shh;Gli3! mutant! mice! that! display! the! same! polydactilous! phenotype!
(Huy!and!Joyner,!1993;!Litingtung!et%al.,%2002;!te!Welscher!et%al.,%2002),!suggested!
that! one! the! of! major! roles! of! Shh! during! limb! development! was! to! avoid! Gli3!
processing.!!
!
1.3.2.2*Digit*patterning**
Apart! from! the! classical! morphogen! model,! several! models! have! been!
proposed!in!order!to!explain!how!digit!patterning!(number!and!identity)!occurs.!In!
chick,!the!sequence!in!which!extra!digit!were!induced!in!mirror?image!duplications!
obtained! after! application! of! Shh!in! the! anterior! limb! mesenchyme,! led! to! the!
formulation! of! the! Promotion[morphogen* gradient* model.! This! model! is! a!
modification!of!the!morphogen!gradient!model!in!which!apart!of!the!concentration,!
the! exposure! time! to! Shh! is! also! critical! for! digit! identity! specification.! Anterior!
digits!are!specified!first!and!then!promoted!to!more!posterior!fates!as!the!exposure!
time!increases.!This!model!suggests!that!specification!of!posterior!digits!requires!a!
longer!exposure!time!to!higher!concentrations!of!Shh!than!anterior!ones!and!also!
Introduction!
!
!
42!
that! lower! concentrations! of! Shh! even! if! maintained! for! longer! are! not! able! to!
specify!posterior!digits!(Yang!et%al.,!1997).!
Later,! fate! map! experiments! determined! that! Shh! descendants’!cells!
contributed!to!the!formation!of!digits!4!and!5!and!half!of!digit!3.!In!addition,!it!was!
shown! that! these! digits! transcribed! Shh!for! longer! in! a! progressive! posterior!
fashion!and!also!that!they!have!been!exposed!to!Shh!for!a!longer!period!of!time!in!
an! autocrine! manner.! Thus,! the! most! posterior! digit! is! the! one! in! which!its!
precursors! have! transcribed! Shh!for! longer.! Based! on! these! observations,! the!
Temporal[spatial*gradient*model!was!proposed.!According!to!this,!a!gradient!of!
Shh!signalling!is!established!along!the!AP!axis,!where!longer!exposure!time!to!Shh!
signalling!is!critical!for!posterior!digit!identity.!Digit!1!specification!is!independent!
of! Shh!signalling,! digit! 2! requires! a! spatial! gradient! of! Shh!signalling,! digit! 3!
requires!a!spatial!and!temporal!gradient!and!digit!4!and!5!have!transcribed!Shh!for!
longer!and!requires!only!a!temporal!gradient!(Harfe!et%al.,!2004).!The!generation!of!
a!mutant!mice!with!normal!lasting!but!reduced!Shh!expression,!where!digit!2!was!
lost!but!digit!4!and!5!were!specified!supported!this!model!(Scherz!et%al.,!2007).!
More!recently,!the!analysis!of!mutant!mice!in!which!Shh!transcription!was!
arrested!at!different!time!points!without!affecting!its!level!of!expression!led!to!the!
proposal!of! the! Biphasic* model.! These! experiment! demonstrated! that! the!
reduction!in!digit!number!was!proportional!to!the!stage!at!which!Shh!expression!
was!arrested.!The!analysis!of!digit!condensations!in!this!mutants!showed!that!the!
order!in!which!digit!were!lost!(d3,!d5,!d2!and!d4)!was!the!inverse!to!normal!digit!
formation!(d4,!d2,!d5!and!d3).!According!to!this,!the!model!proposed!that!Shh!is!
required! for! the! specification! of! digit! identity! in! an! early! and! transitory! phase,!
while! the! expansion! of! the! digital! plate! requires! continuous! Shh!expression! in!
order!to!generate!sufficient!digit!precursor!cells!(Zhu!et%al.,!2008).!!
In! addition,! recent! studies! in! chick! led! to! the! transformation! of! the!
Promotion?morphogen!gradient!model!into!a!new!Growth[morphogen*model!in!
Introduction!
!
43!
which!Shh!through!the!control!of!cell?cycle!regulators!integrates!proliferation!and!
specification!of!digit!precursor!cells.!In!chick,!progressive!inhibition!at!early!stages!
of!Shh!signalling!with!the!use!of!the!Smo!inhibitor!Cyclopamine,!resulted!in!loss!of!
more!posterior!digits!and!confirmed!that!shorter!exposures!to!Shh!are!required!for!
the!specification!of!anterior!digits!in!comparison!to!posterior!digits!(Scherz!et%al.,!
2007;! Towers! et% al.,!2008).! In! addition,! blocking! proliferation! through! the!
application! of! Trichostatin! A! (TSA)! resulted! in! reduction! in! limb! size! with! only!
posterior! structures! present.! Application! of! TSA! abolished! Shh!expression,! but,!
when! the! effect! of! the! TSA! was! ended! Shh!expression! was! recovered! and!
maintained! as! in! non?manipulated! circumstances,! though! temporally! shifted,!
implying!that!Shh!expression!was!controlled!by!cell!proliferation!and!also!that!the!
recovery! of! Shh!signalling! after! TSA! application! was! enough! to! determine!
posterior!digit!identity!(Towers!et%al.,!2008).!
!
1.3.3*Dorso[Ventral*axis*and*the*non[AER*ectoderm*
The!presence!of!morphological!distinguishable!characteristics!in!the!dorsal!
and!ventral!regions!of!the!limb!reflects!its!established!DV!polarity.!In!mouse!limbs,!
hair!only!appears!on!the!dorsal!surface!of!the!autopod!and!nails!are!present!in!the!
dorsal!surface!of!digit!tips,!while!footpads!and!eccrine!glands!are!developed!in!the!
ventral!surface.!Moreover,!the!internal!DV!organization!is!also!appreciable!due!to!
the!dorsal!location!of!extensor!muscles!and!ventral!location!of!the!flexor!ones.!!
DV! patterning! establishment! is! a! complex! process! involving! interactions!
within!the!ectoderm!but!also!between!the!mesoderm!and!the!ectoderm.!The!stage!
at! which! the! limb! acquires! its! DV! polarity! is! not! fully! determined.! Surgical!
manipulation! experiments! in! chick! demonstrated! that! DV! polarity! of! both!
ectoderm! and! mesoderm! is! already! established! before! the! initial! bud! emerges!
(Altabef!et%al.,!1997;!Altabef!and!Tickle,!2002;!Kimmel!et%al.,!2000;!Michaud!et%al.,!
1997).!Two!different!phases!can!be!defined.!The!earliest!one!where!mesodermal!
Introduction!
!
!
44!
influences!impart!the!DV!limb!polarity!to!the!ectoderm,!whereas!a!latest!one!when!
this!information!has!been!acquired!by!the!ectoderm!and!this!impose!it!polarity!to!
the! limb! bud! mesoderm! (Kieny! et% al.,!1971;! Saunders!&! Reuss,! 1974;! Geduspan!
and!MacCabe,!1989).!!
!
1.3.3.1*Polarizing*the*limb*ectoderm*
Whether!the!information!required!for!the!establishment!of!DV!limb!polarity!
resides!in!the!mesoderm!remains!controversial.!Experiments!carried!out!by!Kieny!
and! colleagues! in! which! DV! inversion! of! presumptive! limb! mesoderm! grafted!
under! the! flank! ectoderm! resulted! in! limbs! with! the! same! DV! polarity! of! the!
ipsilateral!non?manipulated!ones,!suggested!that!influences!from!the!environment!
were! responsible! for! limb! DV! polarity! (Kieny! et% al.,!1971).! However,! similar!
grafting!experiment!carried!out!by!Saunders!and!Reuss!suggested!that!from!very!
early! stages! the! ability! to! establish! the! DV! polarity! of! the! limb! resides! in! the!
presumptive! limb! mesoderm.! In! these! experiments,! DV! inversion! of! 12HH! wing!
presumptive!mesoderm!transplanted!under!the!flank!ectoderm,!resulted!in!limbs!
with!reverse!DV!polarity.!In!contrast!to!Kienys!results,!this!DV!inversion!led!to!the!
assumption!that!DV!information!resides!in!the!presumptive!limb!mesoderm!by!this!
stage! and! that! the! mesoderm! was! able! to! impose! its! polarity! to! the! covering!
ectoderm!(Saunder!and!Reuss,!1974).!
In!view!of!the!controversial!results!obtained!in!the!experiments!mentioned!
above,! further! experiments! in! chick! where! bidorsal! limbs! developed! either! by!
positioning! the! limb! prospective! field! between! two! rows! of! somites! or! by! the!
insertion! of! a! barrier! between! the! prospective! limb! field! and! the! lateral!
somatopleure,!confirmed!the!hypothesis!that!signals!emanating!from!the!somitic!
mesoderm!and!the!LPM!were!responsible!for!the!DV!polarity!of!the!limb!between!
stage!13!and!15HH.!Le!Douarin!and!colleagues!proposed!that!molecules!emanating!
from! the! somitic! mesoderm! dorsalized! the! limb! ectoderm,! whereas! inductive!
Introduction!
!
45!
signals! emanating! from! the! somatopleure! exerted! a! ventralizing! effect! over! the!
limb! ectoderm! (Michaud! et% al.,! 1997).! These! experiments! led! to! a! possible!
explanation! for! the! different! results! obtained! by! previously! mentioned!
experiments!regarding!DV!polarity!establishment.!Since!the!ectoderm!covering!the!
somites! could! be! the! source! of! dorsalizing! factors! responsible! for! limb! dorsal!
identity,! it! is! possible! that! in! grafting! experiments! carried! out! by! Reuss! and!
Saunders!a!portion!of!somites!attached!to!the!presumptive!limb!mesoderm!when!
grafted!into!the!flank!led!to!the!reversed!DV!polarity!of!the!grafts.!Nevertheless,!
the!molecular!mechanisms!that!mediate!mesodermally!derived!signals!responsible!
for!limb!DV!polarity!remains!unknown.!Bmps!in!the!ventral!LPM!and!its!antagonist!
Noggin!in!the!somitic!mesoderm!were!proposed!as!candidates!for!polarizing!the!
ventral!and!dorsal!limb!ectoderm!respectively!(Michaud!et%al.,!1997).!!
Regarding! later! stages! in! development,! Geduspan! and! MacCabe´s!
experiments!in!chick!demonstrated!that!by!15/16HH!when!limb!bud!starts!to!be!
appreciable,! the! ability! to! polarize! the! limb! has! been! already! acquired! by! the!
ectoderm.! In! chick! 180º! rotation! of! the! limb! ectoderm! after! 16HH! led! to! DV!
inverted!limb!polarity,!while!inversion!before!this!stage!had!no!effect!implying!that!
the!information!required!for!DV!establishment!resides!in!the!ectoderm!by!16HH!
and!that!the!ectoderm!is!able!to!imposed!its!polarity!to!the!mesoderm.!Hence,!from!
this!stage!on!the!ectoderm!is!responsible!for!limb!DV!polarity.!!
!
1.3.3.2*The*non[AER*ectoderm**
The!ectoderm!covering!the!dorsal!and!ventral!sides!of!the!limbs!responsible!
for!DV!pattern!establishment!constitute!the!signalling!centre!called!the!non?AER!
ectoderm.! The! restricted! domains! of! expression! of! Wnt7a,% a! secreted! signalling!
molecule!member!of!the!Wnt!family,!expressed!in!the!dorsal!ectoderm!(Dealy!et%al.,!
1993;! Riddle! et% al.,! 1995)! and! the! homeodomein! transcription! factor! En1!
expressed! in! the! ventral! limb! ectoderm! (Davis! et% al.,! 1991;! Gardner! and! Baral,!
Introduction!
!
!
46!
1992),! are! responsible! for! the! DV! limb! pattern! establishment! and! impose! its!
polarity!to!the!underlying!mesoderm!(Fig.!8)!(Riddle!et%al.,!1995;!Vogel!et%al.,!1995;!
Parr! and! McMahon,! 1995;! Rodriguez?Esteban! et! al.,! 1997;!Cygan! et% al.,! 1997;!
Loomis!et%al.,!1996;!Loomis!et%al.,!1998).!!
Wnt7a%expression!in!the!dorsal!ectoderm!has!been!shown!to!be!required!for!
the! expression! of! the! LIM! homeodomain! transcription! factor! (Lmx1b)% in! the!
underlying!distal!dorsal!limb!mesenchyme!and!it!is!both!necessary!and!sufficient!
for!dorsal!limb!specification!as!revealed!by!gene!inactivation!studies!in!mouse!and!
misexpression!experiments!in!chick!(Parr!and!McMahon,!1995;!Riddle!et%al.,!1995;!
Vogel!et%al.,!1995;!Rodriguez?Esteban!et!al.,!1997).!!
!
!
Figure*8.*DV* pattern* establishment.*A)! Pattern! of! expression! of! the! genes!
involved! in! DV! pattern! establishment! in! ectodermal! and! mesodermal!
compartments.! B)! Schematic! model! of! the! genetic! interaction! responsible! for!
DV!pattern!establishment.*
!
Null! mutations! in! the! En1!gene! leads! to! dorsalization! of! the! ventral! limb!
ectoderm! due! to! ectopic! activation! of! Lmx1b! in! the! ventral! limb! mesoderm!
triggered!by! ectopic! Wnt7a! expression! in! the!ventral!ectoderm.!Remarkably,!the!
phenotype! of! the! compound! Wnt7a;En1! mutant! displayed! a! double! ventral! limb!
phenotype!very!similar!to!that!of!Wnt7a!mutant!(Wurst!et%al.,!1994;!Loomis!et%al.,!
1996;!Cygan!et%al.,!1997;!Loomis!et%al.,!1998).!In!addition,!En1!overexpression!in!
Introduction!
!
47!
chick! limb! dorsal! ectoderm! was!shown! to! repress! Wnt7a!expression! with! the!
subsequent!downregulation!of!Lmx1b!in!the!mesoderm!(Logan!et%al.,!1997;!Laufer!
et% al.,! 1997).! Hence,! En1! expression! in! the! ventral! ectoderm! confers! ventral!
identity!through!the!restriction!of!Wnt7a!to!the!dorsal!ectoderm!that!is!responsible!
for! limb! dorsal! identity! through! its! requirement! for! ! Lmx1b!expression! in! the!
underlying!dorsal!limb!mesoderm!(Fig.!8B)(Davis!et%al.,!1991;!Gardner!and!Barald,!
1992;!Parr!and!McMahon*et%al.,!1995).!
! Manipulation!experiments!and!gene?targeting!analysis!in!chick!and!mouse!
respectively!suggest!that!additional!molecules!to!Wnt7,!En1!and!Lmx1b!might!be!
involved! in! DV! pattern! establishment! (Pautou! and! Kieny,! 1973;! MacCabe! et% al.,!
1974;! Geduspan! and! MacCabe,! 1989;! Akita,! 1996).! Interestingly,! Lmx1b!is!
expressed!all!over!the!dorsal!mesoderm,!but!only!the!expression!on!the!distal!part!
of!the!limb!depends!on!Wnt7a!expression!in!the!dorsal!ectoderm,!as!revealed!by!
the!lack!of!Lmx1b!expression!in!the!most!distal!part!of!the!limb!in!Wnt7a!mutant,!
from! E11.5!on!(Cygan! et% al.,! 1997;! Loomis! et% al.,! 1996;! Loomis! et% al.,! 1998).! In!
addition,!while!Wnt7a!dependent!Lmx1b!expression!is!restricted!to!the!most!distal!
part! of! the! limb,! ectopic! expression! of! Wnt7a! in! the! ventral! ectoderm! of! En1!
mutant!mice!leads!to!ectopic!expression!of!Lmx1b!all!over!the!ventral!mesoderm!
(Cygan!et%al.,!1997;!Loomis!et%al.,!1996;!Loomis!et%al.,!1998).!Finally,!after!16HH!in!
chick!the!later!the!ectoderm!is!inverted,!the!more!distally!restricted!DV!inversions!
are!developed,!implying!the!ability!of!the!ectoderm!to!polarize!only!the!distal!limb!
mesoderm!(Geduspan!and!MacCabe,!1989).!
!
1.3.3.3*Dorso[Ventral*boundaries*in*the*limb*ectoderm*
Chick?quail!chimeras!and!cell!labeling!experiments!in!chick,!as!well!as!gene?
targeting!manipulation!assays!in!mouse,!revealed!the!existence!of!three!different!
DV! boundaries! located! at! different! levels! in! the! AER! that! are! essential! for! its!
proper!development!(Fig.!9)(Michaud!et%al.,!1997;!Altabef!et%al.,!1997;!Altabef!and!
Tickle,!2002;!Kimmel!et%al.,!2000).!!
Introduction!
!
!
48!
Experiments! in! chick! demonstrated! the! existence! of! a! boundary! that!
operates!along!the!presumptive!limb!and!flank!ectoderm.!The!boundary!is!present!
at! least! from! the! anterior! part! of! the! wing! to! the! posterior! part! of! the! leg.! The!
presence! of! this! boundary! prevents! mixing! of! cells! and! splits! the! ectoderm! into!
dorsal! and! ventral! domains.! This! boundary! is! supposed! to! mark! the! location! at!
which! limbs! arise.! As! the! limbs! emerge! and! the! AER! develops,! the! boundary!
localize!into!the!DV!middle!extent!of!the!AER!(Michaud!et%al.,!1997;!Altabef!et%al.,!
1997;! Altabef! and! Tickle,! 2002).! However,! cell! lineage! restriction! along! this!
boundary! remains! quite! controversial.! While! chick?quail! graft! experiments!
supported!the!existence!of!a!sharp!boundary!localized!in!the!middle!DV!extent!of!
the! AER! avoiding! cell! mixing! between! dorsal! and! ventral! ectoderm,! DiI! cell!
marking!experiments!in!chick!showed!that!dorsal!AER!cells!extended!all!over!the!
AER!while!ventral!ectodermal!cells!were!restricted!to!the!ventral!half!of!the!AER!
(Michaud!et%al.,!1997;!Altabef!et%al.,!1997;!Altabef!and!Tickle,!2002).!!
The! use! of! Cre?LoxP! based! fate! maps! and! retroviral! cell?marking!
experiments!carried!out!in!mouse!supported!the!existence!of!a!boundary!located!at!
the! middle! of! the! AER.! The! boundary,! separates! En1!expressing! cells! that!
constitutes!the!ventral!half!of!the!AER!from!the!dorsal!half!of!the!AER!and!dorsal!
ectoderm! that! does!not! express! En1! (Kimmel! et% al.,! 2000).! Interestingly,! this!
boundary! is! established! previous! to! and! independently! of! En1!activity! in! the!
ectoderm!(Altabef!and!Tickle,!2002;!Kimmel!et%al.,!2000).!In!addition,!the!boundary!
starts! to! disappear! when! the! AER! reaches! its! maximum! height! by! E11.5.! Thus,!
based! on! this! transient! property! of! the! boundary! its! role! was! related! with! AER!
integrity,!by!the!stage!the!boundary!disappears!the!AER!starts!to!regress!(Kimmel!
et%al.,!2000).!Based!on!this!observation,!the!authors!proposed!that!the!controversy!
behind!the!experiments!carried!out!in!chick!in!which!dorsal!ectodermal!cells!were!
found! in! the! ventral! half! of! the! AER! in! DiI! cell! marking! experiments! could! be!
explained!due!to!later!analysis!compared!to!chimera!analysis.!
!
Introduction!
!
49!
Additional! experiments! in! mice! demonstrated! two! additional! boundaries!
present!in!the!AER.!At!very!early!stages!a!boundary!appears!at!the!dorsal!edge!of!
the!AER.!This!boundary!avoids!mixing!between!cells!expressing!Wnt7a!located!in!
the!dorsal!ectoderm!and!cells!that!do!not!express!Wnt7a.!The!other!one!appears!in!
the! ventral! edge! of! the! AER! when! the! AER! acquires! morphologically!
distinguishable!characteristics!(Kimmel!et%al.,!2000).!
!
*
Figure* 9.* Illustration* showing* the*
compartment* boundaries* and* also* gene*
expression* within* mouse* and* chick* AER.!
Cell!lineage!compartments! defined!by!mouse!
and! chick! experiments.! The! dashed! line!
represents! the! DV! midline! of! the! AER.! The!
extent!of!the!ventral!compartment!correlates!
with! En1!expression,! Fgf8! ! is! expressed!
through! the!entire!ridge! and! Wnt7a!is!
expressed! in! the! dorsal! ectoderm! whereas!
Radical% fringe!is! expressed! in! the! dorsal!
ectoderm!and!the!AER.!From!Tickle!2001.!
!
!
*
1.4.*AER*development*
Studies!in!chick!and!mouse!limb!buds!have!characterized!the!dynamics!of!
AER! morphology! (Todt! and! Fallon,! 1984;! Bell! et% al.,! 1998;! Loomis! et% al.,! 1998).!
Several!phases!can!be!considered!during!AER!development:!!AER!induction,!AER!
maturation!and!AER!regression.!
Introduction!
!
!
50!
1.4.1*AER*induction*
The!induction!of!the!AER!corresponds!to!the!specification!of!AER!precursor!
cells,!termed!the!“pre?AER”!cells.!In!this!phase!Fgf8!is!induced!in!a!patchy!pattern!
in!the!limb!ectoderm.!The!“pre?AER”!cells!are!defined!as!cells!that!express!Fgf8!but!
are! not! yet! anatomically! distinguishable! from! the! rest! of! the! neighbouring!
ectodermal!cells!(Bell!et%al.,!1998,!Loomis!et%al.,!1998).!By!E9!and!16HH!in!mice!
and!chick!respectively,!a!patchy!pattern!of!Fgf8!expression!becomes!appreciable!in!
the!limb!ectoderm.!While!in!mouse!this!initial!expression!of!Fgf8!is!restricted!to!
the!ventral!ectoderm!(Loomis!et%al.,!1998;!Kimmel!et%al.,!2000)!(Fig.10A),!in!chick!
it!is!induced!distally!in!both!dorsal!and!ventral!ectoderm!opposite!to!somites!15?
20! (Crossley! et% al.,! 1996,! Ohuchi! et% al.,! 1998).! As! previously! mentioned,! in! the!
current! model! Fgf10!signalling! from! the! LPM! is! absolutely! required! for! the!
induction!of!Fgf8!in!the!limb!ectoderm,!through!the!induction!of!Wnt3a!or!Wnt3!in!
chick!and!mouse!limb!ectoderm!respectively!(Ohuchi!et%al.,!1997;!Min!et%al.,!1998;!
Sekine! et% al.,! 1999;! Soshnikova! et% al.,! 2003;! Barrow! et% al.,! 2003),! in! which! Bmp!
signalling!plays!also!a!critical!role!(Ahn!et!al.,!2001;!Pizette!et%al.,!2001;!Soshnikova!
et%al.,!2003;!Pajni?Underwood!et%al.,!2007).!
*
1.4.2*AER*maturation*
Once!the!AER!is!induced!and!the!limb!develops,!a!“mature?AER”!is!formed.!
Morphogenetic!movements!of!the!ectoderm!compact!the!pre?AER!cells!towards!the!
DV!boundary!of!the!limb!bud.!In!mice,!by!E10!movement!and!compaction!of!“pre?
AER”! cells! located! first! in! the! ventral! ectoderm! developed! into! a! bi?layered!
epithelium! to! form! a! pronounced! thickening! of! the! ectoderm! close! to! the! DV!
interface!(Crossley!and!Martin,!1995;!Loomis!et%al.,!1998)(Fig.!10D).!At!E10.5?11!a!
mature?AER!is!first!appreciable!confined!along!the!DV!boundary!of!the!developing!
limb!and!formed!a!polystratified!epithelium!composed!of!3?4!cell!layers!(Meyer!et%
al.,!1997;!Bell!et%al.,!1998).!In!chick,!by!18HH!the!columnar!shape!of!AER!cells!in!
Introduction!
!
51!
the!wing!bud!ectoderm!makes!them!distinguishable!from!the!neighbouring! non?
AER!cells!that!exhibit!cuboidal!morphology.!Later!on!by!20HH!this!columnar!cells!
form!a!pseudostratified!epithelium,!the!mature!AER.!
!
!
Figure*10.*Fgf8*expression*during*early*mouse*limb*development.!Fgf8!is!first!detected!in!
the!ventral!limb!ectoderm!by!E9.!(A),!Fgf8!expression!spreads!along!the!ventral!limb!ectoderm!
occupying!a!broad!territory!(B?C)!and!becomes!confined!to!the!distal!tip!of!the!limb!by!E10.5!(D).!
From!Fernandez?Teran!and!Ros,!2008.!
!
1.4.3*AER*regression*
Finally,! regression! of! the! AER! occurs.! By! E11! once! the! AER! reaches! it!
maximum! height,! it! begins! to! regress! to! the! point! that! no! AER! descendants! are!
present!at!birth!as!revealed!by!cell!lineage!analysis!in!mice!(Guo!et%al.,!2003).!Fgf!
expression!in!the!AER!decays!starts!first!over!the!interdigital!spaces!and!remains!
over!the!digits!until!the!last!phalanges!are!laid!down!(Pizette!and!Niswander!et%al.,!
1999;!Ganan!et%al.,!1996;!Khokha!et%al.,!2003;!Zuñiga!et%al.,!1999;!Wang!et!al.,!2004;!
Pajni?Underwood!et%al.,!2007).!
!
*
Introduction!
!
!
52!
1.4.4*AER*maintenance*
Once!the!AER!is!induced!two!regulatory!feedback!loops!established!in!the!
limb!became!critical!for!its!maintenance.!The!first!is!the!previously!mentioned!loop!!
between! Fgf10! in! the! mesoderm! and! Fgf8! in! the! limb! ectoderm! responsible! for!
AER!induction!(Min!et%al.,!1998;!Sekine!et%al.,!1999;!Xu!et%al.,%1998;!Arman!et%al.,!
1999;!Girovodsky!et%al.,!2003;!Lu!et%al.,!2008;!Soshnikova!et%al.,!2003;!Barrow!et%al.,!
2003).!Since!removal!of!any!component! of!this!loop!once!the!AER!is!established!
resulted! in! AER! regression,! demonstrated! its! requirement! also! for! AER!
maintenance!(Lu!et%al.,!2008).!!
The!other!regulatory!feedback!loop!is!related!to!the!role!that!Bmp!exerts!in!
the!AER!after!its!induction.!Bmp!signalling!becomes!detrimental!for!the!AER!and!a!
regulatory!loop!between!Shh!from!the!ZPA!and!Fgf!from!the!AER!is!responsible!for!
blocking! the! negative! effects! that! Bmps! exert! over! the! AER,! required! for! the!
correct!outgrowth!and!patterning!of!the!limb.!Shh!emanating!from!the!ZPA!induces!
the!Bmp!antagonist!Gremlin!(Grem1)!in!the!limb!mesenchyme.!Grem1!blocks!the!
negative!effect!that!Bmp!exerts!over!the!AER!allowing!Fgf!expression!in!the!AER!
(Pizette!and!Niswander!et%al.,!1999;!Ganan!et%al.,!1996;!Khokha!et%al.,!2003;!Zuñiga!
et% al.,! 1999)(Fig.! 11).! Subsequently,! Fgf! maintains! Shh! expression! in! the! ZPA!
establishing!the!regulatory!feedback!loop.!Remarkably,!the!correct!cessation!of!this!
loop!is!determinant!for!the!correct!development!of!the!limb.!The!fact!that!cells!that!
have!expressed!Shh!are!not!able!to!express!Grem1!generates!a!gap!between!these!
two!domains.!As!the!limb!grows!this!gap!becomes!bigger!allowing!the!regression!of!
the!AER!mediated!by!Bmp!signalling!(Scherz!et%al.,!2004).!On!the!other!hand,!it!has!
been!proposed!that!high!levels!of!Fgf!expression!inhibit!Grem1!expression.!Thus,!as!
the!limb!bud!grows!and!the!Fgf!expression!level!in!the!AER!becomes!higher,!the!
gap! between! these! two! domains! becomes! bigger! allowing! Bmp! function! and!
subsequent!regression!of!the!AER!(Verheyden!and!Sun!2008).!!
*
Introduction!
!
53!
*
Figure* 11.* Illustration*of* regulatory*
interactions* involving* the* process* of*
AER* maintenance.!The! arrows! indicate!
induction! and! the! bars! repression.! The!
color! code! is! indicated! in!the!bottom,! for!
more! detail! see! the! text.!Taken! from!
Fernandez?Teran!and!Ros!2008.!
*
*
*
*
*
1.4.5*Split*hand/foot*malformation*
Split!hand/foot!malformation!(SHFM)!is!a!human!congenital!malformation,!
affects!to!the!distal!portion!of!the!upper!and!lower!limbs!that!is!characterized!by!
the!absence!of!central!digits!and!fusion!of!the!remaining!ones.!It!is!believed!that!it!
is!the!consequence!of!a!failure!in!the!maintenance!of!the!medial!region!of!the!AER!
that! lacks! Fgf8! expression! (Temtamy! and! McKusick,! 1978;! Sifakis! et% al.,!
2001)(Fig.12).! The! incidence! of! SHFM! is! about! 1:18,000! live! births! and! can!
appear!as!an!isolated!entity!or!as!part!of!a!syndrome.!In!human!at!least!6!loci!have!
been!associated!to!non?syndromic!SHFM.!Isolated!forms!of!SHFM!are!commonly!
inherited! in! an! autosomic! dominant! fashion,! with! incomplete! penetrance! and!
variable! expressivity! (Scherer! et% al.! 1994),! with! the! exception! of! SFHM! type! VI!
that! is! inherited! in! an! autososmal! recessive! fashion! and! SHFM! type! II! that! is!
linked!to!the!X!chromosome!(Ahmad!et%al.,%1987;!Faiyaz?Ul?Haque!et%al.!1993).!!
Introduction!
!
!
54!
Figure* 12.* AER* defect* leading* to*
Split* hand/foot* malformation*
(SHFM).!Normal!development!of!the!
autopod!(top)!and!SHFM!phenotype!
(bottom).! A! failure! to! maintain!AER!
activity!(red)!in!the!developing!limb!
bud!leads! to! the! absence! of! the!
central! rays!and! causes! an! SHFM!
phenotype.! AER,! apical! ectodermal!
ridge;! PZ,! progress! zone;! ZPA,! zone!
of! polarizing! activity.!From! Duijf! et%
al.,!2003.*
!
SHFM!type!I!is!commonly!linked!to!minimal!deletion!of!DSS1!in!human!
and!to!distalFless%homeobox!genes!Dlx5!and!Dlx6!in!mouse!(Simeone!et%al.,!1994;!
Scherer!et%al.,!1994;!Crackower!et%al.,!1996).!The!Dlx5;Dlx6!double!KO!resembles!
the! limb! phenotype! characteristic! of! SHFM! (Robledo! et% al.,!2002;! Merlo! et% al.,!
2002),!confirming!the!implication!of!the!human!orthologs!DLX5!and!DLX6!in!this!
pathology.! Based! on! the! phenotype! of! the! Dactylin! (Dac)! mutant! mice! and!
posterior!mapping!analysis!the!Dac!gene!was!proposed!as!the!gene!responsible!
for!SHFM!type!III,!although!it!is!not!fully!clear!(Johnson!et%al.,!1995;!Sidow!et!al.,!
1999;!de!Mollerat!et%al.,!2003).!In!humans!mutations!in!Tp63,!the!homolog!of!the!
cell?cycle!regulator!p53,!have!been!shown!to!be!the!cause!of!the!SHFM!type!IV.!
Finally,!the!HoxD!cluster!has!been!related!to!SHFM!type!V!and!Wnt10b!to!SHFM!
type! VI!whereas! no! gene! has! been! associated!to! SHFM! type! II.! Despite! the!
identification!of!6!loci!involved!in!SHFM,!only!Tp63!(SHFM!IV)!and!Dlx5!and!Dlx6!
(SHFM! I)! have! been! unequivocally! involved! in! this! malformation.! Disruption!
analyses!in!mice!revealed! that!Tp63!is! required! for! AER!formation!(Mills!et% al.,!
Introduction!
!
55!
1999;!Yang!et%al.,!1999).!Further!analysis!of!the!role!of!Tp63!and!Dlx!genes!helped!
to!clarify!the!epistatic!relation!between!them.!It!has!been!shown!that!Tp63!acts!
upstream!of!the!Dlx!genes,!for!proper!AER!development!(Lo!Iacono!et%al.,!2008;!
Kouwenhoven!et%al.,!2010).!!
*
1.4.6*AER*induction*and*DV*pattern*establishment*
As! mentioned,! mouse! pre?AER! cells! are! induced! in! a! patchy! pattern! of!
expression!in!the!ventral!limb!ectoderm!by!the!time!the!DV!polarity!is!acquired!by!
the! ectoderm! (Kimmel! et% al.,! 2000).! In! contrast,! chick! Fgf8!expressing! cells! are!
initially! detectable! in! a! wider! domain! along! the! limb! DV! border! (Crossley! and!
Martin,!1995;!Crossley!et%al.,!1996).!As!the!limb!bud!develops,!Fgf8!expressing!cells!
committed!to!form!the!AER!are!confined!to!the!DV!limb!interface,!at!the!distal!tip!
of! the! limb! bud.! Chick?quail! chimera! experiments! revealed!that! the! whole!
ectoderm! covering! the! prospective! limb! field! forms! the! AER.! In! addition! these!
experiments! demonstrated! that! the! ectoderm! overlaying! the! somites! and! the!
intermediate!mesoderm! give! rise! to! the! dorsal! ectoderm! of! the! limb,! while! cells!
from! the! ventral! ectoderm! are! originated! in! the! ectoderm! overlying! the! lateral!
somatopleure!(Michaud!et%al.,!1997).!However,!while!cell!lineage!analysis!in!chick!
confirmed! the! origin! of! dorsal! and! ventral! ectoderm,! whether!these!cells!
contributed!to!the!AER!remains!controversial.!In!contrast!to!chimera!experiments,!
DiI! cell?labeling! experiment! in! chick! supported! a! mingled! origin! of! the! AER,!
constituted!by!ventral!and!dorsal!ectodermal!cells!mixed!with!AER!cells!(Altabef!et%
al.,!1997).!In!addition,!cell?marking!experiments!in!mouse!indicated!that!only!part!
of! the! initial! pre?AER! cells! were! committed! to! form! the! mature! AER! (Guo! et% al,!
2003;!Kimmel!et%al.,!2000).!!
The! fact! that! AER! induction! and! DV! pattern! establishment! occurs!
concomitantly!and!also!the!positioning!of!the!mature!AER!at!the!DV!interface,!lead!
Introduction!
!
!
56!
to!the! hypothesis! that! these! two! processes! are! under! common! regulatory!
mechanisms.! This! is! supported! by! the! fact! that! ectopic! limbs! developed! after!
application!of!Fgf!soaked!beads!in!the!flank!ectoderm!arise!at!the!DV!boundary,!in!
precise! alignment! with! normal! limbs! independently! of! the! position! of! the! bead!
(Cohn!et%al.,!1995;!Vogel!et%al.,!1996;!Ohuchi!et%al.,!1997).!
This!notion!came!from!the!study!of!the!chicken!mutant!limbless!that!fails!to!
develop!an!AER!as!a!possible!consequence!of!a!failure!in!DV!pattern!establishment!
(Ros!et%al.,!1996;!Grieshammer!et%al.,!1996;!Noramly!et%al.,!1996).!Limbless,!is!an!
autosomic!recessive!mutation!in!chick!affecting!the!ectodermal!component!of!the!
limb.!It! is! characterized!by! the!absence! of! Fgf8!expression! in! the! limb! ectoderm!
and!lack!of!the!AER!that!leads!to!the!regression!of!the!limb!bud!by!19HH.!Limbless!
limb!buds!lack!En1!expression!in!the!ventral!ectoderm!with!the!subsequent!ectopic!
Wnt7a!expression!in!the!ventral!ectoderm,!leading!to!the!development!of!bidorsal!
limb!buds.!It!has!been!suggested!that!the!failure!in!DV!polarity!establishment!in!
limbless!limb!buds!could!be!the!reason!for!the!absence!of!Fgf8!expression!in!the!
limb!ectoderm!and!the!subsequent!bud!regression!(Ros!et%al.,!1996;!Noramly!et%al.,!
1996;!Grieshamer!et%al.,!1996).!In!addition,!grafting!experiments!in!chick!in!which!
confrontation! of! ventral! and! dorsal! limb! ectoderm! resulted! in! the! induction! of!
ectopic! AERs! supported! the! requirement! of! the! DV! interface! for! AER! induction!
(Laufer!et%al.,!1997;!Tanaka!et%al.,!1997).!
In! contrast,! eudiplopodia,% another! recessive! mutation! in! chick,! it! is!
characterized! by! the! development! of! ectopic! AERs! in! the! dorsal! limb! ectoderm.!
Interestingly,!the!digits!developed!from!these!ectopic!AERs!are!characterized!by!a!
double!dorsal!phenotype!(Goetinck,!1964),!denoting!the!absence!of!DV!patterning,!
implying!that!DV!pattern!establishment!is!not!a!prerequisite!for!AER!induction.!It!
was!further! supported! by! the! development! of! an! AER! in! double! dorsal! limbs!
developed! when! prospective! limb! mesoderm! was!grafted!between! two! rows! of!
somites,!or!even!when!a!filter!was!placed!proximal!to!the!somatopleure!(Michaud!
et%al.,!1997).!
Introduction!
!
57!
!In!addition,!if!limb!DV!polarity!is!a!prerequisite!for!AER!induction,!it!would!
be! expected! that! mice! lacking! DV! specification! markers! such! as! Wnt7a!or! En1!
should! develop! defects! in! AER! induction.! Nevertheless,! AER! induction! is! not!
affected! in! either! Wnt7a!or! En1! mutant! mice,! neither! in! the! double! mutant!
Wnt7a;En1.! Interestingly,! in! mice! lacking! En1,! ectopic!Wnt7a!expression!in! the!
ventral! ectoderm! prevents! the! compactation! of! the! AER,! leading! to! a! ventrally!
extended!AER!(Cygan!et%al.,!1997;!Loomis!et%al.,!1996;!Loomis!et%al.,!1998;!Parr!and!
McMahon,!1995).!
!
!
Figure* 13.* Illustration* for*the* morphogenetic* movements* during*
the*formation*of*the*AER*in*mouse.*Sequential!convergent!movements!
of!the!ectoderm.!An!initial!wave!of!lateral!morphogenetic!movements!of!
the! ectoderm! confined! AER! precursor!cells!into! the! ventral! ectoderm.!
Afterwards,!a!second!wave!compresses!these!cells!to!the!distal!1/3!of!the!
ventral!ectoderm!and!finally!the!last!wave!constricts!the!cells!at!the!DV!
limb! boundary.! Lack! of! En1!leads! to! ventrally! extended! AER.! From!
Loomis!et%al,.%1998.*
!
The!phenotypes!in!which!AER!maturation!defects!arise!due!to!lack!of!En1!
expression!in!the!ventral!ectoderm!supports!the!notion!that!Bmps!emanating!from!
the!LPM!are!the!molecules!required!for!the!establishment!of!ventral!limb!polarity!
Introduction!
!
!
58!
and!proper!AER!positioning!(Michaud!et%al.,!1997;!Ahn%et%al.,%2001;!Soshnikova!et%
al.,! 2003;! Pajni?Underwood! et% al.,! 2007;! Maatouk! et% al.,! 2009;! Choi! et% al.,!2012).!
Based!in!the!ventrally!extended!AER!morphology!of!En1!mutant!mice,!Loomis!and!
colleagues!proposed!a!model!in!which!convergent!morphogenetic!movements!are!
required! for! the! mature! AER! morphology.! In! this! model,! lateral! convergent!
movements! would! initially! compact! the! pre?AER! cells! in! the! ventral! ectoderm.!
Afterward,! morphogenetic! movements! from! the! ventral! ectoderm! compacts! the!
AER! cells! and! confine! them! to! the! DV! interface.! En1!has! been! proposed! as! the!
candidate!responsible!for!the!morphogenetic!movements!required!to!confine!the!
ventrally! located! AER! cells! to! the! DV! boundary! (Loomis! et% al.,! 1998)(Fig.! 13).!
However,! AER! maturation! defects! present! in! En1% mutant! mice! are! rescued! by!
additional!removal!of!Wnt7a,!suggesting!that!neither!En1!nor!Wnt7a!are!required!
for! proper! AER! induction! and! positioning! at! the! DV! limb! interface! (Parr! and!
McMahon,!1995;!Cygan!et%al.,!1997;!Loomis!et%al.,!1996;!Loomis!et%al.,!1998).!
In!contrast,!En1!misexpression!experiments!in!chick!led!to!the!proposal!that!
a! boundary! between! cells! expressing! En1!and! non?expressing! could! be! an!
indispensable! prerequisite! for! AER! induction.! While! low! levels! of! En1!
missexpresion!in!the!dorsal!ectoderm!led!to!the!development!of!additional!AERs,!
high!levels!of!missexpression!abolished!endogenous!AER!formation!(Laufer!et%al.,!
1997;!Logan!et%al.,!1997;!Rodriguez?Esteban!et%al.,!1997;!Pizette!et%al.,!2001).!Thus,!
it!was!proposed!that!En1!could!be!responsible!for!the!specification!of!the!ventral!
ectoderm! through! cell! lineage! restriction! in! the! ventral! ectoderm.! Nevertheless,!
either!retroviral!overexpression!in!the!chick!ectoderm!or!its!missexpression!with!
the!use!of!the!Msx2FCre!line!in!mice!that!drives!expression!all!over!the!AER!did!not!
affect! the! DV! cell! lineage! boundaries! present! in! the! limb! ectoderm! (Altabef! and!
Tickle,! 2002;! Kimmel! et% al.,! 2000).! Hence,! it! is! possible! that! common! signalling!
mechanisms! responsible!for!DV! pattern! establishment! and! AER! induction! lies!
upstream!of!En1.!
!
Introduction!
!
59!
The! common! parallelism! between! vertebrates! AER! and! Drosophila´s!wing!
margin,! both! arise! at! the! DV! boundary! and! are! required! for! PD! growth! (Diaz?
Benjumea!et%al.,!1993),!led!to!the!proposal!that!Radical%Fringe%(RFfng)!in!the!chick!
performed!the!same!role!to!Fringe!in!Drosophila!that!is!responsible!for!PD!growth!
and!DV!positioning!of!the!wing!margin!(Brook!et%al.,!1996).!RFfng!is!expressed!in!
the! limb! dorsal! ectoderm! reaching! the! ventral! boundary! of! the! AER! and! it! was!
proposed! that! the! boundary! between! RFfng!expressing! and! non?expressing! cells!
was!responsible!for!the!correct!positioning!of!the!wing!margin!at!the!DV!boundary!
(Laufer!et%al.,!1997;!Rodriguez?Esteban!et%al.,!1997).!However,!limb!developmental!
defects!were!not!appreciable!after!its!disruption.!
!
1.4.6.1*The*role*of*the*Bmp*signalling*pathway*
In!agreement!with!Le!Douarin!and!colleagues´!proposal!that!Bmp!signalling!
in! the! LPM!could! act! as! a! ventralizing! factor! providing! ventral! identity! to! the!
ventral!limb!ectoderm,!En1!has!been!shown!to!be!a!downstream!effector!of!Bmp!
signalling!pathway! in! ventral! limb! specification! (Pizette! et% al.,! 2001;! Ahn! et% al.,!
2001).!!
Bmps! are! a! large! family! of! growth! factors! members! of! the! Transforming!
Growth!Factor?β!superfamily!(TGF?β)!together!with!TGF?βs,!Activins/Inhibins,!and!
Müllerian! Inhibiting! Substance! (Kingsley,! 1994).! Bmps! are! implicated! in! several!
processes! governing! vertebrate! limb! development,! such! as! chondrogenesis! or!
programmed! cell! death! (Dudley! et% al.,! 1995;! Dunn! et% al.,! 1997;! Kawakami! et% al.,!
1996;!Luo!et%al.,!1995;!Pizette!and!Niswander,!1999;!Pizette!and!Niswander,!2000;!
Zou! and! Niswander,! 1996).! Intracellular! transduction! of! the! Bmp! signalling! is!
transmitted!by!the!type!I!and!type!II!receptors.!The!Bmp!ligands!have!been!shown!
to!signal!through! three!different!types!II!receptors,! the! BmprII,! the! ActR?IIa! and!
the! ActR?IIb! (Yamashita! et% al.,! 1995;! Hoodless! et% al.,! 1996).! In! addition,! four!
different! type!I! receptors! have! been! described! for! Bmp! signal! transduction,!
Introduction!
!
!
60!
BmprIa,! BmprIb,% ALK2% and% ALK1.! BmprIa!is! a! ubiquitously! expressed! type! Ia!
receptor! also! termed! activin! receptor?like! kinase?3! or! ALK?3! and! BmprIb,% also!
termed!ALK?6,!is!responsible!for!cartilaginous!condensations!(Zou!et%al.,!1997;!ten!
Dijke! et% al.,! 1994;! Koenig! et% al.,! 1994).! Remarkably,! Bmp2,! Bmp4!and! Bmp7!are!
ligands! binding! the! type! Ia! receptor! and! are! expressed! in! the! limb! during!
development!(Yamaji!et%al.,!1994).!However,!two!of!them,!Bmp2!and!Bmp7,!have!
been!also!shown!to!bind!to!the!activin!type!II!receptors!(Yamashita! et%al.,!1995;!
Hoodless!et%al.,!1996).!
Both!Bmpr,%type!I!and!II,!are!required!for!intracellular!signal!transduction.!
Binding!of!Bmp!ligands!to!the!single!pass!transmembrane!sherine?treonine!kinase!
receptors!type!II,!phosphorylates!and!activates!the!type!I!receptors!which!in!turns!
phosphorylates! Smad! transcription! factors! for! subsequent! nuclear! translocation!
and!activation!or!repression!of!target!genes!(Heldin!et%al.,!1997;!Massagué,!1998;!
Attisano! and! Wrana,! 1998;! Derynck! et% al.,! 1998;!Padgett! et% al.,! 1997;! Whitman,!
1997).! Three! major! classes! of! Smads! have! been! described.! One! is! the! receptor!
regulated! Smads! (R?Smad).! They! are! the! substrates! of! the! kynase! receptors! and!
are!critical!for!biological!response.!TGF?ß!and!activin!receptors!are!responsible!for!
Smad2! and! Smad3! phophorylation! for! transduction! of! their! specific! signal,!
whereas!Bmp!signalling!is!propagated!through!Bmpr!mediated!phosphorylation!of!
Smad1,! Smad5! and! Smad8.! After! phosphorylation! of! the! R?Smad! a! heteromeric!
complex! with! the! second! class! of! Smad,! the! common! mediator! (co?Smads)! is!
formed.! In! mammals! only! co?Smad4! has! been! described.! Once! the! heteromeric!
complex!(R?Smad/co?Smad)!is!formed!translocates!into!the!nucleus!and!regulates!
gene! expression.! Finally,! Smad6! and! Smad7! belong! to! the! inhibitor! Smads! (I?
Smads)!class!that!prevents!the!access!or!phosphorylation!of!R?Smads.!
Even!though!intensive!studies!focused!in!the!role!of!Bmp!signalling!in!limb!
induction,! its! role! is! not! fully! understood.! Missexpresion! in! chick! of! the!
constitutively!active!form!of!the!BmprIa!led!to!either!ectopic!AER!development!or!
disruption!of!the!endogenous!AER!induction,!suggesting!that!a!boundary!of!Bmp!
Introduction!
!
61!
signalling!and!non?signalling!was!required!for!AER!induction!(Pizzete!et%al.,!2001).!
Further!analysis!revealed!that!missexpression!of!the!constitutively!active!BmprIa!
in!the!dorsal!limb!ectoderm!induced!En1!expression!and!also!the!Msx1!and!Msx2!
transcription!factors,!transcriptional!mediators!of!the!Bmp!signalling!(Suzuki!et%al.,!
1997;!Pizette!and!Niswander,!1999;!Pizzete!et%al.,!2001).!!
BmprIa!overexpression! led! to! the! expression!of! Fgf8% triggered! by! the!
induction!of!either!En1!or!Msx1/2.!Moreover,!missexpression!of!En1!or!Msx1/2!led!
also!to!the!development! of! ectopic!AERs.!However,!the! etymology! of!the!ectopic!
AER! developed! after! missexpression! of! either! Msx!or! EnF1!was! different.! It! was!
suggested! that! ectopic! AER! developed! after! En1! missexpression! in! limb! dorsal!
ectoderm! arise! as! a! misplacement! of! the! endogenous! AER,! whereas! the! ectopic!
AERs! resulting! from! Msx! missexpression! arise! in! a! patchy! pattern! scattered!
through!the!dorsal!ectoderm!independently!of!the!endogenous!AER!(Pizette!et%al.,!
2001).!Thus,!it!was!suggested!that!Bmp!signalling!through!the!BmprIa!could!link!
DV! patterning! and! AER! induction! through! the! activation!of!En1!and! Msx!
transcription!factors!respectively!(Fig.!14)(Pizette!et%al.,!2001).!!
!
*
*
*
Figure*14.*Role*of*Bmp*signalling*in*
the* ventral* limb* ectoderm.!BMP!
through! the! induction! of! En1!is!
responsible! for!ventral! patterning,!
while! its! role! in! AER! induction!is!
mediated! by! the! Msx! transcription!
factors.!From!Pizette!et%al.,!2001.!
!
Introduction!
!
!
62!
In! contrast! to! missexpression! experiments! in! chick,! the! analysis! of! the!
double!Msx1;Msx2! knock! out! mutant! that! exhibited!a! disruption! of! the! anterior!
region! of! the! AER! and!a! failure!in!DV! boundary! establishment! at! that! region,!
consistent!with!a!role!for!the!Msx!transcription!factors!in!DV!pattern!establishment!
rather!than!in!AER!induction!(Lallemand!et%al.,!2005).!
Early! lethality! due! to! defects! in! gastrulation! in! mice! lacking! BmprIa%
(Mishina% et% al.,!1995),! forced! researchers! to! investigate! its! role! in! limb!
development!using!conditional!approaches!with!the!use!of!the!Cre/LoxP!system.!
Conditional! removal! of! BmprIa! in! the! limb! has! been! performed! with! the! use! of!
different! Cre! lines.! Removal! of! the! BmprIa! from! the! limb! mesoderm! has! been!
performed!with!the!use!of!the!Prx1Cre!line.!This!line!drives!Cre!activity!in!the!limb!
mesoderm! (Logan! et% al.,!2002).! Prx1Cre!dependent! BmprIa! removal! led! to! AP!
defects,! decrease! in! growth! of! the! autopod! and! minor! DV! defects! with!slight!
ectopic! expression! of! Lmx1b! in! the! ventral! limb! mesoderm! (Ovchinnikov! et% al.,!
2006).!!
Regarding! the! ectoderm,! removal! of! the! BmprIa!has! been! also! performed!
with!the!use! different! Cre!lines!(Ahn! et% al.,!2001;!Shosnikova!et% al.,! 2003;!Pajni?
Underwood!et%al.,!2007).!The!use!of!the!Msx2?Cre!line!that!drives!Cre!expression!in!
the!ventral!limb!ectoderm!and!the!AER,!before!AER!induction!in!the!hindlimb!(19!
Somites)! and! after! its! induction! in! the! forelimb! (21! Somites)(Sun! et% al.,! 2000;!
Barrow! et% al.,! 2003),! suggested! a! dynamic! role! for! Bmp! signalling! during!
development.!At!early!stages!it!is!required!for!AER!induction!while!at!later!stages!
mediates!AER!regression!(Fig.!15)(Pajni?Underwood!et%al.,!2007).!!
Msx2FCre!dependent!BmprIa%removal!at!early!stages,!prior!to!AER!induction!
in!the!hindlimb,!resulted!in!absence!of!Fgf8!induction!and!lack!of!the!AER!with!the!
subsequent!complete!agenesis!of!the!hindlimbs,!implying!its!requirement!for!AER!
induction! (Pajni?Underwood! et% al.,! 2007).! Requirement! that! was! first!
demonstrated!with!the!use!of!the!Brn4FCre!deleter!line!that!drives!expression!of!
Introduction!
!
63!
Cre! in! the! dorsal! and! ventral! ectoderm! of! the! limb! buds! by! E9.75,! with! highest!
expression! levels! in! the! AER! (Ahn! et% al.,! 2001;! Soshnikova! et% al.,! 2003).!
Noteworthy,! while! Msx2FCre! removal! of! the! BmprIa!led!to! agenesis! of! the!
hindlimbs!in!all!affected!individuals,!the!use!of!the!Brn4Cre!line!resulted!in!a!wide!
range!of! phenotypes,!ranging! from! agenesis! of! the! hindlimbs! with! no! detectable!
Fgf8! expression,! to! grossly! malformed! limbs! with! severely! reduced! Fgf8!
expression!levels.!The!differences!in!the!phenotypes!of!both!conditional!mutants!
are!explained!due!to!the!specific!kinetics!of!each!Cre!line!used!(Ahn!et%al.,!2001;!
Pajni?Underwood! et% al.,! 2007).! Interestingly,! while! Brn4FCre! mediated! BmprIa%
removal! exhibited! variable! penetrance! in! AER! disruption,! the! penetrance! in! DV!
patterning!establishment!defects!was!complete,!implying!that!AER!induction!and!
DV!pattern!seems!to!be!independent!processes!(Ahn!et%al.,!2001).!!
!
!
Figure*15.* Skeletal* phenotype* of* Msx2)Cre;BmprIAflox$conditional*
mutant* mice.*Skeletal! preparations! of! normal! (A,C,G)! and! mutant!
newborns(B,D,H)!and!dorsal!view!of!gross!morphology!of!normal!(E)!and!
mutant! (F)! forelimbs.! In! mutants,! hindlimb! elements! are! absent! and!
pelvic!bones!are!reduced!or!absent,!while!all!the!forelimb!elements!are!
developed.!The!autopd!shows!interdigital!webbing!(F)!and!broader!digit!
tips! with! double?dorsal! phenotype! (asterisks! in! H).! From! Pajni?
Underwood!et%al.,!2007.!!
Introduction!
!
!
64!
The!role!of!Bmps!at!later!stages!once!the!AER!is!formed,!is!related!to!AER!
regression.! At! later! stages! of! limb! development! Bmp! signalling! becomes!
detrimental!for!the!AER!and!regulates!the!cessation!of!Fgf8!expression!in!the!AER!
(Zuñiga!et%al.,!1999;!Pizette!et%al.,!2001;!Pajni?Underwood!et%al.,!2007;!reviewed!in!
Fernández?Teran!and!Ros,!2008).!Msx2FCre!removal!of!the!BmprIa!led!to!soft!tissue!
syndactyly!in!forelimbs!as!a!consequence!of!reduced!programmed!cell!death!in!the!
interdigital! tissue! mediated! by! prolonged! Fgf! expression! in! the! AER! (Fig.! 15F).!
Noteworthy,!soft!tissue!syndactyly!present!in!BmprIa;Msx2FCre!conditional!mutant!
forelimbs!was!rescued!by!additional!removal!of!Fgf8!and!Fgf4!from!the!AER!(Pajni?
Underwood! et% al.,! 2007).! According! to! this! results,! Brn4FCre;BmprIa!conditional!
mutant! forelimbs! exhibited! subtle! defects! (Ahn! et% al.,! 2001).! Interestingly,! DV!
pattern!was!properly!established!in!these!forelimbs,!as!assessed!by!expression!of!
Lmx1b,!En1!and!Wnt7a!(Ahn!et%al.,!2001).!Absence!of!DV!pattern!defects!in!these!
forelimbs!implies!that!the!DV!pattern!is!already!established!before!E10,!when!the!
BmprIa!has!been!widely!removed!and!also!that!once!it!is!established!Bmps!are!no!
longer! required! for! proper! DV! pattern! (Wong! et% al.,! 2012;! Ahn! et% al.,! 2001).! In!
contrast,! Msx2FCre! dependent! removal! in! forelimbs! resulted! in! digit! tips! with!
double! dorsal! phenotypes! implying! its! continuous! requirement! for! proper! DV!
pattern!establishment!(Pajni?Underwood!et%al.,!2007)(Fig.!15F,H).!!
Based!on!the!kinetics!of!the!different!Cre!deleter!lines!used!to!abolish!Bmp!
signalling! through! the! removal! of! the! BmprIa% in! the! limb! ectoderm,! a! temporal!
window! between! E9.5! and! E9.75! was!suggested! for! the! requirement! of! Bmp!
signalling!in!the!hindlimb!ectoderm!for!both!AER!induction!and!establishment!of!
the! ventral! limb! polarity! (Ahn% et% al.,% 2001;! Soshnikova! et% al.,! 2003;! Pajni?
Underwood!et%al.,!2007).!Based!on!this!hypothesis,!Bmp4,!and!Bmp7%expressed!in!
the! LPM!were!proposed! as! candidate! molecules! emanating! from! the! lateral!
somatopleure! responsible! for!BmprIa% activity! in! the! ventral! limb! ectoderm!
required!for!the!DV!patterning!of!the!limb!ectoderm!through!the!induction!of!EnF1!
in!the!ventral!ectoderm!(Ahn%et%al.,%2001).!
Introduction!
!
65!
The! functional! redundancy! and! the! overlapping! expression! domains!
between!the! Bmp! ligands!expressed! in! the!limb! makes! it!difficult! to! analyze!the!
role! of! particular! Bmps! during! limb! development.! In! order! to! assess! their! role,!
several! combinations! of! conditional! removals! of! the! different! Bmp! ligands!
expressed!in!either!the!limb!ectoderm!or!mesoderm!have!been!performed!(Selever!
et%al.,!2004;!Bandyopadhyay!et%al.,!2006;!Maatouk!et%al.,!2009;!Choi!et%al.,%2012).!
However,! none! of! the! conditional! deletion! of! the! Bmp! ligands! in! either! the! limb!
ectoderm! or! mesoderm! mimicked! the! amelic!hindlimb! phenotype! obtained! with!
the!Msx2FCre!conditional!removal!of!the!BmprIa!from!the!limb!ectoderm.!!
Single!KO!mice!for!either!Bmp2!or!Bmp4!died!at!early!stages!prior!to!limb!
bud!induction!(Winnier!et%al.,!1995;!Zhang!and!Bradley,!1996),!while!polidactylous!
hindlimbs! arise! in! the! Bmp7! null! mice! (Dudley! et% al.,!1995;! Luo! et% al.,!1995;!
Hofmann!et%al.,!1996).!Bmp4!conditional!removal!from!the!mesoderm!with!the!use!
of!the!Prx1Cre!line!resulted!in!polydacytilous!limbs!with!defects!in!both,!the!AER!
and! DV! pattern! establishment.! AER! defects! corresponded!to! ventrally! expanded!
Fgf8!expression,!as!a!result!of! the!absence!of!En1!expression!and!ectopic! Wnt7a!
expression!in!the!ventral!limb!ectoderm,!followed!by!ectopic!Lmx1b!expression!in!
the!ventral!mesoderm!(Selever!et%al.,!2004).!Chondrogenic!condensations!failed!to!
form! when! Bmp2!was! additionally! removed! in! addition! to! a! longer?lived! AER!
without! reported! DV! patterning! defects.! Finally,! removal! of! Bmp2!in! a! null!
background!for!Bmp7!exhibited!subtle!malformation!(Bandyopadhyay!et%al.,!2006).!
!Removal! of! different! Bmp! ligands! in! the! limb! ectoderm! was! performed!
with!the!use!of!the!Msx2FCre.!Conditional!deletion!of!Bmp2!and!Bmp4,!resulted!in!
polydactilous!limbs!with!double?dorsal!digit!tips!due!to!lack!of!En1!expression!in!
the!ventral!ectoderm!and!ectopic!expression!of!Lmx1b!in!the! ventral! mesoderm.!
These! mutants! also! displayed! elongated! and! thinner! AER! as! judged! by! CD44!
expression!and!expanded!expression!of!Fgf4!and!Fgf8!along!the!DV!and!the!AP!axes!
(Maatouk!et%al.,! 2009).!Triple! deletion! of! Bmp2,! Bmp4!and!Bmp7!resulted! in! the!
same!DV!an!AER!defects!with!an!ectrodactylous!limb!phenotype!(Choi!et%al.,%2012).!!
Introduction!
!
!
66!
Unexpectedly,! conditional! removal! of! the! Bmps! expressed! in! the! AER!
resulted!in!a!more!severe!forelimb!phenotype!than!that!of!BmprIa!removal,!even!
though!both!are!removed!with!the!use!of!the!Msx2FCre!line.!The!downregulation!of!
Msx1/2!expression!observed!in!both!the!mesoderm!and!the!ectoderm!when!Bmps!
are!removed!from!the!ectoderm,!downregulation!not!appreciable!when!BmprIa!is!
removed,!could!be!explained!due!to!additional!receptors!apart!form!the!BmprIa!by!
which!Bmp!signalling!maintains!the!expression!of!Msx1/2!(Choi!et%al.,!2012)!
In! conclusion,! early! removal! of! the! Bmp! ligands! expressed! in! either! the!
ectoderm!or!the!mesoderm!did!not!result!in!agenesis!of!the!hindlimbs!shown!in!
Msx2FCre!dependent!conditional!BmprIa!removal.!One!possible!explanation!is!that!
removal! of! the! Bmp! ligands! expressed! in! the! ectoderm! could! be! masked! by! the!
expression! of! those! ligands! in! the! underlying! mesoderm! and! vice! versa! or! by!
additional! ligands! apart! form! Bmp2/4! and! 7! signalling!through! the! BmprIa.!
However,! as! mentioned! it! has! been! demonstrated*that! Bmp! signalling! and! its!
downstream! effectors! are! required! for! proper! DV! establishment! and! AER!
induction! (Ahn% et% al.,% 2001;! Soshnikova! et% al.,! 2003;! Selever! et% al.,! 2004;!
Bandyopadhyay!et%al.,!2006;!Pajni?Underwood!et%al.,!2007;!Maatouk!et%al.,!2009;!
Choi!et%al.,!2012).!Nevertheless,!AER!induction!and!DV!pattern!establishment!relies!
on!intricate!interactions!between!different!signalling!pathways.!Disruption!of!Bmp!
signalling!in!the!ectoderm!is!not!the!only!signalling!pathway!that!results!in!loss!of!
Fgf!signalling!from!the!AER!and!failure!of!DV!polarity!establishment!(Barrow!et%al.,%
2003;!Soshnikova!et%al.,!2003).!
!!
1.4.6.2*The*role*of*the*WNT*signalling*pathway*
Disruption! of! the! Wnt/ß?catenin!signalling!pathway! led! also! to! both,!
limbless! phenotypes,! due! to! failure! of! AER! induction! and! DV! patterning! defects!
(Soshnikova! et% al.,! 2003;!Barrow! et% al.,! 2003).! The! vertebrate! Wnt! ligands!
homologs! of! wingless! in! Drosophila,! represent! a! large! family! of! signalling!
Introduction!
!
67!
molecules.!Through!the!control!of!cell!proliferation!and!differentiation!it!regulates!
several! aspects! of! limb! development! (Eastman! and! Grosschedl,! 1999;! Huelsken!
and!Burchmeier,!2001).!!
Wnt! signalling!transduction! is! carried! out! by! a! number! of! seven! pass!
transmembrane! receptors! of! the! Frizzled! family! (Fzd).! Depending! on! the! co?
receptor!associated,!either!Lrp5!and!Lrp6!or!Knypek!and!orphan!receptors!like!Ror!
and!Ryk,!the!binding!of!Wnt!ligands!to!Fzd!receptor!leads!to!the!activation!of!either!
canonical!or!non?canonical!pathways!respectively!(Pinson!et%al.,!2000;!Tamai!et%al.,!
2000;!Wehrli!et%al.,!2000;!Halford!et%al.,!2000;!Topczewski!et%al.,!2001;!Yoda!et%al.,!
2003).!!
Canonical!Wnt!signalling!transduction!through!the!Fzd/Lrp!complex!results!
in!the!stabilization!of!ß?catenin.!Binding!of!the!ligand!leads!to!the!activation!of!the!
Dishevelled! (Dvs)! protein!and!results! in! the! inhibition! of! the! docking! proteins!
Axin,! APC! (adenomatous! polyposis! coli)! and! GSK?3beta! (Glycogen! Synthetase!
Kinase!3beta)!complex.!In!absence!of!Wnt!signalling!this!complex!phosporylates!ß?
catenin! in! its! N?terminus! and! promotes! its! ubiquitination! for! posterior!
degradation!via!proteasome!(Zeng!et%al.,!1997;!Korinek!et%al.,!1997;!Liu!et%al.,!2002;!
Schwarz?Romond!et%al.,!2002).!Hence,!activation!of!Dsv!leads!to!the!stabilization!of!
ß?catenin! through! the! inhibition!of! its! phosphorylation! that! allows!its!
translocation!to!the!nucleus.!Once!in!the!nucleus,!interactions!with!Lef1!(Lef)!and!
Tcf1%(Tcf),!members!of!the!lymphoid!enhancer!factor!and!the!T!cell!factor!families!
of!transcription!factors!respectively,!activates!target!genes!(Behrens!et%al.,!1996;!
Galceran!et%al.,!1999).!
Ectopic! induction! of! Fgf8! and! subsequent! limb! outgrowth! after!
misexpression!of!Wnt3a,!ßFcatenin!or!Lef1!in!the!chick!limb!ectoderm!was!the!first!
evidence! for! the! requirement! of! the! Wnt/ß?catenin! signalling! pathway! for! limb!
outgrowth.! Moreover,! misexpression! of! a! Lef1! construct! lacking! DNA! binding!
domain,!used!as!dominant!negative!form!that!competes!with!the!endogenous!Lef1!
Introduction!
!
!
68!
for! ß?catenin,!led!to! AER! disruption! (Kengaku!et% al.,! 1998).! The! finding!that!
induction!of!Fgf8!expression!in!the!limb!ectoderm!was!preceded!by!the!expression!
of! Wnt3a! in! the! limb! ectoderm,! and! that! Wnt3a!was! able! to!induce! Fgf8!when!
missexpressed,! positioned! Wnt3a! upstream! of! Fgf8!induction! in! limb! bud!
development!(Kengaku!et%al.,!1998).!!
In! mouse,! disruption! of! Wnt3a! stunted! trunk! development! caudal! to! the!
forelimb,!but!did!not!interfere!with!limb!development!(Takada!et%al.,!1994;!Greco!
et%al.,!1996).!However,!the!lack!of!limbs!in!the!lef1;Tcf1!double!KO!supported!the!
implication!of!the!canonical!Wnt!signalling!in!mouse!limb!development!(Galgeran!
et%al.,!1999).!Nevertheless,!the!ubiquitous!expression!of!these!transcription!factors!
in! ectodermal! and! mesodermal! compartments,! did! not! allow! to! determined!
whether!they!were!specifically!required!in!either!the!mesoderm!or!the!ectoderm.!!
The!finding!that!conditional!removal!of!Wnt3!in!the!limb!ectoderm!resulted!
in!an! amelic! phenotype!indicated! that!the!function!described!for!Wnt3a!in! chick!
was!performed!by!Wnt3!in!the!mouse!(Barrow!et%al.,!2003).!Wnt3!is!expressed!all!
over!the!limb!ectoderm!at!least!from!E9.5!to!E11.5.!Because!Wnt3!null!homozygous!
mice! fail! to! develop! due! to! defects!in! gastrulation,! conditional! approaches! were!
undertaken! with! the! aim! to! unravel! its! requirement! during! limb! development!
(Roelink!and!Nusse,!1991;!Barrow!et%al.,!2003).!RARFCre!and!Msx2FCre!deleter!lines!
were! used! to! remove! Wnt3! from! the! limb! ectoderm.! The! Msx2FCre!line! drives!
expression!of!the!Cre!in!the!limb!ectoderm!before!and!after!induction!of!the!AER,!
in!hindlimb!and!forelimb!respectively,!whereas!the!RARFCre!drives!Cre!activity!in!
the!forelimb!ectoderm!and!mesoderm!prior!to!AER!induction!(Moon!and!Capecchi!
2000;! Barrow! et% al.,! 2003).! As! expected,! the! forelimb! phenotype! of! RARFCre!
conditional!mutants!was!much!more!severe!than!that!of!the!Msx2FCre!due!to!the!
earlier!activation!of!Cre!activity.!Variable!defects!ranging!from!oligodactyly!of!one!
digit!and!absence!of!the!ulna,!to!development!of!three!digits!and!normal!zeugopod!
were!obtained!with!the!RARFCre!forelimbs,!while!removal!with!the!Msx2FCre!line!
exceptionally!resulted!in!absence!of!digit!5!in!the!forelimb,!whereas!the!hindlimb!
Introduction!
!
69!
phenotype!was!quite!variable!ranging!from!completely!normal!to!total!absence!of!
the!hindlimbs!(Barrow!et%al.,!2003).!!
The!analysis!of%RARFCre!mutant!forelimbs!revealed!that!the!lack!of!anterior!
and!posterior!digits!correlated!with!lack!of!Fgf8!expression!and!lack!of!thickened!
ectoderm! in! those! regions,! similarly! to! Msx2FCre! mutant! hindlimbs.! Cre!
monitorization! of! both! RARFCre!and! Msx2FCre!with! the! use! of! Rosa26! reporter!
(R26R)!line!that!bears!a!LoxP!flanked!DNA!STOP!sequence!preventing!expression!
of! the! downstream! LacZ,! in! the! R26R!locus,! until! Cre! mediated! recombination!
(Soriano,!1999),!revealed!that!lack!of!Fgf8!expression!was!restricted! to!domains!
were! Cre! expression! was! active! in! both! dorsal! and! ventral! ectoderm.! Therefore,!
they! demonstrate! that! removal! of! Wnt3% from! dorsal! and! ventral! abolished! Fgf8!
induction.!However,!despite!ubiquitous!Wnt3!expression!in!the!limb!ectoderm,!it!
was!suggested!that!its!signalling!was!restricted!to!the!pre?AER!cells!(Barrow!et%al.,!
2003).! Remarkably,! human! mutations! in! Wnt3! result! in! a! tetramelic! phenotype!
(Niemann!et%al.,!2004).!
To! further! assess! the! implication! of! Wnt/ß?catenin!signalling! in! the! limb!
ectoderm,! conditional! removal! of! ßFcatenin!was! performed! with! the! use! of! the!
Msx2FCre!and! the! Brn4FCre!lines! (Fig.! 16)(Barrow! et% al.,% 2003;! Soshnikova! et% al.,%
2003).! Removal! of! ßFcatenin! prior! to! AER! induction! led! to! limbless! phenotypes!
with!no!detectable!expression!of!Fgf8,!Bmp4%and!Bmp2%(Fig.!16B.D,E).!In!addition,!
DV!pattern!establishment!defects!with!loss!of!En1!and!ventrally!expanded!Wnt7a!
expression!were!also!appreciable.!Moreover,!its!removal!after!AER!induction!led!to!
truncated!limbs!and!double!dorsal!forelimb!implying!that!canonical!Wnt!signalling!
is! required! for! AER! induction,! but! it! is! also! continuously!required! for! AER!
maintenance!and!DV!pattern!establishment!(Barrow!et%al.,%2003;!Soshnikova!et%al.,%
2003).!!
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*
Introduction!
!
!
70!
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Figure* 16.*Limb* phenotype* of* loss* of* function* of*
β
)catenin$ with* the$Msx2)Cre$
driver*line.!Newborn!gross!morphology!and!skeletal!phenotype!of!control!(A,C,F)!and!
mutant! mice! (B,D,E,G,H).!
β
Fcat,%
β
Fcatenin!loss! of! function;%cont,! control;! u,! ulna;! h,!
humerus;!r,!radius;!a!autopod.!From!Barrow!et%al.,!2003.%
!
Consistent! with! its! role! in! AER! induction,! ßFcatenin!gain! of! function!(GOF)!
mutation! with! the! use! of! the! Brn4FCre!exhibited!ventrally! expanded! AERs! with!
expanded! expression! of! Bmp4!and! Fgf8,! while! DV! defects! were! not! appreciable!
(Soshnikova!et%al.,!2003).!Remarkably,!Msx2FCre!dependent!ßFcatenin!GOF!rescues!
the!stunted!limb!development!due!to!the!removal!of!Fgfr2!from!the!limb!ectoderm.!
Hence,! this! GOF! approaches! confirmed! that! Wnt/ß?catenin!was!absolutely!
required!and!sufficient!for!Fgf8!induction,!downstream!of!Fgf10!(Soshnikova!et%al.,!
2003,!Barrow!et%al.,!2003;!Lu!et%al.,!2008).!!
!
1.4.6.3*Epistatic*relationship*between*Wnt*and*Bmp*signalling*pathways*
The! epistatic! relation! between! Wnt! and! Bmp! signalling! pathways! in! DV!
pattern!establishment!and!AER!induction!remains!quite!controversial!(Soshnikova!
et%al.,!2003;!Barrow!et%al.,!2003).!Based!on!overexpression!experiments!in!chick!in!
Introduction!
!
71!
which!the!dominant!active!form!of!ßFcatenin!resulted!in!the!induction!of!the!Fgf8,%
Bmp2/4% and! 7% together! with! lack! of! expression! of! these! ligands! when! Wnt/ß?
catenin!signalling!is!disrupted,!led!to!the!proposal!that!Wnt/ß?catenin!signalling!
lies!upstream!of!Bmp!signalling!in!the!limb!ectoderm!in!both,!AER!induction!and!
DV!patterning!(Barrow!et%al.,!2003).!
!
!
Figure*17.*Epistatic*relationship*between*Wnt/β[catenin*and*Bmp*signalling*pathways*in*
DV*pattern*establishment*and*AER*induction.*A)!Wnt/β?catenin!acts!upstream!or!in!parallel!to!
Bmp!signalling!in!DV!establishment!as!assessed!by!En1!(A,C,E,G!and!I)!and!Wnt7a!(B,D,F,H!and!J)!
expression!in! wild!type! (wt)!(A,B),!
β
Fcatenin!loss! of! function!(
β
Fcatflox)!(C,D),!
β
Fcatenin!Gain!of!
function!(
Δ
NF
β
Fcat)(E,F),%BmprIA!mutant!(BmprIAflox)!(G,H)!and!Gain!of!function!of!
β
Fcatenin!in!
the! absence! of! BmprIA!(
Δ
NF
β
Fcat;BmprIAflox)! (I,J).! En1!expression!is! lost! from! the! ventral! limb!
ectoderm!and!Wnt7a!is!ectopically!expressed!in!the!ventral!ectoderm!at!30!somites!stage!in!both!
β
Fcatflox!and!BmprIAflox!mutants!and!it!is!not!rescued!in!the!
Δ
NF
β
Fcat;BmprIAflox.!B)!Wnt/β?catenin!
acts!downstream!of!Bmp!signalling!in!AER!induction!(See!scheme!at!right)!as!assessed!by!Fgf8!
(A,C,E,G! and! I)! and! Bmp4!(B,D,F,H! and! J)! expression! in! wild! type! (wt)! (A,B),!
β
Fcatenin!loss! of!
function!(
β
Fcatflox)!(C,D),!BmprIA!mutant!(BmprIAflox),!
β
Fcatenin!Gain!of!function!(
Δ
NF
β
Fcat)(E,F),%
(G,H)!and!Gain!of!function!of!
β
Fcatenin!in!the!absence!of!BmprIA!(
Δ
NF
β
Fcat;BmprIAflox)!(I,J).!Gain!
of! function!mutation! of! Wnt/β?catenin! in! absence! of! the! BmprIA!rescues!Fgf8!and! Bmp4!
expression!lost!in!BmprIA!mutants.!From!Soshnikova!et%al,!2003.!
Introduction!
!
!
72!
However,! the! observation! that! the! ßFcatenin!GOF! was! able! to! rescue! the!
limb!phenotype!of!the!BmprIa!mutant!suggested!that!Wnt/ß?catenin!signalling!lies!
downstream!of!Bmp!signalling!in!AER!induction!and!in!parallel!or!downstream!in!
DV! pattern! establishment.% ßFcatenin! GOF! mutation! in! absence! of! the! BmprIa!
rescued! the! loss! of! Bmp4!and! Fgf8! expression! in! the! limb! ectoderm! of!BmprIa!
mutants!while!lack!of!En1!expression!and!ventrally!expanded!expression!of!Wnt7a!
were! not! rescued! (Soshnikova! et% al.,!2003)!(Fig.! 17).! Thus,! the! linkage! between!
AER!induction!and!DV!pattern!establishment!must!lie!somewhere!between!Wnt/ß?
catenin!and!Bmp!signalling!pathways,!because!disruption!of!either!of!them!results!
in!both,!lack!of!AER!induction!and!DV!patterning!defects!(Soshnikova!et%al.,!2003;!
Barrow! et% al.,! 2003).! However,! further! analysis! of! the! crosstalk! between! both!
signalling!pathways!should!be!necessary!to!determine!their!epistatic!interactions.!
*
1.5.*The*family*of*the*Specificity*Protein*of*transcription*factors**
The! number! of! transcription! factors! that! belong! to! this! family! is! highly!
variable!amongst!different!species,!ranging!from!two!Sp?members!like!btd%and!DF
SP1!in!Drosophila!to!up!to!13!like!in!same!teleost!fishes!(Wimmer!et%al.,!1996).!In!
mammals,!9!members!of!the!Specificity!Protein!(Sp)!family!of!transcription!factors!
have!been!described!(Zhao!and!Meng,!2005).!There!is!evidence!for!the!relevance!of!
several! members! of! this! family! for! the! correct! development! of! the! embryo.!
Interestingly,! the! human! Sp1!was! the! first! identified! and! cloned! binding?specific!
transcription! factor! (Dynan!and!Tjian,! 1983;! Kadonaga! et% al.,! 1987).!Targeted!
disruptions!of!several!members!of!this!family!have!been!reported.!Animals!lacking!
Sp1!died!at!E10.5!(Marin!et%al.,!1997).!Sp3!null!mice!develop!to!term!and!die!due!to!
a! respiratory! failure!and! exhibited! teeth!and! ossification! defects! (Gollner! et% al.,!
2001;!Bouwman!et%al.,!2000).!Disruption!of!Sp4!led!to!reduced!viability!and!cardiac!
defects! (Supp! et% al.,! 1996;! Nguyen?Tran% et% al.,!2000).! No! overt! phenotype! was!
shown!in!Sp5!mutants!(Harrison%et%al.,!2000).!Disruption!of!Sp6!led!to!mild!limb!
Introduction!
!
73!
defects!restricted!to!the!autopod!and!also!in!the!teeth!and!skin!(Nakamura!et%al.,!
2004;! Hertveldt! et% al.,! 2008;! Talamillo! et% al.,! 2010).! Targeted! mutation! in! Sp7!
resulted! in! impaired! osteoblast! differentiation! (Nakashima! et% al.,! 2002)! and! Sp8!
null!mice!develop!to!term!and!die!due!to!defects!in!the!closure!of!the!neural!tube!
(Bell!et%al.,!2003;!Treichel!et%al.,!2003).!!!
The!distinctive!features!of!the!proteins!of!this!family!are!a!highly!conserved!
carboxy! terminal! DNA?binding! domain,! characterized! by! three! Cys2His2! zinc!
finger!motifs!(which!recognize!a!commonly!known!structures,!GC!and! GT! boxes,!
within!the!promoter!region!of!the!genes)!and!a!Btd!box!located!in!close!proximity!
to! it! (Wimmer! et% al.,! 1996).! In! addition,! all! the! members! of! the! family,! with! the!
exception! of! Sp6,!contain!a! Sp! box! located! in! their! N?terminus! (Zhao%and! Meng,!
2005;! Runko! and! Sagerstrom,! 2003).! The! role! of! the! Sp! box! has! not! been!
determined!but!has!been!related!to!proteolytic!cleavage!or!transcriptional!activity.!
There! is! also! an! amino?terminal! region,! which! is! more! variable! that!includes!
transcriptional!activation!or!repression!domains!(Fig.!18).!
Release!of!the!human!genome,!revealed!the!linkage!between!Sp4!and!Sp8,!
separated!by!545kb!with!their!promoters!located!face!to!face,!similar!to!Sp1?Sp7,!
Sp2?Sp6%and!Sp3FSp9%gene!pairs,!whereas!Sp5%is!located!at!distance!of!2564bp!from!
Sp3,% independently! of! the! rest! of! the! Sp! family! members.! Interestingly,! the! Sp!
transcription! factors! are! located! in! the! same! chromosomes! of! the! Hox!genes.!Of!
most!interest,!genetic!evolutionary!analyses!amongst! different!species!suggested!
the!existence!of!an!ancestral!Sp!cluster!linked!to!the!Hox!cluster!during!metazoan!
evolution!(Schaeper!et%al.,!2010).!!
Recently,! the! group! of! Earnst! Wimmer! based! on! phylogenetic! protein!
domain! structures,! expression! patterns! and! genomic! localization! of! the! Sp!
members!in!different!species!proposed!that!previously!designated!orthologs!for!DF
SP1!and!btd!in!Drosophila,!Sp1!and!Sp8!in!human!respectively,!are!not!directly!!
!
Introduction!
!
!
74!
homologs.! They! proposed! that! DFSP1! is! more! closely! related! to! human! Sp8,! the!
ortholog!of!mouse%Btd,!and!that!btd!is!the!ortholog!of!Sp5%(Schaeper!et%al.,!2010).!!
!
!
Figure*18.*Specificity*Protein*family*of*transcription*factors.*The!Btd!box!and!three!zinc!finger!
domains! located! at! the! C?terminus! are! conserved! in! all! the! members! of! the! family.! In! the! N?
terminus!an!Sp!box!characteristic!of!the!members!of!this!family!is!conserved!in!all!the!members!
with!the!exception!of!Sp6!and!Sp9.!Two!transactivation!domains!(glutamine!(Q)?rich!regions)!and!
serine/!threonine!(S/!T)?rich!regions!for!proteasome?dependent!degradation!are!present!in!some!
members!of!the!family.!From!Zhao!and!Meng,!2005.!
!
So! far! only! two! members! of! the! Specificity! Protein! (Sp)! family! of!
transcription!factors,!Sp6!and!Sp8,!also!known!as!Epiprofin!(Epfn)!and!Buttonhead%
(Btd)!respectively,! have! been! suggested! as! possible! candidates! mediating! the!
Wnt/ß?catenin!dependent!induction!of!Fgf8.!Both!transcription!factors!have!been!
demonstrated!to!function!downstream!of!Wnt/ß?catenin!signalling!and!upstream!
of!Fgf8!in!the!limb!ectoderm!(Kawakami!et%al.,!2004;!Sahara!et%al.,!2007;!Talamillo!
et%al.,!2010).!!
Introduction!
!
75!
Disruption!of!Sp6!prevents!AER!maturation!and!results!in!syndactyly!with!
partial!dorsalization!of! the! digital!tips! whereas! disruption!of! Sp8! results! in! limb!
truncation!due!to!the!premature!regression!of!the!AER.!During!limb!development!
both!are!expressed!in!the!entire!prospective!limb!ectoderm!prior!to!Fgf8!induction!
and!become!progressively!confined!to!the!AER!as!the!limb!bud!develops!(Bell!et!al.,!
2003;!Treichel!et!al.,!2003;!Talamillo!et!al.,!2010).!
!The! role! of! Sp6!and! Sp8! during! limb! development! is! not! completely!
determined.! Several! reports! indicate! that! they! are! required! for! Wnt/β?catenin!
dependent!maintenance!of!Fgf8!expression!in!AER!cells,!but!not!for!its!induction!
(Bell!et%al.,!2003;!Treichel!et%al.,!2003;!Kawakami!et%al.,!2004;!Talamillo!et%al.,!2010;!
Lin%et% al.,!2013).! Based! on! their! overlapping!patterns!of!expression!and!on!their!
LOF!phenotypes,!it!has!been!previously!suggested!that!both!factors!may!act!in!a!
redundant!manner!in!the!induction!and!maintenance!of!the!AER.!In!addition,!the!
fact! that! AER! induction! and! DV! pattern! establishment! occurs! concomitantly,! DV!
defects!present! in!mice! lacking! Sp6! (Talamillo! et% al.,!2010)!and!the! fact!that! the!
orthologs!in%Drosophila!of!the!Sp!family!have!been!shown!to!be!able!to!induce!the!
entire!genetic!network!required!for!ventral!imaginal!disc!development,!including!
the! establishment! of! a! sharp! boundary! of! En1!expression! (Estella! et% al.,!2003;!
Estella! and! Mann,! 2010),! led! us! to! hypothesize! that! Sp6!and! Sp8!could!be! the!
molecules! responsible!for! coordinating! AER! induction! and! DV! pattern!
establishment.!
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2.*Aim*of*the*present*Thesis*
Since! Sp6!and! Sp8! are! close! related! genes! and! have! similar! patterns! of!
expression! during! limb! development,! the! aim! of! the! present! Thesis!is! the!
analysis! of! the! function! and! possible! redundancy! between! the! Sp! family!
members!Sp6!and!Sp8!in!limb!development,!particularly!in!the!induction!of!the!
AER!and!the!DV!pattern!establishment.!Therefore,!we!have!used!mouse!genetics!
to! study! the! requirement! and! the! possible! redundancy! between! Sp6! and! Sp8!
transcription!factors!during!limb!development!through!the!generation!of!double!
Sp6;Sp8! null! mutants! and!also! Sp6null;Sp8! conditional! mutants! using! the! Ap2F
Cre!and!the!Msx2FCre!lines.!!
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3.*MATERIALS*&*METHODS*
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3.*Materials*&*methods*
3.1*Mouse*strains**
The!EpfnFnull!(Sp6),!the! Sp8CreERT2,!the! Sp8flox,! the!Catnblox(ex3),! the! Apα2F
Cre,!the!Msx2FCre,!,!and!the!ROSA26!reporter!(R26R)!mouse!lines!were!used!in!
this! study.! All! animal! procedures! were! conducted! accordingly! to! the! EU!
regulations! and! 3R! principles! and! reviewed! and! approved! by! the! Bioethics!
Committee!of!the!University!of!Cantabria.!All!mice!were!maintained!in!a!mixed!
genetic! background! (C57BL6/CD1)! and! genotyped! based! on! previously!
published!reports.!!
The!Sp6[null*(Sp6)*allele!is!characterized!by!the!absence!of!the!2nd!exon!
of!the!Sp6!gene!that!encodes!the!entire!coding!sequence!(CDS)!of!the!Sp6!gene!
(Nakamura!et%al.,!2004).!
The! Sp8CreERT2*allele*bears! a! tamoxifen! inducible! Cre! recombinase! in!
substitution!of! the! Sp8!allele.!This!line!expresses!the! CreERT2! recombinase!in!
the! Sp8! locus.! The! CreERT2! is! a! modification! of! the! Cre! that! contains! a!
G400V/M543A/L544A!triple!mutation!in!the!Estrogen!Receptor!ligand?binding!
domain! (ER?LBD)! that! makes! it! more! sensitive! to! Tamoxifen! (Tam)! than! the!
mutant!ER!LBD!with!a!single!G521R!substitution.!!
The! Sp8flox*allele!contains!the! Sp8!gene! flanked! by! two! LoxP! sites! that!
enable!conditional!removal!with!the!use!of!Cre!driver!lines.!The!first!LoxP!site!is!
located! 5´! to! exon! 1! and! the! second! one! 3´! to! exon! 3.! Cre! dependent!
rearrangement!between!the!two!LoxP!sites!results!in!the!excision!of!the!3!exons!
of!the!Sp8!gene,!completely!disrupting!it!(Zembricky!et%al.,!2007).!
The!Catnblox(ex3)%(ßFcatenin)%allele%contains!two!LoxP!sites!that!flanks!the!
third!exon! of! ßFcatenin% gene.! Cre! dependent! excision! of! this! exon!avoids!
phosphorylation!of!ß?catenin!and!results!in!stabilization!of!the!protein!(Harada!
et%al.,!1999).!
!
Materials,&,Methods!
!
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84!
The! Ap2α)Cre*driver* line* bears!an! IRESCre! cassette! into! the!
3´untraslated! region! of! the! Ap2α$locus! between! the! Stop! codon! and! the!
polyadenilation! sequence! allowing! the! regulation! of! the! Cre! under! the! Ap2α!
locus! without! affecting! the! Ap2!gene! function!(Macatee! et% al.,!2003).! This! line!
drives! Cre! expression! in! the! limb! ectoderm! prior! to! Fgf8!expression! in! both!
forelimb!and!hindlimb!(Boulet!et%al.,!2004).!!
The! Msx2)Cre* driver* line!is! a! transgenic! line! that! bears!a! 439bp!
fragment!of!the!Msx2!AER!specific!enhancer!followed!by!the!Cre!with!a!nuclear!
localization!sequence!that!allows!specific!Cre!expression!in!the!limb!ectoderm!by!
19!and!21!So!in!hindlimb!and!forelimb!respectively!(Sun%et%al.,%2000).!
The! Rosa26$ reporter* (R26R)* line!bears! a! floxed! 4x! polyadenilation!
sequence! that! avoids! LacZ! expression! in! the! ubiquitously! expressed! ROSA26!
locus! (Soriano,! 1999).! By! mating! these! mice! with! Cre?expressing! transgenic!
mice,!excision!of!the!LoxP!flanked!STOP!sequence!occurs!allowing!expression!of!
the! LacZ! reporter.! This! line! is! a! reporter! for!cells! in! which!Cre! dependent!
rearrangement!has!occurred!and!also!their!descendants.!!
*
3.2*Mouse*mating*strategies*
Sp8CreERT2! heterozygous! mice! were! mated! to! Sp6! heterozygous! mice! for!
the! generation! of! double! heterozygous! mice! (Sp6+/?;Sp8+/CreERT)! for! posterior!
analysis!of!double!Knock!Out!mutant!mice!(Sp6?/?;Sp8CreERT/CreERT)!obtained!from!
crosses!between!double!heterozygous!mice,!with!the!aim!to!unravel!their!role!
and!their!possible!redundancy!in!AER!induction!and!DV!pattern!establishment.!
R26R!heterozygous!mice!were!mated!to!Ap2αCre!heterozygous!to!define!
the! expression! pattern! of! the! Cre! under! the! Ap2α! promoter,! to! determine! its!
suitability!for! conditional!removal!of!Sp8!from!the! limb! ectoderm.!In!addition,!
R26R!heterozygous!mice!were!matted!to!Sp8CrERT2!heterozygous!mice!to!analyze!
it!suitability!for!temporally!deletion!of!genes!from!the!limb!ectoderm.!
!
Materials,&,Methods!
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85!
Conditional!removal!of!the!Sp8flox!allele!was!performed!with!two!different!
deleter! lines! the! Ap2αCre!line! and! the! Msx2FCre!line.! Sp8! limb! conditional!
mutants,! in! an! Sp6!null! background,! were! generated! by! mating! double!
heterozygous!females!(Sp6+/?;Sp8+/flox)!with!double!heterozygous!males!carrying!
the! Cre! recombinase! under! the! Ap2α! promoter! (Sp6+/?;Sp8+/flox;Ap2α+/Cre)! or!
under! the! Msx2! AER! specific! enhancer! (Sp6+/?;Sp8+/flox;Msx2+/Cre).! The! followed!
matting! strategies! are!shown! below,! in! each! cross! the! expected! Mendelian!
fraction!of!the!genotypes!of!interest!is!indicated!in!brackets.!Both!Cre!lines!are!
active!in!the!germ!cells,!thus!DNA!rearrangement!occurs!between!the!LoxP!sites!
flanking! the! floxed! alleles! leading! to! the! generation! of! deleted! (D)! alleles.! In!
order!to!ubiquitously!ablate!the!floxed!allele,!we!decided!to!bear!the!Cre!in!the!
males!because!the!penetrance!for!Cre!activation!in!the!germ!cells!is!of!the!100%!
(Weng!et%al.,!2008).!
!
*
Materials,&,Methods!
!
!
86!
!Since! Sp6!and! Sp8!have! been! shown! to! act! downstream! of! Wnt/ß?
catenin,!conditional!ßFcatenin%GOF!approaches!were!performed!with!the!use!of!
the! Sp8CreERT2!line! (Talamillo! et% al.,! 2010,! Kawakami! et% al.,!2004).! Catnblox(ex3)!
heterozygous! mice! were! mated! to! Sp8CreERT2!heterozygous! mice! for! the!
generation!of!double!Catnb+/lox(ex3);Sp8+/CreERT2!heterozygous!embryos.!
*
3.3*Mouse*embryos*
Noon!of!the!day!the!vaginal!plug!was!detected!was!design!as!embryonic!
day!(E)!0.5.!Embryos!were!harvested!by!caesarean!section!from!pregnant!mice!
at!the!desired!developmental!stage!and!dissected!in!Phosphate!Buffered!Saline!
(PBS?!137mM!NaCL,!2.7mM!KCL,! 10mM! Na2HPO4,! 2mM!KH2PO4)!for! posterior!
fixation!in!Paraformaldehyde?4%!(PFA)!between!4!hours!(h)!to!overnight!(ON)!
at!4ºC.!Genotyping!was!performed!with!DNA!obtained!from!the!yolk!sac!or!from!
tail!biopsies.!
*
3.4*DNA*extraction*
Mice! were! genotyped! by! the! polymerase! chain! reaction! (PCR)! analysis.!
The!DNA!was!obtained!from!tail!biopsies!or!from!embryonic!membranes.!
*
3.4.1*DNA*extraction*form*tail*biopsies*
For! tail! DNA! extraction,! 0.5cm! length! tail! biopsies! were! placed! in! a!
solution! containing! 400! μl! Tail! buffer! (10! mM! Tris.Cl! pH8.0,! 50mM! EDTA,!
100mM! NaCl,! 5%SDS)! and! 20! μl! of! proteinase! K! (stock! 20mg/ml,! Merck,! #!
24568)!and!incubated!at!55ºC!ON.!The!next!day,!once!the!tissue!was!digested!the!
volume! was! brought! to! 750μl! adding! Tail! buffer! and! additional!250μl! of! NaCl!
6M.! Samples! were! then! centrifuged! 10! minutes! (min)! at! 12.000G,! at! room!
temperature! (RT).! Then,!750μl! of! the! supernatant! were! transferred! to! a! new!
tube!for!posterior!DNA!precipitation.!500!μl!of!isopropanol!were!added!and!spin!
down! for! 10! min! at! 4ºC.!The! supernatant! was! discarded! and! the! pellet! was!
Materials,&,Methods!
*
!
87!
washed!with!1ml!of!70%!ethanol!(EtOH)!and!centrifuged!for!5!min!at!4ºC.!Then,!
the! pellet! was! resuspended! in! 500μl! TE! buffer! (10! mM! Tris.Cl! pH8.0,! 100! μM!
EDTA)! and! incubated! at! 55ºC! for! 20! min! with! the! lid! open! the! first! 5! min! to!
evaporate!traces!of!EtOH.!
In! general,! 1µl! of! extracted! DNA! obtained! from! tail! biopsies! and!
resuspended!in!TE!was!used!for!the!PCR!reaction.!
*
3.4.2*Yolk*sac*DNA*extraction*
For!yolk!sac!DNA!extraction,!the!yolk!sac!was!dissected!and!transferred!
to!an!eppendorf!tube!containing!100μl!of!lysis!buffer!(50mM!KCl,!10mM!Tris!pH!
8.3,!2!mM!MgCl2,!0,1mg/ml!gelatin,!0,45%NP?40,!0,45%!Tween!20)!and!0,5!μl!of!
proteinase!K!(stock!20mg/ml)!and!incubated!ON!at!55ºC.!Then,!the!proteinase!K!
was! heat!inactivated! at! 95ºC! for! 10! min! and! 1µl! of! this! solution!was! used!
directly!for!mice!genotyping!by!the!PCR!reaction.!
*
3.5[*Mice*Genotyping*by*the*PCR*reaction*
Mice! were! genotyped! following! standard! PCR! protocols,! varying! the!
annealing! temperature! and! the! annealing! time! according! to! the! melting!
temperature! (Tm)! of! the! primers! used.! The! PCR! reaction! was! performed! in! a!
total! volume! of! 25µl! with! the! use! of! ReadyMixTM!Taq! PCR! Reaction! Mix! with!
MgCl2! mastermix! (Sigma?Aldrich).! The! primers! were! used! in! a! final!
concentration!of!200nM.!PCR!products!were!run!in!a!1%!agarose!gel!containing!
Red!Safe!for!DNA!staining!and!visualized!in!the!Gel?Doc!under!ultraviolet!light!
and! the! use! of! the! Quantity–One! software! (Bio?Rad).! Finally,! the! molecular!
weight!of!the!products!was!determined!with!the!use!of!MassRuler!DNA!Ladder!
(Fermentas).!The!set!of!primers!used!to!genotype!each!mouse!strain!is!specified!
below!and!it!is!indicated!into!5'!to!3'!orientation.!
%
%
Materials,&,Methods!
!
!
88!
Msx2FCre%driver!line!
Msx2FCre?F:!AAC!AAC!TCT!GCT!GAC!TGC!TCC!TG!
Msx2FCre?R:!CCT!GGC!GAT!CCC!TGA!ACA!TGT!CC!
Ap2αCre%driver!line!
Ap2αCre?F:!TGGAAAATGCTTCTGTCCGTTTGC!
Ap2αCre?R:!AACGAACCTGGTCGAAATCAGTG!
R26R%line!!
R26R?F:!ACC!CTG!GCG!TTA!CCC!AAC!TT!!
R26R?F:!CTG!TCC!CCG!TAA!CCG!AC!!
Sp8CreERT2%allele!!
Sp8CreERT!wt?F:!CAA!GCA!CGT!GAA!GAC!ACA!CAG!T!
Sp8CreERT!wt?R:!TGT!GTG!CTA!CTT!ACT!GTC!CAC!ATC!C!
Sp8CreERT!mt?F:!GAA!GTC!TCT!GGA!AGA!GAA!GGA!CCA!T!
Sp8flox!allele!!
Sp8Flox?F:!CCA!ATG!GGA!GAA!AAA!CAC!ACC!CCC!TCT!TAC!TCC!TC!
Sp8Flox?R:!CCA!GCT!TCC!TGG!ACT!CTT!TCA!GTA!TAG!TTT!TGA!AG!
Epfn!(Sp6)!allele!!
Epfn!mt?F:!GCTTCCTCGTGCTTTACGGTATC!
Epfn!wt?F:!GCTGGAAACCGTGAAGGAAAGG!
Epfn!R:!GGGTTAGGGGTCATAAGGGATAGG!
Catnblox(ex3)!allele!!
Catnblox(ex3)?F:GACACCGCTCTGGACAATG!
Catnblox(ex3)?R:GTGGCTGACAGCAGCTTTTCTG!
*
3.6*Skeletal*preparations*
Newborn! mice! were! collected! and! dissected! in! PBS.! Once! the! skin! was!
removed!newborns!were!eviscerated!and!fixed!in!EtOH?96%!ON.!For!cartilage!
staining,!EtOH?96%!was!replaced!by!Alcian!Blue!Staining!solution!(80%!EtOH?
96%,!20%!glacial!acetic!acid!and!0.3mg/ml!Alcian!Blue)!for!the!next!2?3!days.!
Materials,&,Methods!
*
!
89!
Next,! excess! staining! was! removed! through! successive! changes! in! EtOH?96%.!
Once! the! replaced! EtOH! appeared! clear! Etoh! was! replaced! by!Potassium!
Hydroxide!1%!(KOH)!for!3?4h.!For!bone!staining,!once!the!bones!become!visible,!
KOH!1%!was!replaced!by!Alizarin!Red!staining!solution!(0.05mg/ml!Alizarin!Red!
in!KOH?1%)!for!about!20!h.!Once!the!bones!acquired!the!desired!Alizarin!Red!
staining,! the! samples! were! cleared! for! approximately! 24h! in! 20%! KOH?1%! in!
glycerin! and! followed! by! additional!successive! changes! of! EtOH?
70%/glycerin/H20! (1:2:7,! 3:3:4,! 4:4:2! and! 5:5:0),! every! 24! h.! Skeletal!
preparations! were! observed! with! the! use! of! an! Olympus! SZX12! stereoscopic!
scope!and!photographs!were!taken!with!a!Nikon!DS?Fi1!camera.!!
*
3.7*RNA*probe*synthesis*
3.7.1*Transformation*
Specific!DNA!sequences!for!RNA!probe!synthesis!are!cloned!in!plasmidic!
vectors.! For! transformation,! 1?3µl!of! the! vector! bearing! the! target! DNA!was!
added! to! an! eppendorf! containing! 30! μl! of! Dh5α! (E.% coli)! competent! bacteria.!
Bacteria!were!incubated!on!ice!for!30!min!and!transformed!by!heat!shock!for!20!
seconds!at!37ºC.!!
!
3.7.2*Mini[prep*
For!amplification!of!target!DNA!transformed!bacteria!were!grown!in!LB!
at!37ºC!for!one!hour!before!being!plated!in!a!Petri!dish!containing!LB?Agar!(LB!
20mg/ml,!Agar!15mg/ml,!X?gal!0.04mg/ml!and!IPTG!0.04!mg/ml)!and!cultured!
ON!at!37ºC.!Antibiotic!resistant!gene!encoded!by!the!plasmids!enables!selective!
growth! of! transformed! bacteria,! depending! on! the! plasmid! used! the!
corresponding! antibiotic! was! added! to! the! medium,! whereas! X?gal! and! IPTG!
were! used! to! differentiate! between! transformed! bacteria! with! plasmids!
containing!an!insert!(white!colonies)!from!those!with!an!empty!plasmids!(Blue!
colonies)!when!colony!screening!by!PCR!was!performed.!The!next!day,!a!positive!
colony! was! selected! from! the! Petri! dish! and! transferred! to! an! Erlenmeyer!
Materials,&,Methods!
!
!
90!
containing! 10! ml! LB! (Amp! 1/1000)! and! incubated! ON! at! 37ºC! in! a! rocking!
platform.! Finally,! plasmidic! DNA! extraction! was! performed! using! the! “Quiakit!
Spin!Mini?prep!Kit”!(Quiagen)!following!manufacturers!recommendation.!
*
3.7.3*Plasmid*linearization*
The!target!DNA!is!cloned!into!the!polylinker!region!of!the!plasmid!flanked!
by!Sp6,!T3!or!T7!RNA!polymerase!promoters.!Purified!plasmidic!DNA!containing!
the!target!DNA!sequence!for!probe!synthesis!was!linearised!for!“inFvitro“%sense!
and! antisense?transcription!using! the!appropriate! specific! restriction! enzymes!
(RE).!!
*
3.7.4*In*vitro*transcription*
Once!the!plasmid!containing!the!target!DNA!sequence!for!probe!synthesis!
was! lienarised,! “inFvitro“% sense! and%antisense?transcription!in! presence! of!
digoxigenin! labeled!ribouridiltriphospahate! nucleotides! (Dig?UTP)! were!
performed!using!the!appropriate!specific!polymerase!for!each!plasmid.!!
*
3.8*Whole*mount*in*situ*hybridization**
Whole!mount!in!situ!hybridization!(ISH)!was!performed!to!analyze!gene!
expression!in!mutant!embryos.!After!fixation!in!PFA!between!4h!to!ON!at!4ºC,!
embryos! were! rinse! in! PBS! 5! min! (twice)! for! removal! of! the! fixative.! Before!
incubation! in! hybridization!buffer! containing! digoxigenin! labeled!antisense!
mRNA!probe!several!steps!for!permeabilization!were!performed.!Embryos!were!
first!washed!for!5!min!in!PBS?Tween!(PBT?!0.1%Tween!in!PBS)!and!dehydrated!
by! successive! changes! in! rising! methanol! (MetOH)! concentrations! diluted! in!
PBT;!25%,!50%,!75%,!and!two!of!100%!prior!to!store!ON!at!?20ºC.!The!period!of!
time! spent! in! each! dehydration! steps! varies!depending! on! the! size! of! the!
embryo,!for!example!5!and!10!min!for!E9.5!and!E10.5!embryos!respectively.!
Materials,&,Methods!
*
!
91!
Embryos! were! rehydrated! by! successive! changes! in! decreasing! MetOH!
concentrations!diluted!in!PBT;!100%,!75%,!50%,!25%!and!rinsed!in!PBT!twice!
for!5!min!per!step.!Afterwards,!samples!were!bleached!in!hydrogen!peroxide?6%!
diluted! in! PBT! for! 60! min.! Then,! embryos! were! rinsed! in! PBT! for! 5! min! and!
incubated!in!proteinase! K! buffer! (50mM! Tris! pH! 7,4,! 5mM! EDTA)! containing!
proteinase! K! (10µg/ml).! The! incubation! time! varies! among! different!
developmental! stages! and! also! between! the! ectoderm! and! the! mesoderm.! To!
check!gene!expression!in!the!ectoderm!we!have!performed!5!min!digestion!of!
proteinase!K!(5µg/ml).!Proteinase!K!was!replaced!by!PBT!for!the!next!5!min!and!
post?fixed! with! Glutaraldehyde?0,2%! in! PFA?4%! for! 20! min.! After! fixation,!
embryos!were!rinsed!twice!in!PBT!for!5!min!each.!Finally,!PBT!was!replaced!by!
hybridization!buffer!(50%!formamide,!5x!SSC,!2%!blocking!powder,!0.1%!Triton!
X?100;!0.1%!Chaps,!1mg/ml!tRNA,!50ug/ml!Heparin!pH4.5,!500mM!EDTA!pH8)!
and!embryos!were!prehybridazed!ON!at!65ºC.!
The! next! day,! embryos! were! frozen!at! ?20ºC! at! least! for! 6h!and! then!
hybridized.! For! hybridization,! previous! hybridization! buffer! was! replaced! by!
hybridization! buffer! containing! the! desired! amount! of! probe! for! each!
experiment!and!hybridized!ON!at!65ºC.!Before!being!replaced,!the!hybridization!
buffer!containing!the! probe!was! previously! heated! at! 90ºC! for! 2! min! to! avoid!
possible!secondary!structures!present!within!the!probe.!
The! following! day,! to! get! rid! of! unspecific! binding! post?hybridization!
washes! were! performed.! Hybridization! buffer! was! replaced! by! Chaps?0.1%! in!
2xSSC!(1M!NaCL,!100mM!Sodium!Citrate,!0.1%!Chaps)!and!washed!3!times!for!
30!min!each!at!65ºC.!Then,!embryos!were!rinsed!three!times!in!Chaps?0.1%!in!
0.2xSSC! (10mM! NaCl,! 1mM! Sodium! Citrate,! 0.1%! Chaps)! for! 1h! and! 10! min.!
Afterwards,! embryos! were! washed! twice! for! 10! min! in! KTBT! buffer! at! RT!
(50mM! TrisHCL! pH7,4,! 150mM! NaCL,! 10mM! KCL,! triton?1%)! for! posterior!
preblock! with! 20%! sheep! serum! in! KTBT! for! 6h.! For! the! next! step,! embryos!
were! incubated! at! 1/2000! anti?Digoxigenin! Alkaline! Phosphatase! (anti?DigAP)!
antibody!(Ab.)!diluted!in!20%!sheep!serum!in!KTBT!and!rock!ON!at!4!ºC.!!
Materials,&,Methods!
!
!
92!
For!post?antibody!washes!embryos!were!rinsed!at!RT!in!KTBT!for!1h,!5!
or!more!times!and!then!ON!at!4ºC.!The!next!day,!embryos!were!rinsed!twice!in!
NTMT!buffer!(100mM!TrisHCL!pH9.5,!50mM!MgCl2;!100mM!NaCL,!Triton?1%)!
for! 15! min.! Finally,! detection! of! alkaline! phophatase! activity! was! obtained!
incubating!the!embryos!in!the!dark!with!NTMT!solution!containing!3!µl/ml!of!
NBT! and! 2,3! µl/ml! of! BCIP! stock! solution.! When! the! desired! signal! level! was!
obtained,! embryos! were! rinsed! in! KTBT! for! 5! min! and! fixed! in! PFA?4%.!The!
embryos!were!observed!with!the!use!of!the!Olympus!SZX12!stereoscopic!scope!
and!photographs!were!taken!with!a!Nikon!DS?Fi1!camera.!
*
3.9*Paraffin*embedding*and*histological*sections*
Embryos! were! embedded! in! paraffin! for! posterior! sectioning,! either! to!
analyze! the! LacZ! staining! in! more! detail,! to! perform! ISH! in! sections,! TUNEL!
assay! or! inmunohistochemistry! (IHC).! After! fixation,! embryos! were! rinsed! in!
PBS!twice!for!removal!of!the!fixative!solution!and!progressively!dehydrated!in!
rising! EtOH! concentrations! in! PBS! (50%,! 70%,! 96%! and! 100%! twice).! The!
embryos! were! cleared! with! two! changes! of! Xilol! (10! min! each)! and! finally!
embedded!in!paraffin!at!60ºC!for!1h!30!min.!A!Leica!RM2125RT!microtome!was!
used!to!perform!7!μm!embryo!sections.!!
!
3.10*Araldite*embedding*and*semithin*sections*
Araldite! embedding! was! performed! following! standard!procedures.!
Briefly,! tissue! was! fixed! ON! at! 4ªC! in! a! fixative! solution! containing!PFA?4%,!
glutaraldehyde?0.016%,!CaCl2!0.002%!and!phosphate!buffer!0.12M.!For!removal!
of!the!fixative,!tissue!was!washed!for!several!times!with!0.4M!phosphates!buffer!
and! post?fixed!in! osmium! tetroxide!(Glucose! 0,04M,! phosphate! buffer! 0,14M!
Cl2Ca! 0,0125%! and! O4Os! 2%).! After! 2h,! osmium! tetroxide! was! removed! and!
tissue!was! washed!ON.!Then,!tissue!dehydration!was! performed! by! the!use! of!
rising! acetone! concentrations.! Afterwards,! tissue! was! embedded! in! 50%!
Materials,&,Methods!
*
!
93!
araldite?50%!anhydride!acetone!and!finally!kept!in!100%!araldite!for!3!days!at!
60ºC!to!allow!araldite!polymerization!for!posterior!semithin!sectioning.!
*
3.11*In*Situ*Hybridization*in*sections*
To!perform!ISH!in!paraffin!sections,!paraffin!was!removed!from!the!tissue!
sections!by!two!washes!of!Xilol!of!10!min!each!and!routinely!rehydrated.!Next,!
permeabilization!with!proteinase!K!(10!µg/ml)!was!performed!for!7!min!and!30!
seconds.!Slides!were!immediately!rinsed!in!PBS!for!5!min!and!post?fixed!for!20!
min!in!PFA?4%.!Afterwards,!the!slides!were!rinsed!in!PBS!for!5!min!twice.!PBS!
washes!were!followed!by!an!acetylation!step!(0,1M!Triethanolamine,!0,066mM!
Acetic!Anhydride)!of!10!min!to!reduce!background.!Then,!the!slides!were!rinsed!
twice!in!PBT!for!5!min!each!and!washed!for!additional!5!min!in!distilled!water!
(dH2O)!before!allowing!them!to!air!dry.!!
Once! the! slides! were! dried,! hybridization! buffer! containing! the! desired!
amount! of! probe! for! each! experiment! was! added! and! a! coverslip! was! applied!
over!the!slides!for!posterior!hybridization!ON!at!65ºC,!in!a!humid!chamber.!The!
hybridization! buffer! containing! the! desired! amount! of! probe! was! previously!
heated!at!90ºC!for!2!min!to!avoid!secondary!structures.!
The! next! day,! slides! were! rinsed! in! 5xSSC! at! RT! (2.5M! NaCL,! 250mM!
Sodium!Citrate)!until!the!covers!were!removed!and!post?hybridization!washes!
were!then!performed!to!remove!unspecific!bindings.!Slides!were!washed!for!30!
min! in! 1xSSC/Formamide! (500mM! NaCL,! 50mM! Sodium! Citrate! and! 50%!
formamide),!afterwards!20!min!in!2xSSC!(1M!NaCL,!100mM!Sodium!Citrate)!and!
two! additional! 20! min! washes! in! 0.2xSSC! (10mM! NaCL,! 1mM! Sodium! Citrate)!
were!performed.!All!the!post?hybridization!washes!were!done!at!65ºC.!Finally,!
three! washes! of! 5! min! each! at! RT! in! MABT! pH7,5! (NaCL! 150mM,! Maleic! Acid!
100mM,! 0.04%! Tween)! were! performed! previous! to! incubation! with! blocking!
solution!(20%!sheep!serum!in!MABT)!for!1h,!at!RT.!The!blocking!solution!was!
replaced!by!5%!sheep!serum!in!MABT,!containing!1/2500!dilution!of!the!anti?
DigAP.!Ab.,!for!posterior!incubation!ON!at!4ºC!in!a!humid!chamber.!
Materials,&,Methods!
!
!
94!
Finally,!for!post?antibody!washes!embryos!were!rinsed!at!RT!in!MABT!for!
5!min,!3!times.!Then,!MABT!was!replaced!by!NTM!pH9.5!for!10!min.!Detection!of!
alkaline! phophatase! activity! was! obtained! incubating! the! embryos! in! the! dark!
with!NTM!containing!3! µl/ml!of!NBT!and!2,3!µl/ml!of!BCIP!stock!solution.!When!
the! desired! signal! level! was! obtained,! embryos! were! rinsed! in! PBS! for! 5! min!
treated!with!osmium!tetroxide?0.02%!for!1!min,!washed!5!min!in!PBS!and!fixed!
in! PFA?4%.! After! 20! min,! slides! were! washed! in! PBS! for! 5! min,! then! in! dH2O!
other! 5! min! and! stained! with! eosin! for! 3! min! for! posterior! progressive!
dehydration!using!changes!of!10!min!in!rising!EtOH!concentrations!(50%,!70%,!
96%,! 100%! twice! and! Xilol! twice).! Cytoseal! was! used! as! mounting! media.!
Samples!were!observed!with!the!use!of!the!Nikon!ECLIPSE!80i!microscope!and!
photographs!were!taken!with!a!Nikon!DS?Ri1!camera.!
RNA!probes!used!in!this!study:!
Fgf8% (Crossley! et% al.,! 1996),%Bmp4%(Bandyopadhyay! et% al.,!2006),! Tp63%
(Mills!et%al.,!1999),%Dlx5%(Robledo!et%al.,!2002),!Wnt7a%(Cygan et%al.,!1997),!En1%
(Loomis! et% al.,! 1996),% Msx2% (Robert! et% al.,! 1991),%Shh% (Lopez?Martinez! et% al.,!
1995),!Fgf10%(Sekine!et%al.,!1999),%Sp6%(Nakamura!et%al.,!2004),!Sp8%(Treichel!et%
al.,!2003).!
!
3.12*LacZ*Staining*
LacZ!staining!was!performed!to!visualize!Cre!dependent! removal! of! the!
STOP!codon!that!avoids!LacZ!expression!in!R26R!mice.!Embryos!were!dissected!
in!cold!PBS!and!transferred!to!a!numbered!2!ml!eppendorf!tube!containing!PBS!
and! keep! in! ice! until! all! embryos! were! collected.! Then,! the! PBS! solution! was!
replaced! by! fixative! solution! (1%! Formaldehyde,! 0.2%! Glutaraldehyde,! 0.02%!
NP40,! 0.01%! Sodium! deoxycholate! in! PBS)!for! 20?30! min! (depending! on! the!
stage)!at!4!ºC!on!a!rocking!platform.!After!fixation,!the!embryos!were!rinsed!with!
two!changes!of!PBS!for!10!min!each!and!washed!with!two!changes!of!LacZ!buffer!
pH! 7,3! (Sodium! phosphate! dibasic! 75mM,! Sodium! phosphate! monobasic!
monohydrated! 30mM,! MgCl2! 2mM,! 0.1%! sodic! deoxichalate! (Merck,! reference!
Materials,&,Methods!
*
!
95!
1.06504.0250),!0.2%!igepal!(Aldrich,!reference!542334),!Tris!HCL!20mM)!for!10!
min!each,!at!37ºC.!LacZ!buffer!was!replaced!by!the!staining!solution!(potassium!
ferrocyanide!(K4Fe!(Cn6))!5mM,!potassium!ferrocyanide!(K3Fe!(Cn6))!5mM,!X?Gal!
0,1%)! and! the! embryos! were! kept! in! this! solution! for! 90! min! on! a! rocking!
platform!at!RT.!Staining!reaction!was!stopped!replacing!the!staining!solution!by!
PBS.!After!several!washes!in!PBS,!the!embryos!were!fixed!in!PFA?4%!and!stored!
at! 4ºC.! The! embryos! were! observed! with! the! use! of! the! Olympus! SZX12!
stereoscopic!scope!and!photographs!were!taken!with!a!Nikon!DS?Fi1!camera.!
*
3.13*Tamoxifen*administration*
Tamoxifen! administration! was! used! to! activate! nuclear! translocation! of!
the!CreERT2!enzyme!in!experiments!were!the!Sp8CreERT2!line!was!mated!either!to!
R26R! mice,! to! evaluate!it! use! for! temporally! controlled! deletion! of! genes!
specifically!from!the!limb!ectoderm,!or!to!Catnblox(Ex3)%mice,!to!generate!a!GOF!of!
mutation!
β
Fcatenin.!40mg!Tam!were!dissolved!in!400μl!of!absolute!EtOH.!This!
required!extensive!vortex!to!obtain!complete!solution.!Then!3.6!ml!of!autoclaved!
sunflower! oil! were! added.!To!evaluate!the! use! of! the! Sp8CreERT2!line! for!
temporally! controlled! deletion! of! genes! specifically! from! the! limb! ectoderm,!
intraperitoneal! administration! of! 1mg! (100! μl)! to! pregnant! mice! with! a!
tuberculin!syringe!and!25?gauge!needle,!at!E7.5,!was!performed.!Embryos!were!
obtained!by!caesarean!section!48h!later!(E9.5).!Tam!oral!gavage!was!performed!
for!the!analysis!of!the!GOF!mutation!of!ßFcatenin%with!the!Sp8CreERT!line.!For!oral!
gavage! 0.05! Tam! mg! per! mice! gram! weight! were! administrated.! Three!
consecutive! tamoxifen! oral! administrations! every! 24h! were! performed! from!
E7.5!to!E9.5.!and!embryos!were!obtained!by!cesarean!section!of!pregnant!mice!at!
E10.5.!
!
3.14*mRNA*extraction*
Mouse! E10.5! mRNA! was! used! for! the! quantitative! analysis! of! the!
expression! levels! of! Sp8!and! Sp6! in! the! limb! ectoderm! by! quantitative?PCR!
Materials,&,Methods!
!
!
96!
analysis!(q?PCR),!as!well!as!for!the!generation!of!Fgf8!and!Bmp4!probes!by!PCR!
and!to!clone!Sp8.!
For! mRNA! extraction! tissue! weight! was! measured! and! 500! µl! of! Trizol!
(Invitrogen)! per! 100mg! were! added! to! each! sample! for! posterior!
homogenization!of!the!tissue.!Once!homogenized,!samples!were!laid!for!5!min!at!
RT! and! incubated! in! 0.1! ml! of! chloroform! per! volume! of! Trizol.! After! 3! min,!
samples!were!centrifuged!at!12.000G!for!15!min!at!4ºC.!After!centrifugation,!the!
supernatant!was!collected!and!incubated!with!0.5ml!isopropanol!per!volume!of!
Trizol.!After!10!min,!samples!were!centrifuged!at!12.000G!for!10!min!at!4ºC.!The!
supernatant! was! discarded! and! the! pellet! washed! with! EtOH?70%! and!
resuspended! in! RNA?ase! free! water! after! the! EtOH! was! evaporated.! Samples!
were!then!stored!at!?80ºC!before!measuring!the!concentration!at!the!Nanodrop!
(Nanodrop!technologies!ND?1000).!
*
3.15*Retro*Transcription**
cDNA! was! use! as! template! for! q?PCR,! PCR! probes! generation! and! Sp8!
cloning.! To! perform! the! retro?transcriptase!PCR! (rt?PCR),! two! mix! were!
prepared.!In!the!first,!1ug!of!total!RNA,!obtained!from!E10.5!mouse!embryos,!was!
diluted!in!10.5µl!RNA?ase!free!water!and!heated!at!65ºC!for!5!min.!Afterwards,!
9.5µl! of! the! second! mix! containing! the! reaction! buffer! (DTT! (0,1M),! dNTP,!
random!primers,!RNA?ase!inhibitor!and!M?MLV!retro?transcriptase!(Invitrogen))!
were!added!and!immediately!incubated!for!50!min!at!42ºC!and!additional!5!min!
at!70ªC,!before!storing!at!?20ºC.!
*
3.16*Quantitative*PCR**
Mouse! E10.5! limb! RNA! samples! were! used! for! rt?PCR! and! posterior!
analysis! of! Sp6!and! Sp8!expression! level! by! q?PCR.! E10.5! embryos! were!
harvested! in! PBS! and! forelimbs! and! hindlimbs! were! removed.! RNA! was!
extracted!separately!from!3!pools!of!8!forelimbs!or!8!hindlimbs!each,!and!then!
Materials,&,Methods!
*
!
97!
retrotranscribed!as!described!above.!3!pools!were!analyzed!for!either!forelimbs!
or!hindlimbs!and!two!replicates!for!each!pool!were!performed.!!
After! rt?PCR! cDNA! concentration! was! measured! with! the! Nanodrop!
(Nanodrop!technologies!ND?1000)!and!adjust!to!1µg/µl.!q?PCR!was! performed!
with!the!use!of!the!Mx3005P!cycler!and!MxPro!software!analysis!(Stratagene).!
For! q?PCR! reaction,! 40µl! reaction! mix! containing! 2µg! of! cDNA,! 20µl! of!
SYBRgreen! mix! and! 18µl! of! water! was! prepared.! Final! concentration! of! the!
specific!primers!for!each!gene!analyzed!was!of!125nM.!Afterwards,!the!samples!
were!divided!in!two,!obtaining!two!18µl!replicates!of!each!sample.!!
The! housekeeping! gene! used! as! a! control! to! normalize! the! expression!
levels!of!each!one!of!the!genes!measured!in!this!experiment!was!the!18sRNA.!The!
relative! expression! levels! were! calculated! using! the! equation! 2?∆Ct!(Livak! and!
Schmittgen,!2001),!were!the!threshold!for!each!cycle!(Ct)!is!defined!as!the!cycle!
in!which!the!fluorescence!reaches!a!stable!threshold!level.!!
The! primers! used! to! analyze! Sp6,! Sp8!and! 18sRNA!expression! levels! by!
qPCR!SYBRGreen!are!indicated!below:!
Sp6!qPCR!primers!(5´to!3´orientation):!
Sp6?F:!tgctaaccgctgtctgtgg!
Sp6?R:ctggtatgtctggagaggttgc!
Sp8!qPCR!primers!(5´to!3´orientation):!
Sp8?F:!ttatctccaaggtgcacacg!!
Sp8?R:gcttgaaccaggactcatacg!
18sRNA!qPCR!primers!(5´to!3´orientation):!
18sRNA?R:!ttggcaatgtttcgctc!
18sRNA?F:!cgccgctagaggtgaaattt!
!
3.17*Generation*of*antisense*probes*for*ISH*by*PCR!!
A! 384bp! length! Fgf8!and! a! 597bp! length! Bmp4!specific! probes! for! ISH!
were! generated! by! PCR,! using! cDNA! samples! obtained! from! E10.5! embryos.!
Materials,&,Methods!
!
!
98!
Specific! primers! for! PCR! amplification! of! Fgf8!and! Bmp4! from! cDNA! samples!
were!designed!using!on!line!bioinformatics!tools!such!as!sequence!analysis!and!
BLAST! programs,! together! with! the! sequence! manipulation! suite! website!
(www.bioinformatics.org.).! Once! amplified,! the! PCR! product! was! run! in! 1%!
agarose! gel! and! gel! purified! following! manufacturer! instructions! of! the!
Quiaquick!gel!extraction!kit!(Quiagen).!Addition!of!recognition!sites!for!the!Sp6!
polymerase! to! 5´ends! of! specific! reverse! primers! used! to! amplify! each! of! the!
genes,! enables! direct! “inFvitro“% transcription! of! the! antisense! probe! for! ISH!
directly! from! gel! purified! PCR! products.! Primer?sequences! used! are! specified!
below,!whereby!Sp6!polymerase!promoter?sequence!is!underlined!and!in!upper!
case.!
Fgf8!antisense!PCR!probe!primers!(5´to!3´orientation):!
Fgf8?F:!gcacttgctggttctctgc!
Fgf8?R:!ATTTAGGTGACACTATAGAAggtagttgaggaactcgaagc!
Bmp4!antisense!PCR!probe!primers!(5´to!3´orientation):!
Bmp4?F:!tcattccggattacatgagg!
Bmp4?R:!ATTTAGGTGACACTATAGAAcggcagttcttattcttcttcc!
*
3.18*Isolation*and*cloning*of*the*Sp8*coding*sequence*
1516bp!fragment!corresponding!to!the!CDS!of!the!Sp8!gene!was!amplified!
from!cDNA!samples!from!E10.5!embryos!with!the!use!of!PCR!extender!system!
(5Prime)! using! the! primers! specified! below.! The! PCR! product! was! run! in! 1%!
agarose!gel!for!posterior!purification!with!the!use!of!the!Quiaquick!gel!extraction!
kit! (Quiagen)!following! manufacturers! recommendations! and! cloned! with! the!
used!of!the!TOPO?TA!cloning!kit!(Invitrogen)!into!provided!pCR2.1?Topo!vector.!!
For!plasmid!amplification,!4µl!of!the!pCR2.1?Topo!vector!bearing!the!PCR!
product!were!used!for!transformation!of!Dh5α!(E.%coli)!competent!bacteria.!After!
incubation!of!transformed!bacteria,!colony!PCR!screening!of!individual!colonies!
was!performed!with!the!use!of!M13!primers!that!recognized!flanking!regions!of!
the!polylinker!from!the!pCR2.1?Topo!vector.!Among!the!screened!colonies,!three!
Materials,&,Methods!
*
!
99!
colonies!that!carried!an!insert!of!the!correct!size!were!sequenced.!The!sequence!
confirmed!100%!homology!to!Sp8.!Once!the!CDS!of!the!Sp8!gene!was!confirmed,!
it!was!cloned!into!the!expression!vector!pCIG!(Megason!and!McMahon,!2002),!a!
pCAGGS! modified! vector! containing! an! internal! ribosomal! entry! site! (IRES)!
followed! by! a! cDNA! encoding! EGFP! (pCAGG?IRES2?nucEGFP),! useful!for!
electroporation!in!chick!and!mouse.!
To! release! the! Sp8! CDS! from! the! pCR2.1?Topo! vector,! the! vector! was!
linearised!with!the!use!of!the!BamHI!RE.!Then,!fill!in!of!the!3´end!was!performed!
with! the! use! of! the! Klenow! Fragment! (Thermoscientific)! before! releasing! the!
insert!performing!a!second!digestion!with!XhoI!RE.!Once!the!insert!was!released!
the!digestion!product!was!run!in!1%!agarose!gel!and!purify!with!the!Quiaquick!
gel!extraction!kit!(Quiagen)!and!phenol!chloroform!precipitated.!The!PCIG!was!
linearised! performing! a! double! digestion! with! SmaI! and! XhoI! and!
dephosphorylated! with! the!use! of! Shrimp! Alkaline! Phosphatase!
(Thermoscientific).!Different! proportions! of!linearised! PCIG!and! gel! purify!and!
phenol!chloroform!precipitated!insert!containing!the!CDS!of!the!Sp8!gene!(1:0,!
1:1,! 1:3! and! 1:5)! were! incubated! ON! at! 14ºC! for! ligation! in! the! presence! of!
polyethilengycol! (PEG)! and! the! T4?ligase.! The! next! day,! bacteria! were!
transformed! and! grown! as! explained! previously.! Lastly,! a! couple! of! colonies!
containing!the!target!insert!as!verified!by!colony!PCR!screening!were!grown!and!
plasmidic! DNA! was! purified! with! the! use! of! Quiaquick! gel! extraction! kit!
(Quiagen)!and!finally!sequenced.!
M13!primers!used!for!colony!PCR!(5´to!3´orientation):!
M13?F:!GTAAAACGACGGCCAG!
M13?R:!CAGGAAACAGCTATGAC!
Primers! used! to! amplify! Sp8! from! mouse! E10.5! RNA! samples! (5´to!
3´orientation):!
Sp8?F:!GGG!AAC!GCG!AGA!AGA!ACC!
Sp8?R:!GCC!CAG!TCT!AGG!TAC!ACA!AGC!
*
*
Materials,&,Methods!
!
!
100!
3.19*TUNEL*assay!
The! ApopTtag! Direct! Fluorescein! In! Situ! Apoptosis! Detection! kit!
(Milipore,! S7110)! was! used! to! perform! terminal! deoxynucletidyl! transferase!
mediated! deoxyuridinetriphosphate! nick! end! labeling!assay! (TUNEL)!for! the!
analysis! of! cell! death.! It! was! performed! following! manufacturer!
recommendations.!
The!terminal!deoxynucleotidyl!transferase!enzyme!(TdT)!provided!in!the!
kit! catalyzes! a! template?independent! addition! of! fluorescein! (FITC)! tagged!
triphosphate! nucleotides!to! the! free! 3´?OH! ends! of! single! stranded! or! double?
stranded!DNA.!Interestingly,!while!the!low!number!of!free!3´?OH!ends!present!in!
normal!and!proliferative!nuclei!are!not!stained,!the!kit!allows!specific!detection!
of!the!high!concentrated!free!3´?OH!ends!characteristics!of!apoptotic!cells,!as!a!
result!of!DNA!fragmentation.!
7µm!paraffin! sections!were! deparafined,!rehydrated! and! permeabilized.!
Proteinase!K!digestion!(10ug/ml)!was!performed!for!7min!and!30!seconds.!Then,!
samples!were!rinsed!twice!in!PBS!for!5!min!and!incubated!with!the!Equilibration!
Buffer! provided! by! the! Kit.! After! 10! seconds! approximately,! 1h! and! 30! min!
incubation! in! the! working! strength! TdT! enzyme! was! performed.! Enzymatic!
activity!was!stopped!applying!the!Stop/Wash!buffer!for!10! min.!After!washing!
three!times!in!PBS!for!5!min!per!wash,!samples!were!mounted!using!Vectashield!
containing! DAPI! for! nuclear! counter! stain! and!were!visualized! and!
photographied!with! the! SP?5! laser?scan! confocal! microscope! (Leica!
Microsystems).!!
*
3.2O*Immunohistochemistry**
Laminin?b! (Laminin?REF#111575Abcam?)! and! E?cadherin! (Cdh1F
REF#600182Byoscience?)! were! detected! by! immunohistochemitry! (IHC)! in!
paraffin! sections,! to! distinguish! between! ectodermal! and! mesodermal!
compartments! of! the! limb! bud.! To! carry! out! the! double! IHC,! sections! were!
hydrated! and! permeabilized! with! proteinase! K! and! incubated! with! blocking!
Materials,&,Methods!
*
!
101!
serum! (Tris! 50mM,! NaCL! 150mM,! HCL! 0.05N,! 1%! goat! serum,! 1%! BSA,! 0.1%!
Triton?X)!for!1h!at!RT.!Then,!samples!were!incubated!with!Laminin!and!Cdh?1!
primary!Abs! (1/300!and! 1/200! diluted! in! blocking! serum,! respectively)! ON! at!
4ºC.!The!next!day,!tissue!sections!were!rinsed!twice!in!PBS!for!5!min!each!and!
incubated! with! the! fluorescensce! secondary! Abs.! Alexa! 488! and! TexasRed! Ab!
(Jackson!Lab.)!(1/2000!and!1/75!in!PBS,!respectively)!for!1h!and!30!min.!Finally,!
after! three! washes! of! 5! min! each! in! PBS! samples! were! mounted.! Vectashield!
containing! DAPI! for! nuclear! counter! stain! and! were! visualized! and!
photographied!with! the! SP?5! laser?scan! confocal! microscope! (Leica!
Microsystems).!
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105!
4.*Results*
Several!studies! have! positioned! the! transcription! factors! Sp6! and! Sp8!
downstream! of! Wnt/β?catenin%and! upstream! Fgf8! in! the! limb! ectoderm!
(Talamillo! et% al.,! 2010;! Treichel! et% al.,! 2003;! Bell! et% al.,! 2003;! Kawakami! et% al.,!
2004).!Moreover,!the!ability!of!Sp8!to!induce!Fgf8!supports!its!possible!role!in!
Fgf8! induction! (Kawakami! et% al.,! 2004;! Sahara! et% al.,!2007).! However,! single!
disruption!of!either!Sp6!or!Sp8!did!not!result!in!absence!of!Fgf8!induction!in!the!
limb!ectoderm!(Talamillo!et%al.,!2010;!Treichel!et%al.,!2003;!Bell!et%al.,!2003).!Sp6!
and!Sp8! are! expressed! in! the! entire! prospective! limb! ectoderm! prior! to! Fgf8!
induction! and! progressively! become! confined! to! the! AER! as! the! limb! bud!
emerges!(Talamillo!et%al.,!2010;!Treichel!et%al.,!2003;!Bell!et%al.,!2003).!Individual!
genetic!inactivation!of!Sp6!results!in!soft!tissue!syndactyly!of!digits!2!and!3!in!the!
forelimb!and!synostosis!of!digits!3!and!4!in!the!hindlimb!(Talamillo!et%al.,!2010),!
whereas!disruption!of!Sp8!results!in!limb!truncation!at!a!variable!level!but!most!
commonly!at!the!level!of!the!elbow/knee!(Treichel!et%al.,!2003;!Bell!et%al.,!2003).!
The! mild! phenotype! observed! in! the! Sp6! mutant! may! result! from! an!
altered! formation! of! the! digital! plate! probably! as! a! consequence! of! AER!
maturation! defects! (Talamillo! et% al.,! 2010),! while! the! severe! limb! truncation!
observed!in!Sp8!mutants!is!due!to!the!premature!regression!of!the!AER!(Treichel!
et% al.,! 2003;! Bell! et% al.,! 2003).! These! studies! demonstrated! that! individual!
inactivation!of!either!Sp6!or!Sp8!does!not!interfere!with!the!induction!of!Fgf8!in!
AER! cells! (Talamillo! et% al.,! 2010;! Treichel! et% al.,! 2003;! Bell! et% al.,! 2003).! Both!
transcription!factor!acts!downstream!of!Wnt/ß?catenin!signalling!and!upstream!
of! Fgf8% in! the! limb! ectoderm! (Kawakami! et% al.,! 2004;! Sahara! et% al.,!2007;!
Talamillo!et%al.,!2010).!This,!together!with!their!similar!overlapping!patterns!of!
expression! led! us! to! propose! that! Sp6!and! Sp8!could! function! in! a! redundant!
manner!mediating!the!Wnt/ß?catenin!dependent!induction!of!Fgf8.!To!test!our!
hypothesis!we!decided!to!perform!the!Sp6F/F;Sp8CreERT2!/CreERT2!double!mutant!and!
also! the! Sp8! conditional! mutant! in! an! Sp6!null! background! with!the! use!the!
Ap2
α
FCre%and!the!Msx2FCre!deleter!lines.!
Results!
!
!
106!
4.1*Analysis*of*the*expression*of*Sp6*in*the*absence*of*Sp8**
In!support!of!a!possible!functional!redundancy!between! Sp6!and!Sp8,!it!
was!shown!that!Sp8!expression!was!maintained!in!the!absence!of!Sp6%(Talamillo!
et%al.,!2010).!Hence,!we!decided!to!analyze!if!Sp6!expression!was!also!maintained!
in! mice! lacking! Sp8,! with! the! use! of! the! Sp8CreERT!null! allele.! In! the! limb,! Sp6!
expression! is! first! detected! in! dorsal! and! ventral! pre?limb! ectoderm! of! both!
hindlimb! and! forelimb! buds! at! E9,! prior! to! Fgf8!induction,! and! later! its!
expression!progressively!declines,!first!from!the!dorsal!and!then!from!the!ventral!
ectoderm!to!get!predominantly!confined!to!the!AER!(Talamillo!et%al.,!2010).!!
Sp6!expression! in! Sp8! mutant! embryos! was! analyzed!at! different!
developmental!stages,!from!E9.5!to!E11.5!(Fig.!19).!At!E9.5!Sp6!was!detected!in!
the!mutant!limb!ectoderm,!but!at!lower!levels!in!comparison!to!control!animals!
(Fig.! 19A,! B).! At! E10.5,! Sp6!expression! was! still! detectable! but! a! considerable!
decrease! was! appreciable! in! comparison! to! control! embryos! (Fig.! 19.D,! E),!
similarly! to! the! progressively! decrease! of! Fgf8!expression!previously! reported!
for!Sp8!mutants!(Treichel!et%al.,!2003;!Bell!et%al.,!2003).!At!E11.5,!residual!Sp6!
expression!was!detected!in!correlation!with!the!regressing!AER!(Fig.!19.C,!F).!!
The!expression!of!Sp6!in!Sp8!mutants!and!vice!versa,!together!with!their!
overlapping! patterns! of! expression,! supports! our! hypothesis! that! both! factors!
could!function!in!a!redundant!manner!mediating!the!Wnt/ß?catenin!dependent!
Fgf8!induction!in!the!limb!ectoderm.!Hence,!we!decided!to!perform!the!double!
Sp6;Sp8!mutant.!
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Results!
!
107!
!
!
Figure* 19.* Sp6*expression* was*initiated* in* Sp8*mutants* but* it* was*not*
maintained.*Lateral!views!of!control!(A,D,C)!and!Sp8!mutant!embryos!(B,E,F)!
at!the!stage!indicated.!Note!that!Sp6!expression!was!initiated!in!absence!of!Sp8!
but! with! a! considerable! decrease! in! comparison! to! control! embryo! and! was!
progressively!lost,!in!correlation!with!the!regressing!AER!in!Sp8!mutants.!!
*
4.2*Limb*phenotype*of*Sp6;Sp8*compound*mutants*
To!obtain!Sp6;Sp8!dKO,!we!used!the!Sp6?null!(Nakamura!et%al.,!2004)!and!
the! Sp8CreERT2!null! alleles.! Progeny! from! crosses! between! Sp6;Sp8!double!
heterozygous!mice!were!analyzed.!Double!Sp6+/F;Sp8+/CreERT2!heterozygous!mice!
showed! normal! limb! patterning! and! skeletogenesis! yet! displayed! subnormal!
fertility.! When! performing! these! crosses,! we! found! a! reduced! frequency! of!
pregnancies!in!double!heterozygous!females.!Table!1!shows!the!Mendelian!rates!
of! newborns! obtained! from! crosses! between! double! heterozygous! mice.! From!
the! 78! recovered,! the! expected! Mendelian! rates! were! obtained! for! all! the!
genotypes! with! the! exception! of! double! mutants! that! were!represented!
significantly!below!the!expected!1/16!rate!(Table!1).!!
Results!
!
!
108!
Table* 1.Expected* and* obtained* Mendelian* rates* from* crosses* between*
double*Sp6+/);Sp8+/CreERT2*heterozygous*mice.*!
Genotype*
Mendelian*frequency*
Expected*
obtained*
Sp6+/+;Sp8+/+$
1/16!
5!
6!
Sp6+/);Sp8+/+$
1/8!
10!
14!
Sp6+/+;Sp8+/CreERT2$
1/8!
10!
11!
Sp6+/);Sp8+/CreERT2$
1/4!
20!
22!
Sp6)/);Sp8+/+$
1/16!
5!
5!
Sp6+/+;Sp8CreERT2/CreERT2$
1/16!
5!
4!
Sp6)/);Sp8+/CreERT2$
1/8!
10!
8!
Sp6+/);Sp8CreERT2/CreERT2$
1/8!
10!
8!
Sp6)/);Sp8CreERT2/CreERT2$
1/16!
5!
1!
Total*
!
!
78!
!
!
Skeletal! preparations! of! newborns! from! the! whole! mutant! allelic! series!
were!prepared!to!analyze! the! limb!phenotype.!Sp6!mutant! limbs! exhibited! the!
previously! described! phenotype! (Talamillo! et% al.,! 2010).! Briefly,! limb! defects!
were!restricted!to!the!autopods,!soft!tissue!syndactyly!of!digits!2?3!in!forelimb!
and!synostosys!of! digits!3?4! in!hindlimbs! (Fig.!20E,! F).!The! penetrance! was! of!
60%!with!variable!expressivity.!Interestingly,!the!phenotype!was!stronger!on!the!
left!side!with!individuals!showing!only!defects!in!that!side.!DV!defects!over!the!
central!digits!were!also!present.!The!hyponychium,!the!ventral!structure!of!the!
nail,! presented! a! keratinized! morphology! typical! of! the! dorsal! nail! plate!
conferring!a! double! dorsal! phenotype! to! the! distal! tips! of! central! digits!
(Talamillo!et%al.,!2010).!!
Results!
!
109!
!
Figure* 20.* External* morphology*and* skeletal* staining* of* Sp6*and* Sp8*
mutant*newborns.! The! genotype! is! indicated! at!the!top.! (A,D,G)!Gross!
morphology!of!newborn!and!fore?!and!hindlimb!skeletal!staining!as!indicated.!
Control!embryo!(A?C),!Sp6!mutant!(D?F)!and!Sp8!mutant!(G?I).!Limb!defects!in!
the!Sp6!mutant!are!restricted!to!the!autopod!with!the!presence!of!soft!tissue!
syndactyly!of!digits!2!and!3!in!the!forelimb!and!synostosis!of!digits!3!and!4!in!
the!hindlimb!(F).!Lack!of!Sp8!results!in!severely!truncated!limbs!at!the!level!of!
the!elbow/knee!(H,I).!
!Mice! lacking! Sp8! term! to! developed! and! died.! This! mutants! exhibited!
exencephaly! due! to! defects! in! neuropore! closure! and! also! caudal! defects! as!
previously! reported! (Treichel! et% al.,! 2003;! Bell! et% al.,! 2003).! Elongation! of! the!
body!along!the!craneal?caudal!axis!appeared!truncated!at!the!level!of!the!sacral!
region.! Regarding! the! limb,! both! the! forelimb! and! the! hindlimb! exhibited!
truncations!at!variable!level!along!the!PD!axis!as!previously!reported,!but!most!
commonly!at!the!level!of!the!elbow/knee!(Treichel!et%al.,!2003;!Bell!et%al.,!2003).!
In!our!crosses,!5!out!of!the!8!Sp8!mutant!obtained!showed!forelimbs!truncated!at!
the! level! of! the! elbow!with! either! presence! (3/8)! or! absence! (2/8)! of! the!
olecranon,! whereas! in! the! remaining! 3! a! truncated! ulna! was! developed! (Fig.!
20H).! Half! of! the! hindlimbs! analyzed! were! truncated! at! the! level! of! the! knee!
lacking!all!the!elements!distal!to!the!femur!(4/8),!whereas!in!the!other!half!the!
fibula!was!developed!(4/8)!and!in!one!case!a!truncated!tibia!was!also!present!
(Fig.!20I).!
Results!
!
!
110!
Remarkably,!the! double!mutant!specimen!obtained!showed!a!tetramelic!
phenotype! (Fig.!21A?C).! In! this! mutant,! no! skeletal! forelimb! elements! formed!
distal!to!the!scapula!(Fig.!21B).!The! double!mutant!also!exhibited!exencephaly!
due!to!a!failure!in!neuropore!closure!and!defects!in!the!caudal!region.!At!caudal!
level,! the! lumbar! vertebrae! appeared!highly! disorganized! and! the! body! was!
truncated!at!sacral!level!(Fig.!21C),!consistent!with!Sp8!single!mutants.!However,!
the! caudal! defects!appreciable! in! the! double! mutants! were! more! severe! and!
added!the!absence!of!the!hip!bone.!
!
!
Figure*21.*Effects*of*reducing*the*Sp*genes*on*skeletal*development.*Gross!morphology!of!
newborns! (A,D,G)! and! comparison! of! skeletal! preparations! (B,C,E,F,H,I,J,K)! of! the! genotypes!
indicated.! Sp6F/F;Sp8CreERT2/CreERT2! mutants! lacked! all! the! elements! distal! to! the! scapula! in! the!
forelimb! (B)! whereas! the! body! was! truncated! at! the! level! of! the! sacrum! with! the! lumbar!
vertebrae!highly! disorganized!and! only!a! rudimentary!cartilage!element!contributing!to!the!
pelvis! (C).! Sp6+/F;Sp8CreERT2/CreERT2!mutants!presented! the! same! phenotype! to!that! of! Sp6F/F
;Sp8CreERT2/CreERT2! mutants! with! the! difference! that! a! malformed! pelvis! lacking! the! pubis! was!
developed!(E,F).!Sp6F/F;Sp8+/CreERT2!mutants!displayed!a!variable!limb!phenotype!reminiscent!of!
the! human! Split! hand/foot! malformation.! Forelimbs! exhibited!severely! altered! autopods!
lacking! central! digits! with! the! remaining! posterior! ones! fused! and! the! radius!occasionally!
missing! (H,I).! Hindlimbs! displayed! a! misshaped! and! truncated! tibia! with! either! absent! or!
misshaped!fibula!with!a!row!of!skeletal!rod!(J,K).!
Results!
!
111!
All! the! animals! analyzed! in! which! both! copies! of! the! Sp8!gene! and! one!
copy! of! the! Sp6! gene! had! been! removed! (8/8)! exhibited! an! amelic! phenotype!
identical! to! double! mutants! except! the! development! of! a! misshaped! hip! bone!
lacking!the! pubis! (Fig.!21C,F).! As! in! double! mutants,! defects! in! the! neuropore!
closure!that!resulted!in!exencephaly!were!appreciable!together!with!absence!of!
elements!distal!to!the!scapula!(Fig.!21B)!and!with!the!caudal!disorganization!at!
the!level!of!the!sacral!vertebrae!(Fig.!21F).!The!fact!that!both!mutants!displayed!
an! indistinguishable! limb! phenotype! raised! the! question! of! whether! Sp6!is!
expressed!in!Sp6+/F;Sp8CreERT2/CreERT2.!
Skeletal!defects!in!mice!in!which!both!copies!of!the!Sp6!gene!and!one!copy!
of!the!Sp8!gene!had!been!inactivated!were!restricted!to!the!limbs!and!were!quite!
variable! even! in! the! same! animal! (Fig.!21G?K).! In! the! forelimbs,! the! stylopod!
appeared! always! normal! while!the! radius! was! frequently! missing! and! the!
autopods! displayed! severely! altered! and! variable! phenotypes! (Fig.!21H,I).!The!
paw! had! the! "claw?like"! appearance! reminiscent! of! the! human! Split! hand/foot!
malformation! (SHFM).! In! the! most! severe! cases,! the! anterior! or! central! digits!
were!short!or!missing!and!the!posterior!digits!3?4!or!4?5!were!frequently!fused!
(Fig.!21H).! In! less! affected! cases! 5! digits! were!distinguishable! in! spite! of! the!
SHFM!phenotype.! In! the! hindlimb,! the! phenotype! was! constantly! more! severe!
(Fig.!21J,K).!In!the!most!affected!cases,!a!normal!femur!developed!followed!by!an!
incompletely!developed!tibia!and!a!single!thin!digit!was!in!a!few!cases!present!
(Fig.!21K),!while!in!the!less!severe!cases!the!tibia!and!the!ulna!were!normally!
developed!exhibiting!a!single!digit!(Fig.!21J).!Occasionally,!distal!bifurcation!of!a!
digit!was! observed,! in! one!of! the! forelimb! and! one! of! the! hindlimb! analyzed.!
Overall,!the!phenotype!was!compatible!with!the!SHFM!in!humans,!characterized!
by! the! absence! of! central! digits! and! fusion! of! the! remaining! ones.! Of! most!
interest,!the!digits!in!both!fore!and!hindlimbs!of!Sp6F/F;Sp8+/CreERT2!mutants!were!
bidorsal!lacking!ventral!pads!and!exhibiting!circumferential!nails!(Fig.!22B).!The!
phenotype! was! variable! within! a! single! animal,! each! paw! showing! specific!
deficiencies.!However,!a!left!or!right!tendency!for!severity!was!not!identified.!
*
Results!
!
!
112!
Figure* 22.* Sp6)/);Sp8+/CreERT2*exhibited*
double*dorsal*digit*tips.*Gross!morphology!of!
control! (A)! and! Sp6F/F;Sp8+/CreERT2! mutant! (B)!
newborns.!Note!that!Sp6F/F;Sp8+/CreERT2!digit!tips!
exhibited! a! double! dorsal! phenotype! with!
circumferential!nails.!
!
The! only! difference! between! the! double! mutant! and! the! Sp6+/F
;Sp8CreERT2/CreERT2%mutant%was!at! the! level! of! the! pelvic! hip! bone!that! is! more!
affected!in!the!double!Sp6F/F;Sp8CreERT2/CreERT2%!mutant!(Fig.!23B,C)!.!The!pelvic!hip!
bone!is!formed!by!the!fusion!of!three!distinct!skeletal!elements!the!ischium,!the!
ileum!and!the!pubis.!The!only!evidence!of!the!hip!bone!in!the!double!mutant!was!
a! rudimentary! and! unidentifiable! element! (Fig.! 23B),! whereas! Sp6+/F
;Sp8CreERT2/CreERT2% mutants!consistently!showed!a!hip!bone!in!which!the!ischium!
and!the!ilium!were!developed!but!the!pubis!was!absent!(Fig.!23C).!!
!
!
Figure*23.*Pelvic*hip*bone*development*in*mice*lacking*Sp*genes.*The!fusion!
of! three! elements,! the! ileum,! the! ischium! and! the! pubis,! form! the! hip! bone! .(A)!
control.!(B)*Sp6F/F;Sp8CreERT2/CreERT2!mutants!showed!a!caudal!truncation!of!the!body!
at!the!level!of!the!sacrum!with!a!highly!disorganized!lumbar!vertebrae!and!a!single!
rudimentary!cartilage!element!contributing!to!the!pelvis.!(C)!Sp6+/F;Sp8CreERT2/CreERT2!
mutants!presented!a!similar!caudal!phenotype!to!that!of!Sp6F/F;Sp8CreERT2/CreERT2!with!
the!difference!that!a!pelvis!lacking!the!pubis!with!a!misshaped!ileum!and!ischium!
was!developed.!Yellow,!pubis;!orange,!ischium!and!brown!ileum.!
!
Results!
!
113!
Our! genetic! analysis! demonstrates!that! Sp6!and! Sp8!are! together!
absolutely!required!for!limb!development!and!supports!our!hypothesis!that!they!
perform!partially!redundant!functions!in!the!limb!ectoderm,!as!in!their!absence!
no!limbs!formed.!Our!study!of!the!whole!allelic!series!also!shows!that!one!single!
functional!allele!of!Sp6,!in!the!absence!of!other!Sp!alleles!(Sp6!and!Sp8),%is!unable!
to!support!any!limb!development!as!the!limb!phenotype!is!similar!to!that!of!the!
double! mutant.! However,! one! single! functional! allele! of! Sp8,!in! the! absence! of!
other! Sp!alleles! (Sp6!and! Sp8),! permits! a! considerably! degree! of! limb!
development,! as! the! three! segments! of! the! limb! are! developed! although! digits!
exhibited! a! limb! phenotype! reminiscent! of! the! split! hand/malformation! in!
human,!where!central! digits! are!missing!and! the! remaining! posterior!ones! are!
fused.!
!
4.3*Relative*expression*levels*of*Sp6*and*Sp8*in*E10.5*mouse*limbs*
Since! Sp6!and! Sp8! display! similar! temporal! and! spatial! patterns! of!
expression! in! the! limb! ectoderm,! one! possible! explanation! for! this! functional!
difference! is! that! Sp8!has! specific! functions! that! Sp6! cannot! accomplish.!
However,!it!is!also!possible!that!these!functional!differences!rely!on!differences!
in! their! levels! of! expression.! To! check! this! possibility,!we! quantified!the!
expression! levels! of! Sp6!and! Sp8! in! the! limb! ectoderm! by!quantitative! PCR!
analysis!at!E10.5!in!both!forelimb!and!hindlimb!buds!of!wild!type!embryos.!
Our!results!showed!that!the!level!of!expression!of!Sp8,!at!E10.5!in!control!
embryos,! was! 3?fold! times! higher! than! that! of! Sp6! in! the! forelimb! and! 5?fold!
times!higher!in!the!hindlimb.!In!addition,!the!analysis!also!showed!that!both!Sp6!
and!Sp8!expression!occurred!at!higher!levels!in!the!forelimb!than!in!the!hindlimb!
and!that!this!difference!was!higher!for!Sp6.!In!the!case!of!Sp6,!the!expression!was!
4?fold!times!higher!in!the!forelimb!than!in!the!hindlimb,!while!Sp8!exhibited!a!3?
fold!increase!in!the!forelimb!in!comparison!to!the!hindlimb!(Fig.!24).!
Results!
!
!
114!
!
Figure*24.*Sp8*makes*a*more*substantial*contribution*to*
limb* development* than* Sp6.! Relative! expression! levels! of!
Sp6!and!Sp8!in!mouse!E10.5!limbs!from!3!pools!of!8!forelimbs!
or! 8! hindlimbs! each.! Sp8!showed! a! 3?fold! increase! in!
comparison!to!Sp6!in!the!forelimb!and!5?fold!in!the!hindlimb,!
also! showed! that! Sp6!and! Sp8!expression! levels! in! the!
forelimb!are!higher!than!in!the!hindlimb.!!
!!
Our! results! suggest!that! Sp8! makes! a! more! substantial! contribution! to!
limb! development! than! Sp6!because! of! a! higher! level! of! transcription! and!
provides!an!explanation!for!the!phenotypical!differences!when!only!one!copy!of!
them!remains.!
!
4.4*Selection*of*a*limb*ectoderm*specific*Cre*line*and*monitorization*of*its*
activity*
Because! from! the! double! heterozygous! crosses! we! only! got! one! double!
mutant,!we!decided!to!generate!more!specimens!to!the!phenotype!and!evaluate!
possible! genotype! variability.! To! circumvent! the! issues! of! the! reduced! fertility!
and! the! low! recovery! of! double! mutants,! we! used! an! Sp8!conditional! allele! to!
remove!it!specifically!from!the!limb!ectoderm.!Amongst!the!available!lines!with!
Cre! activity! in! the! limb! ectoderm! Msx2FCre,% Brn4FCre;% RARFCre;% Ap2
α
FCre% and%
Results!
!
115!
Mox2FCre,!we!selected!the!Ap2
α
FCre!line!because!it!has!been!reported!to!drive!
very!early!Cre!function!in!both!fore!and!hindlimbs,!at!least!prior!to!Fgf8!initiation!
of!expression!(Boulet!et%al.,!2004).!Because!Sp8!is!already!expressed!in!the!pre?
limb!ectoderm!prior!to!activation!of!Fgf8!in!the!ventral!ectoderm!(Treichel!et%al.,!
2003;! Bell! et% al.,! 2003! and! authors! personal! observations),! we! decided! to!
determine! in! more! detail! the! activity! of! the! Ap2
α
FCre!transgenic! line! prior! to!
limb!bud!emergence!using!the!R26R!strain!and!evaluate!its!suitability!to!perform!
the!conditional!removal!of!Sp8!(Soriano,!1999).!!
The!analysis!of! Rosa26+/tg;Ap2
α
FCre! embryos! showed! that! at! E8.5! lacZ!
staining! was! widely! detected! covering! practically! the! whole! ectoderm! of! the!
embryo,!including!the!pre?limb!ectoderm!(Fig.!25A,!B).!Detection!across!in!the!
pre?limb!area!was!further!confirmed!in!transverse!sections!within!the!pre?limb!
area!(Fig.! 25C).! In! addition,! transverse! sections! also! revealed!the! presence! of!
LacZ! staining! in! the! dorsal! neural! tube,! with! a! weaker! level! of!staining! in! the!
ventral!part!of!the!neural!tube!(Fig.!25C).!!
!
!
Figure* 25.* ROSA26* reporter* activity* for* Ap2
α
)Cre*transgenic* line* in* an* E8.5* mouse*
embryo.*Dorsal!(A)!and!lateral!(B)!views!of!E8.5!embryo.!(C)!Transversal!section!of!the!same!
embryo!at!the!level!indicated!in!B.!Cre!activity!is!detected!in!neural!tube,!with!stronger!level!in!
the!dorsal!part!and!in!the!entire!ectoderm!by!E8.5,!including!the!pre?limb!ectoderm!indicated!
with!a!red!arrow!head!(C).**
!
The!monitorization!of!the!Cre!under!the!Ap2!promoter!indicated!that!Cre!
dependent!DNA!rearrangement!in!the!pre?limb!ectoderm!occurs!at!least!by!E8.5!
previous!to!limb!bud!appearance,!early!enough!to!remove!Sp8!from!the!limb!bud!
ectoderm!before! Fgf8!expression.! Thus,! we! used! the! AP2
α
FCre!line! to!
Results!
!
!
116!
conditionally!inactivate!a!Sp8!floxed!allele!in!an!Sp6!null!background.!In!addition,!
the!detection!of!LacZ!staining!in!the!neural!tube!indicates!that!the!defects!in!the!
closure!of!the!neural!tube!characteristic!of!Sp8!mutants!will!persist!when!using!
this!Cre!line,!due!to!the!overlapping!patterns!of!expression!of!the%Cre,!under!the!
Ap2!promoter,!and!Sp8!in!this!region.!
!
4.5*Conditional* inactivation* of* Sp8*with*the* Ap2
α
)Cre$ line*in*an* Sp6*null*
background:*Morphological*characterization*of*the*compound*mutants*
by*skeletal*staining*
Newborn!skeletal!phenotypes!from!the!whole!allelic!series!obtained!with!
the!use!of!the!Ap2
α
FCre!line!were!similar!to!those!obtained!from!croses!between!
Sp6+/F;Sp8+/CreERT2!double! heterozygous! mice.! As! previously! mentioned! in!
materials!and!methods!(3.2!Mouse!mating!strategies!),!the!Ap2
α
FCre!line!is!active!
in!the!germ!line.!The!activation!of!the!Cre!in!the!germ!line!lead!to!the!removal!of!
the! floxed! alleles! flanking! the! Sp8!gene,! leading! to! the! generation! of! deleted!
alleles! (D).! Therefore,! all! the! Sp8! mutants! obtained! from! this! crosses! bears! a!
floxed!allele,!but!also!a!deleted!allele!for!Sp8!(Sp8f/D).!
With! these! crosses! we! recovered! 2! newborns! that! lacked! both! Sp6!and!
Sp8!in!the!limb,!and!that!showed!in!tetramelic!phenotype!similar!to!that!of!Sp6F/F
;Sp8CreERT2/CreERT2!(Fig.!26A?C).!In!addition!5!conditional!mutants!for!Sp8!bearing!a!
single! functional! allele! of! Sp6! that! displayed! a! similar! phenotype! to! the! Sp6+/F
;Sp8CreERT2/CreERT2%mutants%were! recovered! (Fig.!26D?F).! The! conditional!
phenotype!was!not!restricted!to!the!limb!confirming!a!wide!overlaping!between!
the!expression!of!Ap2!and!Sp8!in!the!neural!tube,!leading!to!defects!in!the!neural!
tube!closure.! However,! the! general! phenotype! was! slightly! improved,! due! to!a!
less! severe! caudal! regresion! present! in! double! conditional! mutants! in!
comparison!to!the!ubiquitous!mutants!(Fig.!26C).!
!!
!
Results!
!
117!
!
Figure* 26.* Ap2
α
)Cre*dependent* removal* of* the* Sp8*floxed* allele* in* an*Sp6*null*
background* mimicked* the* phenotype* obtained* from* ubiquitous*mutants.* Gross!
morphology!of!newborns!(A,D,G)!and!of!skeletal!preparations!(B,C,E,F,H,I,J)!of!the!genotypes!
indicated.!Sp6F/F;Sp8f/D;Ap2
α
FCre%mutants!lacked!all!the!elements!distal!to!the!scapula!in!the!
forelimb! (B)! whereas!only! a! rudimentary! cartilage! element! formed!the! pelvis! (C).!Sp6+/F
;Sp8f/D;Ap2
α
FCre% mutants! exhibited!the! same! phenotype! to!that! of! ;Sp6F/F;Sp8CreERT2/CreERT2!
mutants!with!the!difference!that!the!body!was!truncated!at!the!level!of!the!sacrum!with!the!
lumbar! vertebrae! highly! disorganized! and! a! malformed! pelvis! lacking! the! pubis! was!
developed!(E,F).!Sp6F/F;Sp8+/f!;Ap2
α
FCre%mutants!showed!a!SHFM!like!phenotype!with!a!highly!
variable! phenotype.! Forelimbs! displayed! severely! altered! autopods! lacking! central! digits!
with!the!posterior!ones!fused!and!the!radius!occasionally!missing!(H,I).!Hindlimbs!displayed!
a! misshaped! and! truncated! tibia! with! either! absent! or! misshaped!fibula!with! a! row! of!
skeletal!rods!(J).!
!
Mutants!for!Sp6!in!which!one!copy!of!Sp8!was!conditionally!removed!with!
the!Ap2
α
FCre!line!(n=7),!exhibited!the!same!range!of!phenotypes!described!for!
Sp6F/F;Sp8+/CreERT2!ubiquitous!mutants!(Fig.!26!G?J).!The!defects!were!restricted!to!
the! limbs.! The! forelimbs! exhibited!a! weaker! phenotype! in! comparison! to! the!
hindlimbs!with!defects!restricted!to!the!zeugopod!and!the!autopod,!which!was!
quite! variable! even! in! the! same! animal.! The! styolpod! was! perfectly! developed!
and! the! zeugopod! when! affected! was! mainly! due! to! defects! in! the! radius.! The!
autopods! displayed! severely! affected! phenotypes! with! the! "claw?like"!
Results!
!
!
118!
appearance! typical! of! SHFM!in! which! anterior! or! central! digits! were! short! or!
missing!and!posterior!digits!3?4!or!4?5!were!frequently!fused!(Fig.!26H?I).!The!
hindlimbs! displayed! a! more! severe!phenotype.! The! stylopod! was! correctly!
developed,!whereas!defects!in!the!zeugopod!were!variable.!The!tibia!was!always!
present! but! sometimes! truncated! and! the! fibula,! when! present,! was! truncated!
and!frequently!only!showed!one!or!two!thin!and!unidentifiable!digits!attached!to!
it!(Fig.26J).!This!phenotype!was!compatible!with!the!SHFM!in!humans!mentioned!
above.!In!addition,!as!it!was!the!case!in!previous!Sp6F/F;Sp8+/CreERT2!mutant,!digits!
in! both! fore! and! hindlimbs! were! bidorsal! lacking! ventral! pads! and! exhibiting!
circumferential! nails.! Finally,! due! to! the! activation! of! the! Cre! in! the! germ! line!
mutants! for! Sp6!bearing! a! deleted! allele! of! Sp8!were!also! obtained! (Sp6F/F
;Sp8+/D;Ap2
α
Cre).! This! mutants! displayed! a! similar! phenotype!to! that! of!Sp6F/F
;Sp8+/CreERT2! obtained! from! crosses! between! Sp6+/F;Sp8+/CreERT2!double!
heterozygous!mice.!!
Therefore,!we!confirmed!that!Ap2
α
FCre!dependent!removal!occurs!in!the!
limb! ectoderm! early! enough! to! remove! Sp8! and! mimicked!the! ubiquitous!
removal.!These!results,! further! supported!our! hypothesis!that!both!factors!are!
together! absolutely! required! for! limb! development! and! perform! redundant!
functions! in! the! limb! ectoderm.! In! addition,! confirms! that! a! single! functional!
allele!of!Sp6!in!the!absence!of!Sp8%is!irrelevant!to!support!any!limb!development!
due! to! the! phenotypical! similarity! with! the! double! Sp6;Sp8! mutants.! However,!
one!single!functional!allele!of!Sp8,!in!the!absence!of!Sp6,!permits!a!considerably!
degree! of! limb! development! as! digits! form! although! with! an! ectrodactyly!
phenotype.!!
!
4.6* Conditional* inactivation* of* Sp8*with* the* Msx2)Cre*line* in* an* Sp6*null*
background:*Morphological*characterization*of*the*compound*mutants*
series*by*skeletal*staining!*
Amongst!the! Cre! lines!available!we!decided!to!use!the!Msx2FCre!deleter!
line!to! conditionally!remove!Sp8!from!an!Sp6!null!background!with!the! aim!to!
distinguish! between! the! requirement! of!Sp8!in! the! induction! and! the!
Results!
!
119!
maintenance!of!the!AER.!The!use!Msx2FCre!line!enables!us!to!distinguish!between!
the!requirement!of! this! Sp!factors!in! AER! induction!and! maintenance,! because!
Cre!recombinase!under!the!Msx2!AER!specific!enhancer!is!active!at!19!somites,!
12!hours!prior!to!AER!induction!in!the!hindlimb!ectoderm,!while!in!the!forelimb!
Msx2FCre!is!active!at!21!somites,!in!the!AER!and!the!ventral!limb!ectoderm,!just!
after!AER!induction!(Sun!et%al.,!2002;!Barrow!et%al.,!2003).!!
As!expected!the!hindlimbs!exhibited!a!stronger!phenotype!in!comparison!
to! the! forelimbs! in! all! the! specimens! analyzed,! due! to! an! earlier! onset! of! Cre!
activity.!Conditional!removal!of!Sp8!in!the!absence!of!Sp6!(Sp6F/F;Sp8f/D;Msx2FCre)!
resulted! in! truncated! forelimbs! at! the! level! of! the! elbow! with! the! olecranon!
present,!total!absence!of!hindlimbs!and!a!pelvic!girdle!that!lacked!the!pubis!(Fig.!
27A?C).!
Conditional! mutants! for! Sp8!bearing! a! single! functional! allele! of! Sp6!
(Sp6+/F;Sp8f/D;Msx2FCre)%exhibited!a!considerable!degree!of!variability!even!in!the!
same!animals!with!a!stronger!hindlimb!phenotype!in!comparison!to!the!forelimb!
(Fig.!27D?G).!The!forelimbs!were!truncated!at!the!level!of!the!wrist.!The!humerus!
was!perfectly!developed!and!the!zeugopod!showed!a!truncated!radius!in!half!of!
the!limbs!analyzed!(5/10),!while!in!the!other!half!the!radius!and!the!ulna!were!
correctly!developed.!Regarding!the!autopod,!this!was!practically!missing!except!
in! 4! cases! in! which! carpal! elements! were! also! developed! and! a! cartilaginous!
element! that! resembled! a! digit! was! developed! (Fig.! 27E).! Half! of! the! hindlimb!
lacked!all!the!elements!distal!to!the!femur!(5!/10)!(Fig.!27G).!3!out!of!10!mutant!
hindlimbs!exhibited!an!unidentifiable!element!distal!to!the!femur,!while!in!2!out!
of! 10! hindlimbs! analyzed! the! fibula! was! developed! with! even! smaller!
unidentifiable!element!distal!to!it!(Fig.!27F).!
Sp6!mutants!in!which!one!copy!of!Sp8!was!conditionally!removed!with!the!
Msx2FCre!(Sp6F/F;Sp8f/+;Msx2FCre)!exhibited!a!SHFM!phenotype!as!the!previously!
described!ones!bearing!a!single!functional!allele!of!Sp8!with!the!use!of!the!Ap2
α
F
Cre!line! or! the! ones!obtained! from! crosses! between! double! Sp6+/F;Sp8+/CreERT2!
heterozygous! mice! (Fig.! 27H?K).! In! addition,! germ! line! activation! of! the! Cre!
under!the!Msx2!AER!specific!enhancer,!as!in!the!case!of!the!Ap2
α
Cre%line,!led!the!
Results!
!
!
120!
to! ubiquitous! removal! of! the! floxed! allele! generating! individuals! bearing! null!
alleles!(D)!for%Sp8.!As!expected,!the!Sp6F/F;Sp8+/D!mutant!obtained!in!these!crosses!
exhibited!also!the!SHFM!phenotype!as!the!ones!obtained!in!previous!ubiquitous!
and!conditional!approaches!with!the!Ap2
α
Cre%.!
!
!
Figure* 27.* Msx2)Cre*dependent* removal* of* the* Sp8*floxed* allele* in* an* Sp6*null*
background.* Gross!morphology! of! newborns! (A,D,H,L)! and! comparison! of! skeletal!
preparations!(B,C,E,F,G,I,J,K,M,N,O)!of!the!genotypes!indicated.!Sp6F/F;Sp8f/D;Msx2
α
FCre%mutant!
forelimbs!lacked!all!the!elements!distal!to!the!elbow!(B)!and!resulted!in!a!pelvic!girdle!that!
lacked! the! pubis!with! total! absence! of! the! hindlimbs! (C).!Sp6+/F;Sp8f/D;Msx2
α
FCre!forelimbs!
displayed!a!variable!degree!of!truncations!at!the!wrist!level!associated!to!an!incomplete!digit!
with!the!radius!present!but!often!truncated!(E),!whereas!in!the!hindlimbs!the!femur!appeared!
associated!either!to!an!unidentifiable!element!or!the!fibula!associated!to!a!single!digit!(F,G).!
Sp6F/F;Sp8+/f;Msx2
α
FCre%mutants!forelimbs!displayed!severely!altered!autopods!lacking!central!
digits! with! the! posterior!ones! fused!and! the! radius!occasionally! missing! (I).! Hindlimbs!
displayed!a!misshaped!and!truncated!tibia!with!either!absent!or!misshaped!fibula!with!a!single!
digit!(J,K).!Sp6+/+;Sp8f/D;Msx2
α
FCre%!exhibited!forelimb!defects!restricted!to!the!autopod!with!
only!the!most!posterior!digit!present!(M),!while!the!hindlimbs!showed!a!single!digit!(N)!or!in!
the!most!affected!a!cartilaginous!element!attached!to!the!femur!was!developed!(O).!
!
The!phenotype!of!the!Sp6!mutants!was!identical!as!previously!described!
(Talamillo!et%al.,!2010).!Conditional!mutants!for!Sp8!with!the!use!of!the!Msx2FCre!
deleter! line! exhibited! a! limb! phenotype! milder! than! that! of! the! ubiquitous!
mutant!(Bell!et%al.,!2003;!Treichel!et%al.,!2003)(Fig.!27L).!Forelimbs!defects!were!
restricted!to!the!autopod!in!which!anterior!digits!were!missing!and!only!digit!5!
Results!
!
121!
or! digit! 4! and! 5! were! developed! (Fig.! 27M).! In! hindlimbs,! the! stylopod! was!
always! developed.! In! half! of! them! the! zeugopod! was! absent! and! exhibited! an!
unidentifiable!cartilaginous!element!distal!to!the!femur,!while!in!the!other!half!a!
fibula!and!a!truncated!tibia!associated!to!a!digit!were!developed!(Fig.!27N,!O).!!
In!sum,!the!phenotypes!obtained!with!the!Msx2FCre!line!implies!that!both!
transcription!factors!are!not!only!required!prior!to!AER!induction!but!also!later!
in!development!to!ensure!proper!PD!limb!patterning.!The!range!of!phenotypes!
obtained!from!different!combinations!of!conditional!and!ubiquitous!approaches!
support!a!model!in!which!Sp6!and!Sp8!are!functionally!equivalents!during!limb!
development,! with!Sp8! making! a! more! substantial! contribution! than! Sp6,!
probably! due! to!a! higher! level! of! expression.! Our! results! also! suggest! that! a!
threshold!of!Sp!dosage!may!be!required!and!that!below!that!threshold!limbs!are!
not!formed.!!
!
4.7* Molecular* and* morphological* characterization* of* Sp6;Sp8*double*
mutant*limb*buds*
Gene!expression!analysis!together!with!morphological!characterization!of!
mutant!limb!buds!were!performed!in!embryos!obtained!from!crosses!between!
Sp6+/F;Sp8+/CreERT2! double! heterozygous! mice,! because! the!fraction! of! double!
mutant! recovered! at! E9.5! (Table! 2)! and! E10.5! (Table3)! was! according! to! the!
expected! 1/16! Mendelian! frequency.! At! E9.5,! according! to! the! expected!
Mendelian!rate!6!double!Sp6F/F;Sp8CreERT2/CreERT2!mutant!embryos!were!obtained!
from!a!total!of!95!embryos!recovered!(Table2),!while!at!E10.5!from!153!embryos!
recovered! 9! double! Sp6F/F;Sp8CreERT2/CreERT2! mutants! were! obtained! from! the! 10!
expected!(Table3).!That!indicates!that!the!double!mutant!is!lethal!after!E10.5.!
!
!
!
Results!
!
!
122!
Table*2*Expected*and*obtained*Mendelian*rates*at*E9.5*from*crosses*
between*double*Sp6+/);Sp8+/CreERT2*heterozygous*mice.**
!
!
!
*
*
*
*
*
*
*
*
*
!
Table*3*Expected*and*obtained*Mendelian*rates*at*E10.5*from*crosses*
between*double*Sp6+/);Sp8+/CreERT2*heterozygous*mice.**
!
*
!
*
*
*
!
Results!
!
123!
4.7.1*Molecular*characterization*of*Sp6)/);Sp8CreERT2/CreERT2 double*mutant***
AER*
Since! genetic! removal! of! either! Wnt/ß?catenin!or! AER?Fgfs! resulted! in!
amelic!phenotypes!(Barrow!et%al.,!2003;!Soshnikova!et%al.,!2003;!Sun!et%al.,!2002;!
Boulet! et% al.,! 2004)!identical! to! Sp6F/F;Sp8CreERT2/CreERT2!or! Sp6+/F;Sp8CreERT2/CreERT2!
and!also!because!Sp6!and!Sp8!have!been!suggested!to!be!involved!in!the!Wnt/ß?
catenin!induction!of!Fgf8!in!the!limb!ectoderm!(Kawakami!et%al.,!2004;!Sahara!et%
al.,!2007;!Talamillo!et%al.,!2010;!Lin!et%al.,!2013),!it!seems!reasonable!to!assume!
that!the!amelic!phenotype!of!double!mutants!may!rely!on!a!failure!to!induce!Fgf8!
expression!in!the!limb!ectoderm!(Sun!et%al.,!2002;!Boulet!et%al.,!2004).!Therefore!
we!examined!embryonic!limbs!at!the!stages!when!the!limb!bud!initiates!and!the!
AER!is!formed.!In!the!control!limb!buds,!Fgf8!is!first!detected!in!the!ventral!limb!
ectoderm! at! E9.5! and! become! progressively! confined! to! the! distal! AER! as! it!
matures!(Martin,!1998;!Loomis;!et%al.,!1998;!Bell!et%al.,!1998).!Bmp4!follows!the!
same!dynamics!to!that!of!Fgf8,!with!the!difference!that!Bmp4!is!also!expressed!in!
the!limb!mesoderm!prior!to!and!during!limb!bud!development!(Ahn!et%al.,!2001;!
Selever!et%al.,!2004).!
Induction!of!Fgf8!expression!has!been!reported!to!occur!normally!in!mice!
lacking! Sp6,! however! these! limbs! exhibited! an! expanded! domain! of! Fgf8!
expression! along! the! DV! axis! of! the! limb! that!developed!into! a! double! ridge!
phenotype!in!both!hindlimbs!and!forelimbs!(Talamillo!et%al.,!2010).!In!absence!of!
Sp8,! Fgf8!and! Bmp4!are! correctly! induced! but! are! lost!at! E10.5! when! the! AER!
prematurely! regresses! (Bell! et% al.,!2003;! Treichel! et% al.,!2003).! Therefore,! we!
analyzed! their!expression! at! E9.5! (22?26! somites)! and! found! that! Sp6F/F
;Sp8CreERT2/CreERT2!and!Sp6+/F;Sp8CreERT2/CreERT2%compound!mutants!failed!to!express!
Fgf8! in! the! limb! ectoderm.! Because! both! genotypes! always! showed! the! same!
expression!patterns!for!all!the!genes!analyzed!in!all!the!stages,!in!the!figures!only!
the!results!of!the!Sp6F/F;Sp8CreERT2/CreERT2!mutant!are!shown.!
Expression!of!Fgf8!was!never!detected!from!E9.5!to!E11.5!in!forelimbs!of!
both!Sp6F/F;Sp8CreERT2/CreERT2!and!Sp6+/F;Sp8CreERT2/CreERT2%mutants!(Fig.!29A,!B,!G,!H,!M,!
N),!indicating!that!Sp6!and!Sp8!are!required!for!the!induction!of!Fgf8!in!the!limb!
Results!
!
!
124!
ectoderm.!In!contrast,!Bmp4!expression!was!found!to!be!normal!in!both!the!limb!
ectoderm!and!limb!mesoderm!at!E9.5!(Fig.!29D).!In!addition,!expression!of!Msx2!
a!bonafide!target!of!Bmp!signalling,!confirmed!the!presence!of!Bmp!signalling!in!
the!limb!ectoderm!at!E9.5!(Fig.!29F).!From!E10.5!on,!Bmp4!and!Msx2!were!not!
detected!in! the! limb! ectoderm! confirming!the! absence! of! Bmp! signaling!in! the!
limb!ectoderm,!while!both!were!detected!in!the!limb!mesoderm!(Fig.!29L,J).!At!
E11.5!when!the!limb!practically!regressed,!Bmp4!expression!was!detected!in!the!
limb!bud!mesoderm!but!not!in!the!ectoderm!(Fig.!29O).!!
!
!
Figure* 29.* Gene* expression* analysis* in* Sp6)/);Sp8CreERT2/CreERT2*double* mutant*AER.*In! situ!
hybridization!in!transverse!section!of!control!(A,C,E,G,I,K,M,O)!and!Sp6F/F;Sp8CreERT2/CreERT2!double!
mutant!(B,D,F,H,J,L,N,P)! forelimbs! at! the! stages! indicated.! Fgf8!expression! was! not! detected!in!
mutant!limb!ectoderm!at!any!of!the!stages!analyzed!(B,H,N),!while!Bmp4!was!detected!in!the!limb!
ectoderm!and!mesoderm!at!E9.5!(D)!but!was!lost!from!the!limb!ectoderm!at!E10.5!(J)!and!E11.5!
embryos!(P).!Expression!of!Msx2,!a!bonafide!target!of!BMP!signalling,!was!detected!in!E9.5!(F)!
and! E10.5! (L)! mutant! limbs.! Note! that! initial! budding! occurs! in! Sp6F/F;Sp8CreERT2/CreERT2!double!
mutant!(B,D,F,H,J,L)!and!that!the!bud!has!practically!regressed!by!E11.5!(O,P).!!
!
Results!
!
125!
In!sum,!our!results!are!consistent!with!Sp6!and!Sp8!acting!downstream!of!
Wnt/ß?catenin!in!the!limb!ectoderm!required!for!Fgf8!induction.!In!addition,!the!
fact!that!single!disruption!of!either!Sp6!or!Sp8!leads!to!Fgf8!induction!in!the!limb!
ectoderm,!while!Fgf8!is!never!detected!in!either! double!mutants!or!when!only!
one! functional! allele! of! Sp6! remains,! demonstrated! that! both! transcription!
factors!are!conjointly!absolutely!required!and!act!in!a!redundant!manner!in!the!
induction! of! Fgf8.! However,! in! the! absence! of! Sp6!and! Sp8,! Bmp4!is! initially!
expressed!in!the!limb!ectoderm!by!E9.5,!implying!that!neither!Sp6!nor!Sp8!are!
mediating!the!induction!of!Bmp4!in!the!AER.!!
It!is!important!to!note!that,!disregarding!the!absence!of!Fgf8!expression,!
both! Sp6F/F;Sp8CreERT2/CreERT2!and! Sp6+/F;Sp8CreERT2/CreERT2! mutants! initiated! limb! bud!
development!and!formed!a!small!bulge.!These!emergent!limb!buds!regressed!and!
became! not! visible! between! E10.5! and! E11.! The! current! view! considers! that!
Fgf10!signalling!from!the!limb!mesoderm!through!its!receptor!Fgfr2b!induces!the!
expression! of! a! Wnt! ligand! in! the! limb! ectoderm,! Wnt3a%in! chick! and%Wnt3% in!
mouse,%leading!to!Wnt/ß?catenin!dependent!induction!of!Fgf8!in!the!precursor!
cells! of! the! AER! (Kengaku! et% al.,!1998;! Barrow!et% al.,!2003;! Soshnikova! et% al.,!
2003).!In!turns,!Fgf8!signalling!from!the!ectoderm!is!then!required!to!maintain!
Fgf10! expression! in! the! limb! mesoderm,! establishing! a! positive! regulatory!
feedback! loop! between! Fgf8! expressed! in! the! ectoderm! and! Fgf10!in! the!
mesoderm! that! is! absolutely! required! for! PD! outgrowth! and! patterning! of! the!
limb!(Kawakami!et%al.,!2001).!Therefore,!we!decided!to!analyze!the!expression!of!
Fgf10!in!the!limb!mesoderm!of!Sp6F/F;Sp8CreERT2/CreERT2!mutants.!
Accordingly,! in! Sp6F/F;Sp8CreERT2/CreERT2%double! mutant! limb! buds! Fgf10!
expression! in! the! limb! mesenchyme! was! normally! detected! at! E9.5! in!
comparison!to!control!embryos!(Fig.!30A,B).!However,!at!E10.5!Fgf10!expression!
was!not!maintained!in!the!limb!mesoderm!(Fig.!30D).!Thus,!lack!of!Sp6!and!Sp8!
resulted!in!absence!of!Fgf8!induction!in!the!AER!leading!to!the!failure!in!Fgf10!
maintenance!in!the!limb!mesenchyme.!
!
!
Results!
!
!
126!
!
Figure*30.*Lack*of*Sp6*and*Sp8*resulted*in*loss*of*Fgf10*
maintenance*in*the*limb*mesoderm.*In!situ!hybridization!
in! transverse! sections! of! control!(A,C)!and! Sp6F/F
;Sp8CreERT2/CreERT2!mutant! (B,D)! forelimb! buds! at! the! stages!
indicated.! Fgf10!expression!was! detected! at! E9.5! (C)! in!
mutant!embryos,!but!was!not!maintained!by!E10.5!(D).!
!
Briefly,!our!results!are!consistent!with!Sp6/8!acting!as!mediators!of!the!
Wnt/β?catenin!dependent!induction!of!Fgf8.!We!have!demonstrated!that!Sp6!and!
Sp8!are!absolutely!required!and!act!in!a!redundant!manner!in!the!induction!of!
Fgf8! in! the! limb! ectoderm.! In! addition,!our! results!fits! with! the! existence! of! a!
regulatory!loop!between!Fgf10!in!the!mesoderm!and!Fgf8!in!the!limb!ectoderm!
that!predicts!that!the!failure!of!Fgf8!induction!in!the!limb!ectoderm!will!result!in!
the! downregulation! of! Fgf10!expression! in! the! limb! mesoderm! leading! to! an!
amelic! phenotype,! as! occurred! in! Sp6F/F;Sp8CreERT2/CreERT2!and! Sp6+/F;Sp8CreERT2/CreERT2.!
Finally,! we! have! also! demonstrated! that! initial!Bmp4!and! Msx2!expression! are!
detected!in!absence!of!Sp6!and!Sp8!as!occurs!with!several!AER!markers!in!the!
double! Fgf8/4!(Sun! et% al.,! 2002).! In! contrast,! these! AER! markers! are! not!
expressed! in! mice! in! which! Wnt/β?catenin!has! been! disrupted!(Barrow! et% al.,!
2003;! Soshnikova! et% al.,!2003).! Therefore,!we! provide! further! evidence!
supporting!that!there!is!not!a!simple!linear!relationship!between!Wnt/β?catenin!
in!the!limb!ectoderm!and!Fgf8!in!the!AER.!
!
Results!
!
127!
4.7.2*Cell*death*in*the*developing*limb*bud*in*absence*of*Sp6*and*Sp8*
At! E9.5! an! initial! budding! was! evident! in! Sp6F/F;Sp8CreERT2/CreERT2%mutant!
embryos!but!later!on!this!budding!regressed!resulting!in!an!amelic!phenotype!as!
a! consequence! of! a! failure! in! Fgf8! induction,! reminiscent! of! that! of! Limbless!
chicken!mutant.!Limbless!is!characterized!by!the!development!of!a!limb!bud!that!
undergoes!extensive!apoptosis!and!regresses!leading!to!an!amelic!phenotype,!as!
a!consequence!of!the!inability!to!induce!an!AER!(Fallon!et%al.,!1983;!Carrington!
and! Fallon,! 1988).! Moreover,! surgical! removal! of! the! AER! in! chick,! leads! to!
increased! cell! death! of! the! distal! mesoderm! (Rowe! et% al.,! 1982;! Dudley! et% al.,!
2002)! while!genetic! attenuation! of! Fgf!signalling! from! the! AER! results! in!
proximal! cell! death! both! in! the! mesoderm! and! ectoderm! that! account! for! the!
smaller!size!and!regression!of!the!limb!buds!under!those!circumstances!(Sun!et%
al.,! 2002;! Boulet! et% al.,! 2004).! With! the! aim! to! determine! the! cell! death!
repercussion! due! to! the! removal! of! Sp6!and! Sp8,! we! analyzed! cell! death! by!
TUNEL!assay!in!Sp6F/F;Sp8CreERT2/CreERT2!double!mutant!limb!buds.!
At!E9.5!no!cell!death!was!detected!neither!in!the!control!embryo!nor!in!
the!double!mutant!forelimbs.!However,!by!E10.5!the!entire!limb!bud!undergoes!
massive! apoptosis! in!the!Sp6F/F;Sp8CreERT2/CreERT2%double! mutant,! while! in!
comparison!yo!wild!type!animals!(Fig.!31A,B).!Apoptotic!cells!were!detected!in!
both! ectodermal! and! mesodermal! compartment.! Cell! death! was! extended! all!
over! the! mesodermal! compartment! of! the! limb! bud,! while! in! the! ectodermal!
compartment!apoptotic!cells!were!more!prominent!in!the!proximo?dorsal!and!in!
the!distal?ventral!ectoderm.!
!Our! results! demonstrated! that! in! the! absence! of! both! Sp6!and! Sp8!the!
limb! bud! undergoes! extensive! apoptosis.! The! massive! cell! death! in! Sp6F/F
;Sp8CreERT2/CreERT2%double! mutant! limb! buds,! can! account! for! the! regression! of! the!
limb! bud! leading! to! the! amelic! phenotype! as! reported! for! the! chicken! mutant!
limbless!(Fallon!et%al.,!1983;!Carrington!and!Fallon,!1988).!According!to!this,!Sp6!
and!Sp8!are!required!to!ensure!the!integrity!of!the!limb!bud,!in!order!to!avoid!
cell!death!in!the!developing!limb.!
Results!
!
!
128!
!
Figure* 31.* Cell* death* repercussion* in* Sp6)/);Sp8CreERT2/CreERT2*mutant* limb* buds.*
Transverse!sections!of!control!(A)!and!Sp6F/F;Sp8CreERT2/CreERT2!(B)!mutant!forelimb!buds!
at!E10.5.!On!each!panel,!the!nucleus!is!counter!stained!with!DAPI!(Blue)!and!apoptotic!
cells!(Green)!are!shown.!No!cell!death!was!detected!in!control!embryo!(A).!Extensive!
apoptosis! was! detected! in!both! the! ectoderm! and! the! mesoderm! of! double! mutant!
limb!buds!(B).*!
*
4.7.3* Morphological* characterization* of* Sp6)/);Sp8CreERT2/CreERT2 double*
mutant*AER*
Conditional! removal! of! Fgf8!and! Fgf4! from! the! AER! stunts! limb!
development! due! to! the! absence! of! Fgf!signalling! from! the! AER,! while! a!
morphological!AER!is!still!formed!(Sun!et%al.,!2002;!Boulet!et%al.,!2004).!However,!
disruption!of!Wnt/β?catenin!signalling!in!the!limb!ectoderm!resulted!in!absence!
of!a!morphologycal!AER,!implying!that!the!relation!between!Wnt/β?catenin!and!
Fgf! in! the! limb! ectoderm! is! not! linear! (Sun! et% al.,! 2002;! Barrow! et% al.,!2003;!
Soshnikova!et% al.,!2003).! Therefore,! we! analyzed! in! more! detail! the! thickening!
present! in! the! ventral! limb! ectoderm! of! Sp6F/F;Sp8CreERT2/CreERT2! double! mutants,!
evident!in!TUNEL!assays!due!to!the!massive!cell!death!in!this!area.!!
In! mice,! at! E10.5! morphogenetic! movements! confined! the! AER! cells!
induced!in!the!ventral!limb!ectoderm!to!the!distal!tip!of!the!limb!bud!at!the!DV!
interface!and!adopts!a!polystratified!organization!(Meyer!et%al.,!1997;!Bell!et%al.,!
1998)! (Fig.! 32! A,B).! In! transverse! semithin! sections! of! Sp6F/F;Sp8CreERT2/CreERT2!
mutant! forelimbs! at! E10.5! a! thickening! was! detected! in! the! ventral! limb!
ectoderm! of! Sp6F/F;Sp8CreERT2/CreERT2%double!mutants! with! the! appearence!of! an!
inmature!AER,!sometimes!protruding!into!the!mesoderm!(Fig.!32C,D).!
Results!
!
129!
!
Figure*32.*AER*morphology*of*Sp6)/);Sp8CreERT2/CreERT2*double*mutants*
limb* buds.*Semithin! sections! of! control! (A,B)! and! mutant(C,D)!forelimb!
buds!at!E10.5.!(A)!Control!E10.5!limb!and!(B)!higher!magnification!of!A.!
(C)! Mutant! limb! bud! and! (D)! higher! magnification! of! C,! where! a! slight!
thickening! in! the!ventral! ectoderm! is! appreciable.! Note! that! basement!
membrane!is!marked!with!a!red!dashed!line!in!B!and!D.!
!
To! analyze!in! more! detail! the! thickening! present! in! the! ventral! limb!
ectoderm!of! Sp6F/F;Sp8CreERT2/CreERT2%mutants!limb!buds,!we!decided!to!analyze!the!
presence! of! ectodermal! markers! by! immunhistochemistry.! The! analysis! of!
laminin,! marker! of! the! basement! membrane,!and! E?cadherin!(Cdh1),! a!
transmembrane!protein!specific!of!ectodermal!cells!(Fig.!33A),!led!us!to!verified!
the! contribution! of! ectodermal! cells! to! the! thickening! present! in! the! ventral!
ectoderm!of!Sp6F/F;Sp8CreERT2/CreERT2!mutant!limb!buds.!!
At! E10.5! Cdh1!was! specifically! detected! in! the! forelimb! ectoderm! of!
control! embryos! and! in! the! AER,! while! laminin! was! detected! in! the! basement!
membrane! immediately! under! the! ectoderm! (Fig.! 33A).! In! Sp6F/F;Sp8CreERT2/CreERT2!
double!mutant!limb!buds!Cdh1!was!detected!in!the!limb!ectoderm!and!also!in!the!
ventral!ectodermal!thickening.!In!addition,!laminin!was!detected!below!the!limb!
ectoderm! and! also! below! the! ventral! thickening! implying! that! the! ventral!
ectodermal!thickening!was!of!ectodermal!origin!(Fig!33B).!!
Results!
!
!
130!
!
Figure*33.*Ectodermal*cells*contribute*to*the*ventral*thickening*present*in*Sp6)/)
;Sp8CreERT2/CreERT2*double*mutant*limb*buds.*Immunolabeling!of!E10.5!control!(A)! and!
Sp6F/F;Sp8CreERT2/CreERT2%double! mutant! (B)! forelimb! buds.! Laminin?b! was! detected! below!
the! limb! ectoderm! of! control! embryos,! positive! for! E?cadherin.! Contribution! of!
ectodermal!cells!to!the!ventral!limb!thickening!was!confirmed!due!to!presence!of!Cdh1!in!
the! ventral! thickening! and! also! because! this! thickening! was! above! the! basement!
membrane!as!assess!by!laminin!immunostaining.!Nucleus!are!counter!stained!with!DAPI!
(Blue),!Laminin?b!(laminin;Green),!and!E?cadherin!(Cdh1;!Red).!
!
In! sum,! the! presence! of! ectodermal! markers! in! the! ventral! thickening!
present!in!Sp6F/F;Sp8CreERT2/CreERT2%mutant!limb!buds!demonstrated!that!ectodermal!
cells!are!accumulated!in!the!ventral!limb!ectoderm.!Thus,!in!the!absence!of!both!
Sp6!and!Sp8%AER!morphogenesis!is!initiated,!but!the!process!is!arrested!and!cells!
are!not!pulled!to!the!distal!tip!of!the!limb!and!get!accumulated!in!the!ventral!limb!
ectoderm.!!
*
4.7.4* Dorso[Ventral* pattern* establishment* in* Sp6)/);Sp8CreERT2/CreERT2$
mutant*limb*buds*
Lack!of!Fgf8!expression!and!subsequent!absence!of!AER!development!in!
the!chick!mutant!limbless!has!been!reported!to!occur!associated!to!a!failure!in!DV!
pattern!establishment!due!to!the!absence!of!En1!expression!in!the!ventral!limb!
ectoderm!and! ectopic! expression! of!Wnt7a!in!the!ventral!ectoderm!(Ros!et%al.,!
1996;!Grieshammer!et%al.,!1996;!Noramly!et%al.,!1996).!Moreover,!the!ventrally!
Results!
!
131!
extended! AER! developed! in! En1! mutant! mice! suggested! it! requirement! for!
proper! positioning! of! the! AER! in! the! limb! DV! boundary! (Cygan! et% al.,! 1997;!
Loomis! et% al.,! 1996;! Loomis! et% al.,! 1998).! Additionally,! mice! lacking! ßFcatenin!
exhibited!a!double?dorsal!limb!phenotype!due!to!lack!of!En1!expression!in!the!
ventral! ectoderm! and! ectopic! Wnt7a!expression! (Soshnikova!et% al.,! 2003).!
Therefore,! once! determined! that! both! Sp6!and! Sp8!are! absolutely! required! for!
Fgf8!induction!in!the!limb!ectoderm!together!with!morphological!analysis!that!
confirmed! that! ventral! ectodermal! cells! of! double! Sp6F/F;Sp8CreERT2/CreERT2! mutants!
initiated! AER! morphogenesis,! but! are!not! pulled! to! the! distal! tip! and! get!
accumulated! in! the! ventral! ectoderm,! we! proceeded!to! analyze! DV! pattern!
establishment! in! the! absence! of! both%Sp6% and%Sp8,% through!the! analysis! of! the!
expression! of! the! DV! markers! En1!and!Wnt7a%(Riddle! et% al.,! 1995;! Parr! and!
McMahon,!1995;!Loomis!et%al.,!1996;!Cygan!et%al.,!1997;!Loomis!et%al.,!1998;!Ahn!
et%al.,!2001).!
Previous!analysis!of!Sp6!and!Sp8!single!mutants!revealed!that!DV!pattern!
establishment! was! correctly! initiated! in! both! mutant! limbs! (Talamillo! et% al.,!
2010;!Bell!et%al.,!2003;!Treichel!et%al.,!2003).!Limbs!in!which!Sp6!was!disrupted!
Lmx1b! expression! remained! restricted! to! dorsal! mesoderm! but! by! E14.5!
occasional!patches!of!expression!were!detected!in!the!ventral!distal!mesoderm!of!
either!digit!3,!4!or!5,!without!ectopic!expression!of!Wnt7a.!Authors!argued!that!
double!dorsal!digit!developed!in!central!digits!of!Sp6!mutants!were!independent!
of!Lmx1b!expression!in!the!ventral!mesoderm,!because!Lmx1b!ectopic!expression!
was!detect!also!in!digit!5,!but!this!digit!did!not!exhibited!a!bidorsal!phenotype!
(Talamillo! et% al.,!2010).! In! contrast,! in! mouse! lacking! Sp8!DV! patterning! is!
correctly! initiated! at! E9.5! as! assessed! by! En1!and! Lmx1b!expression,! in! the!
ventral! ectoderm! and! the! dorsal! mesoderm,! respectively.! However,! by! E10.5!
when! limb! development! stunted,! En1!expression! was! lost!(Bell! et% al.,!2003;!
Treichel!et%al.,!2003).!!
The!analysis!of!the!expression!of!DV!markers!in!double!mutant!forelimbs!
was!performed!from!E9.5!to!E11.5!(Fig.!34).!In!control!embryos,!Wnt7a!is!first!
detected! in! the! dorsal! limb! ectoderm! at! E9.5! (Fig.! 34A)! and! at! E10.5! it! is!
expressed! all! over! the! dorsal! ectoderm! up! to! the! dorsal! boundary! of! the! AER!
Results!
!
!
132!
(Fig.!34E).!En1!expression!is!first!detected!at!E9.5!in!the!ventral!limb!ectoderm!
(Fig.!34C)!and!at!E10.5!it!is!expressed!all!over!the!ventral!ectoderm!including!the!
ventral!half!of!the!AER!(Fig.!34G).!!
!
Figure*34.* Analysis* of*DV* markers* in*Sp6)/);Sp8CreERT2/CreERT2*double* mutants*forelimb*
buds.* In! situ! hybridization!for!the! DV! markers! Wnt7a!(A,B,E,F,I,J)! and! En1!(C,D,G,H)! in!
transverse! section! of! control! (A,C,E,G,I)! and! Sp6F/F;Sp8CreERT2/CreERT2!double! mutant! (B,D,F,H,J)!
forelimbs! at! the! stages! indicated.! En1!expression! was! not! detected! in! the! ventral! ectoderm!
(D,H)!and!Wnt7a!expression!was!extended!into!the!ventral!ectoderm!of!Sp6F/F;Sp8CreERT2/CreERT2*
from!E9.5!to!E11.5!(B,F,J).!
!
The! analysis! of! the! DV! markers! Wnt7a!and! En1! in! double! Sp6F/F
;Sp8CreERT2/CreERT2! mutant! forelimbs! revealed! the! absence! of! DV! pattern!
establishment! with! limb! buds! exhibiting! a! double! dorsal! phenotype.! Wnt7a!
expression! was! initially! extended! into! the! ventral! limb! ectoderm! at! E9.5! (Fig.!
Results!
!
133!
34B)! and! its! expression! persisted! over! the! dorsal! and! ventral! ectoderm! of!
mutant!limb!buds!at!E10.5!and!E11.5!(Fig.!34,F,J),!while!En1!was!never!detected!
in!the!limb!ventral!ectoderm!neither!at!E9.5!nor!E10.5!(Fig,!34D,H),!but!it!was!
detected!in!the!flank!ectoderm!ventral!to!the!limb!bud!by!E10.5!(Fig.!34H).!!
Our! results! demonstrated! that!DV! pattern! is! not! established!in! the!
absence! of! Sp6%and! Sp8.!En1!expression! is!not! detected! in! the! ventral! limb!
ectoderm!and!Wnt7a!expression!is!detected!extending!all!over!the!limb!ventral!
ectoderm,! leading! to! the! development! of! double! dorsal! limb! buds.! In! addition,!
besides!normal!expression!of!Bmp4,!in!both!the!ectoderm!and!the!mesoderm!of!
Sp6F/F;Sp8CreERT2/CreERT2%mutant!limb!buds,!En1!is!not!expressed!in!the!absence!of!Sp6!
and! Sp8,! implying! their! requirement! for! proper! ventral! limb! pattern!
establishment.%
!
4.8*Molecular*characterization*of*Sp6)/);Sp8+/CreERT2!mutant*limb*buds**
4.8.1*Gene*expression*analysis*of*the*Sp6)/);Sp8+/CreERT2!mutant*AER*
The! ectrodactylous! limb! phenotype! of! the! Sp6F/F;Sp8+/CreERT2! mutant!
resembles!the!human!malformation!SHFM!that!is!characterized!by!the!absence!of!
central!digits!and!fusion! of! the!remaining!ones!as! a! possible!consequence! of! a!
failure! in! the! maintenance! of! the! medial! region! of! the! AER! that! lacks! Fgf8!
expression! (Temtamy! and! McKusick! 1978;! Sifakis! et% al.,! 2001).! Therefore,! we!
checked!for!Fgf8!expression!in!Sp6F/F;Sp8+/CreERT2!mutant!limbs!to!determine!if!the!
failure!in!Fgf8!expression!in!the!central!region!of!the!AER!could!account!for!the!
limb!phenotype!of!Sp6F/F;Sp8+/CreERT2!mutants.!!
In!Sp6F/F;Sp8+/CreERT2!mutant!limbs,!Fgf8!expression!was!detected!at!E9.5!in!
the!limb!ectoderm!in!a!more!restricted!manner!in!comparison!to!control!limbs!
(Fig.!35A,B).!At!E10.5,!Fgf8!expression!was!confined!to!the!distal!tip!of!the!limb!
in! control! embryos! (Fig.! 35E,I,J).! However,! consecutive! transverse! sections!
throughout!Sp6F/F;Sp8+/CreERT2!mutant!limbs!revealed!an!irregular!Fgf8!expression!
along!the!limb!AP!axis!(Fig.!35F,F´)!that!was!further!confirmed!by!whole!mount!
in! situ! hybridization.! Discontinuous!Fgf8!expression! was! detected! in! the! AER!
Results!
!
!
134!
together!with!a!patchy!pattern!of!Fgf8!expression!in!the!ventral!limb!ectoderm!of!
Sp6F/F;Sp8+/CreERT2! mutants,! that! corresponded! to! an! inmature! AER! (Fig.! 35K,L).!
Bmp4!expression!in!the!AER!followed!the!same!dynamics!and!showed!the!same!
irregular!expression!patterns!of!Fgf8!at!E9.5!and!E10.5!in!Sp6F/F;Sp8+/CreERT2!mutant!
limbs,!as!demonstrated!in!transverse!sections!(Fig.!35D,!H,!H´)!and!whole!mount!
in!situ!hybridization!(Fig.!35M,N).!
!
*
Figure* 35* Gene* expression*analysis* throughout*Sp6)/);Sp8+/CreERT2*mutant* AER.* Fgf8!
(A,B,E,F,FF´)! and! Bmp4!(C,D,G,H,H´)! expression! in! transverse! sections! of!control! (A,C,E,G)! and!
Sp6F/F;Sp8+/CreERT2!(B,D,F,F,´H,H´)!mutant!forelimbs!buds!at!the!stage!indicated!and!dorsal!(I,K,M)!
and!distal!views!(J,L,N)!of!control!(J,K)!and!Sp6F/F;Sp8+/CreERT2!mutant!(K,L,M,N)!E10.5!forelimbs!
hybridized!for!Fgf8!(I,J,K,L)!and!Bmp4!(M,N).!Fgf8!expression!showed!irregular!activation!of!AER!
cells! in!Sp6F/F;Sp8+/CreERT2!mutant! ventral! forelimb! ectoderm.! By! E10.5! the! AER! remained! flat!
displaying! gaps! of! Fgf8!(F,F´,K.L)! and! Bmp4!(H,H´,M,N)! expression! as! shown! in! transverse!
sections!(F,F´,H,H´)!and!whole!mount!in!situ!hybridization!(K,L,M,N)!of!E10.5!forelimb!buds.!Note!
that!F,F´and!H,H´are!consecutive!section!of!the!same!limb.!
!
In!order!to!confirm!the!presence!of!an!immature!AER!in!Sp6F/F;Sp8+/CreERT!
mutants,!we!decided!to!perform!a!double!Laminin;Cdh1!immunolabeling!in!Sp6F/F
;Sp8+/CreERT! mutants! limbs! buds! at! E10.5.! In! wild! type! animals,! laminin! was!
expressed! in! the! basement! membrane! below! the! ectoderm,! while! in! the!
ectoderm!Cdh1!was!specifically!expressed!in!the!limb!ectoderm!(Fig.!36!A,B).!As!
Results!
!
135!
shown!in!Sp6F/F;Sp8+/CreERT%mutant!limb!sections,!the!AER!was!not!confined!to!the!
DV! boundary! of! the! limb! and! did!not! exhibited!the! polystratified! organization!
appreciable!in!control!embryos.!The!ventral!ectoderm!was!slightly!thicker!than!
the!dorsal!ectoderm!as!shown!by!laminin!and!Cdh1!immunolabeling!(Fig.!36A,B),!
consistent! with! ventrally! expanded! expression! of! Fgf8!and! Bmp4! in! the! limb!
ectoderm!of!Sp6F/F;Sp8+/CreERT%mutant.!Therefore,!we!confirmed!the!presence!of!a!
slightly!thicker!ventral!than!dorsal!ectoderm!that!corresponded!to!an!inmature!
AER!morphology!.!
!
!
Figure*36.*Sp6)/);Sp8+/CreERT2*mutant*limb*bud.*Transverse!limb!buds!sections!
counter!stained!with!DAPI!(Blue)!showing!inmunostaining!for!Laminin!(Green)!
and!E?cadherin!(Cdh1)!(Red)!within!control!(A)!and!Sp6F/F;Sp8+/CreERT!(B)!mutant!
limb!buds.!Sp6F/F;Sp8+/CreERT2!mutants!displayed!an!immature!and!flatten!AER.*
!
From!these!results!we!conclude!that!a!single!functional!allele!of!Sp8!in!the!
absence!of!Sp6!is!not!sufficient!to!achieve!normal!AER!morphology!and!leads!to!
irregular!activation!of!Fgf8.%As!the!limb!develops!the!irregular!activation!of!Fgf8%
results! in! gaps! of! Fgf8! expression! along! the! AP! length! of! the! AER! that! could!
account!for!the!absence!of!central!digits.!!
!
!
Results!
!
!
136!
4.8.2*Sp6*and*Sp8*and*the*Tp63*network*leading*to*SHFM*
In! humans,! six! different!loci! have! been! associated!to! SHFM.! This!
syndrome! is! characterized! by! absence! of! central! digits! and! fusion! of! the!
remaining!ones,!possibly!due!to!a!failure!in!the!maintenance!of!the!medial!region!
of!the!AER!that!lacks!Fgf8!expression!(Temtamy!and!McKusick!1978;!Sifakis!et%
al.,! 2001).! The! similar! limb! phenotype! between! the! distinct! forms! of! SHFM!
underlies! the! possible! existence! of! a! regulatory! cascade! involving! the! disease!
genes.! Dlx5/Dlx6!and! Tp63!are! the! only! genes!that! have! been! unequivocally!
shown!to!be!involved!in!SHFM!type!I!and!SHFM!type!IV,!respectively!(Mills!et%al.,!
1999;! Yang! et% al.,! 1999;! Robledo! et% al.,!2002;! Merlo! et% al.,! 2002).! It! has! been!
demonstrated! that! both! phenotypes! arise! due! to! the! disruption! of! the! same!
regulatory! cascade,! in! which!Tp63! acts! upstream! of! the! Dlx!genes! for! proper!
expression!of!Fgf8!in!the!AER!(Lo!Iacono!et%al.,!2008).!!
Since! the! limb! phenotype! of!Sp6F/F;Sp8+/CreERT! mutants! is! similar! to! the!
human!SHFM,!we!proceed!to!analyze!the!expression!of!Tp63!and!Dlx5/6!in!Sp6F/F
;Sp8+/CreERT!and!Sp6F/F;Sp8CreERT/CreERT!mutant!limbs,!in!order!to!determine!if!the!Sp!
transcription!factors!were!components!of!the!Tp63!network!leading!to!SHFM!.!
!Tp63!is!a!key!regulator!of!epidermis!morphogenesis!and!it!is!expressed!in!
the! epithelial! surface! of! the! embryo,! including! the! limb! ectoderm.! Later! on,! at!
E10.5,!it!is!highly!expressed!within!the!AER!(Yang!et%al.,!1999;!Mills!et%al.,!1999).!
At!E10.5,!Tp63!expression!was!detected!in!the!entire!ectoderm!and!the!AER!of!
control! embryos! (Fig.! 37A),! while! Dlx5%expression,! a! direct! target! of! Tp63%(Lo!
Iacono!et%al.,!2008),!was!restricted!to!the!AER!and!Dlx6!(not!shown)!(Fig.!37D).!
Tp63!expression! was! detected! in! the! entire! limb! ectoderm!of! both! Sp6F/F
;Sp8+/CreERT%(Fig.! 37B)! and! Sp6F/F;Sp8CreERT2/CreERT! mutant! limb! buds! (Fig.! 37C,C´),!
while! a! slight!downregulation! of! Dlx5!expression! was! appreciable!in! the!
inmature! and! flatten! AER! of! Sp6F/F;Sp8+/CreERT!(Fig.! 36E)! and! also!in! Sp6F/F
;Sp8CreERT2/CreERT!mutant!limb!buds!(Fig.!37F,F)!.!
!
Results!
!
137!
!
Figure*37.*Analysis*of*the*expression*of*Tp63*and*Dlx5*in*Sp6)
/);Sp8)/CreERT2*and*Sp6)/);Sp8CreERT2/CreERT2*mutant*forelimb*buds.*
Transverse! limb! sections! showing! the! expression! of! Tp63!
(A,B,C,C´)! and! Dlx5! (D,E,F,F´)!genes! in! control! (A,D),! Sp6F/F
;Sp8+/CreERT2!(B,E)! and! Sp6F/F;Sp8CreERT2/CreERT2!(C,C´,F,´F´)! mutant!
limb! buds! at! E10.5.! Tp63!was! detected! in! both! Sp6F/F
;Sp8CreERT2/CreERT2!(B)! and! Sp6F/F;Sp8+/CreERT2!(C,C´)! mutants! while!
Dlx5!(E,!F;!F´)!was!slightly!downregulated!in!both!mutants.!*
!
The!expression!of!Tp63!and!the!Dlx!genes!in!Sp6F/F;Sp8+/CreERT!mutant!limb!
buds!lead!us!to!demonstrate!that!if!the!Sp!transcription!factors!are!components!
of!the!Tp63!network!they!act!downstream!of!the!Dlx%genes.!*
*
4.8.3.* Dorso[Ventral* pattern* establishment* in*Sp6)/);Sp8+/CreERT2!mutant*
limb*buds*
Sp6F/F;Sp8+/CreERT2!newborns!displayed!double!dorsal!digit!tips.!As!we!have!
demonstrated!in!absence!of!both!Sp6!and!Sp8!or!when!a!single!functional!allele!
of!Sp6!remains!in!absence!of!Sp8!DV!pattern!is!not!established.!In!addition,! as!
absence!of!En1!has!been!related!to!AER!maturation!defects!(Loomis!et%al.,!1996;!
Cygan! et% al.,! 1997;! Loomis! et% al.,! 1998),! we! proceed! to! analyze!DV! pattern!
establishment!in!Sp6F/F;Sp8+/CreERT!mutant!limb!buds,!looking!for!the!expression!of!
Wnt7a!and!En1.!!
Results!
!
!
138!
At!E10.5,!Wnt7a!expression!is!restricted!to!the!dorsal!limb!ectoderm!and!
reaches!the!dorsal!AER!border!in!control!embryos!(Fig.!38A).!In!Sp6F/F;Sp8+/CreERT!
mutant!limb!buds,!Wnt7a!expression!was!extremely!variable!even!within!a!single!
limb.!Expression!of!Wnt7a!at!a!variable!ventral!extent!within!a!same!limb!was!
appreciated! as! shown! by! consecutive! transverse! limb! section! of! E10.5! Sp6F/F
;Sp8+/CreERT! mutants! (Fig.! 38B,B´).! Consistent! with! this,! En1!appeared!
downregulated! in! the! ventral! limb! ectoderm! of! Sp6F/F;Sp8+/CreERT! mutants,! in!
comparison! to! wild! type! animals! (Fig.! 38C,D,D´).! Interestingly,! En1!
downregulation! exhibited! a! clear! correlation! with! the! variable! ventral! ectopic!
expression!of! Wnt7a,! correlation! easily! appreciated! when! consecutive! limb!
section!were!hybridized!for!Wnt7a!and!En1!(Fig.!38B,!B´,D,D´).!
!
!
Figure* 38.* Analysis* of* DV* markers* expression* in*Sp6)/)
;Sp8+/CreERT2*mutant*forelimb*buds.*Expression!of!Wnt7a!(A,B,B´)!
and!En1!(C,D,D´)!in!control!(A,C)!and!mutant!(B,B´,D,D´)!forelimb!
transverse! sections! at! E10.5.! DV! establishment! was! disrupted!in!
Sp6F/F;Sp8+/CreERT2!mutant! embryos.! Wnt7a!expression! was!
extended! into! the! ventral! limb! ectoderm! (B,B´)! in! correlation! to!
En1!expression! downregulation!(D,D´),! appreciable! in! limb!
consecutive!sections!bud!(B,B´,D,D´).!
!
In!sum,!our!results!demonstrate!that!when!only!one! functional!allele!of!
Sp8! remains,! in! the! absence! of! other! Sp!alleles! (Sp6!and! Sp8),! DV! pattern!
establishment! is! disrupted.! Besides! normal! expression! of! Bmp4!irregular!
Results!
!
139!
activation!of!En1!in!the!ventral!limb!ectoderm!was!detected,!in!strong!correlation!
to! ventral! ectopic! expression! of! Wnt7a,! leading! to! the! development! of! double!
dorsal!limb!buds!that!could!account!for!the!double!dorsal!digit!tips!developed!in!
Sp6F/F;Sp8+/CreERT!mutant.!
*
4.9*Temporally* controlled*deletion*of* genes*specifically*expressed* in*the*
limb*bud*ectoderm*with*the*Sp8CreERT2*line*
Several! Cre! lines! have! been! described! for! the! conditional! removal! of!
genes!expressed!in!the!limb!ectoderm.!!These!include!the!Msx2FCre!(Lewandosky!
et%al.,!2000),!the!Brn4FCre!(Ahn!et%al.,%2001),!the!Ap2
α
FCre!(Boulet!et%al.,%2004)!
and!the!Mox2FCre!(Delgado!et%al.,!2008)!lines!of!which!any!of!them!is!inducible.!
The!Sp8!null!allele!we!have!used!in!this!work!bears!the!inducible!CreERT2!fusion!
protein!that!drives!the!expression!of!the!Cre!in!the!limb!ectoderm!under!the!Sp8!
locus.! This! line! is! characterized! by! a! modification! of! the! Cre! that! contains! a!
G400V/M543A/L544A!triple!mutation!in!the!Estrogen!Receptor!ligand?binding!
domain! (ER?LBD)! that! makes! it! more! sensitive! to! Tamoxifen! (Tam)! than! the!
mutant! ER! LBD! with! a! single! G521R! substitution.! We! decided! to! evaluate! the!
activity!of!this!line,!using!the!ROSA26!reporter!strain!(R26R;!Soriano,!1999),!as!a!
possible! ectodermal?specific! inducible! deleter! line.! With! this! purpose! we!
analyzed!Rosa26+/tg;Sp8+/CreERT2!embryos!after!administration!of!Tam.!!
Since! it! has! been! previously! shown! that! Cre?mediated! recombination! is!
evident!by!8h!and!complete!by!12h!after!a!single?dose!tamoxifen!intraperitoneal!
injection! (Chen! et% al.,!2008),! we! decided! to! analyze! LacZ! expression! in! E9.5!
embryos!that!had!received!a!single!dose!of!Tam!at!E8.5.!!As!expected,!our!results!
showed! that! the! Cre! recombinase! was! active! in! the! sites! of! Sp8!expression!
replicating!the!pattern!of!expression!described!for!Sp8.!Maximum!staining!was!
observed! in! the! neural! tube,! somites! and! trunk! ectoderm.! ! In! particular,! Cre!
activity! was! observed! through! the! dorsal! and! ventral! limb! bud! ectoderm!
although!in!a!mosaic!fashion!(Fig.!39).!
Results!
!
!
140!
!
Figure*39.* Monitorization* of* the* Cre* activity* under* the* Sp8*locus.* Whole!
mount!LacZ!staining!of!Sp8+/CreERT2;ROSA26+/tg%E9.75!embryo.!(A)!Lateral!view,!(B)!
dorsal! view! and! (C)! forelimb! buds.! After! tamoxifen! administration! the! embryo!
showed! strong! LacZ! expression! in! the! neural! tube,! the! somites,! in! the! forelimb!
ectoderm!and!practically!all!the!caudal!region!of!the!embryo!including!hindlimbs.!
!
In!sum,!the!CreERT2!in!the!Sp8!locus!drives!Cre!activity!in!both!hindlimb!
and!forelimb!ectoderm!early!enough!to!remove!genes!from!the!limb!ectoderm!at!
early!stages.!However,!gene!deletion!is!not!restricted!to!the!limbs!as!shown!by!
strong!LacZ!staining!in!the!other!domains!of!Sp8!expression.!
*
4.10*
β
)catenin*Gain*of*function*with*the*Sp8CreERT2*line*
Sp6! expression! in! the! limb! ectoderm! has! been! shown! to! be! under! the!
control! of! Wnt/β?catenin! signalling! (Talamilo! et% al.,% 2010).! However,! this!
analysis! has! not! been! performed! for! Sp8! although! experiments! in! chick! have!
suggested!that! it!is!also! under!Wnt/β?catenin!control!(Kawakami! et%al.,!2004).!
Therefore,!we!decided!to!analyze!the!expression!of!Sp8!in!mutants!with!GOF!of!
β
F
catenin!with!the!help!of!the!inducible!Sp8CreERT2!line!we!have!just!characterized.!
For! this! experiment! pregnant! females! from! Sp8+/CreERT2!and! CatnFblox(ex3)!
crosses!were!given!Tam!via!intragastric!gavage!every!24h!for!3!consecutive!days!
from! E7.5! to! E9.5.! ! This! procedure! was! followed! to! make! the! recombination!
event!occur!in!all!cells!and!the!embryos!were!recovered!by!cesarean!section!at!
E10.5.!
Results!
!
141!
β
Fcatenin! GOF! mutants! with! the! Sp8CreERT2!line! exhibited! ectopic! Sp8!
expression!in!both!dorsal!and!ventral!forelimb!ectoderm!as!assessed!by!whole!
mount!in!situ!hybridization!of!E10.5!CatnFb+/lox(ex3);Sp8CreERT2/+!mutant!embryos,!
confirming!that!in!the!mouse!Sp8!acts!downstream!of!Wnt/β?catenin!(Fig.!40!D,!
E).! In! addition,! in! correlation! to! Sp8!expression,! Fgf8!was! also! found! to! be!
ectopically!expressed!in!CatnFb+/lox(ex3);Sp8CreERT2/+!mutant!embryos!(Fig.!40F!).!
!
*
Figure* 40.* Gain* of* function* mutation* of*
β
)catenin*with* the*
Sp8CreERT2*led*to*ectopic*expression*of*Sp8*and*Fgf8$in*the*limb*
ectoderm.*Whole!mount!in!situ!hybridization!within!control!(A,!B,!
C)! and! GOF! β?catenin;Sp8+/CreERT2%(D,! E,! F)! mutant! limb! at! E10.5!
showing!the!ectopic!expression!of!both!Sp8!(D,!E)!and!Fgf8!(F).!Note!
that!A!and!D!are!dorsal!views!of!the!limb!while!B,C,E!and!F!are!distal!
views!of!the!limb!bud.!
!
To!further!analyze!the!ßFcatenin!GOF!phenotype!with!the!Sp8CreERT2!line,!
we!decided!to!perform!transverse!sections!within!mutant!limb!buds!at!E10.5!to!
analyze! in! more! detail! the! ectopic! expression! of! Fgf8!and! Sp8.! In! addition,! we!
decided! to! check!Bmp4!and! En1!expression! in! CatnFb+/lox(ex3);Sp8CreERT2/+!
embryos,! because! it! has! been! reported! that! Bmp! signalling!pathway! lies!
downstream!of!Wnt!signalling!for!DV!pattern!establishment!in!the!limb!ectoderm!
(Soshnikova! et% al.,! 2003;! Barrow! et% al.,!2003).! By! E10.5,! Fgf8!and! Bmp4!
expression!is!confined!to!the!distal!tip!of!the!developing!limb!of!control!embryos!
(Fig.!41A),!while!Sp8!at!this!stage!is!predominantly!expressed!in!the!AER!and!in!a!
less!abundant!manner!in!the!ventral!limb!ectoderm!(Bell!et%al.,!2003;!Treichel!et%
Results!
!
!
142!
al.,!2003).!However,!expression!of!Fgf8!and!Bmp4!were!ectopically!detected!in!
both!ventral!and!dorsal!limb!ectoderm!of!E10.5!CatnFb+/lox(ex3);Sp8CreERT2/+!limb!
transverse!sections!(Fig.!41B,!E)!as!it!was!for!Sp8!(Fig.!41C).!Nevertheless,!En1,!
the!most!downstream!effector!for!DV!limb!patterning!downstream!of!the!Bmp!
signalling!pathway! that! it! is! expressed! in! the! ventral! limb! ectoderm!and! the!
ventral!half!of!the!AER!at!E10.5!(Fig.!41!F),!was!completely!downregulated!and!
was! only! detected! in! the! most! proximo?ventral! ectoderm! of! CatnF
b+/lox(ex3);Sp8CreERT2/+!limb!buds!(Fig.!41G).!!
!
!
Figure* 41.* Gene* expression* analysis* in* Sp8CreERT2*mediated*
β
)catenin*
gain*of*function*limb*buds.*Fgf8!(A,B),!Sp8%(C),!Bmp4!(D,E)!and!En1!(F,G)!
expression! in! transverse! sections! within! control! (A,D,F)! and! CatnF
b+/lox(ex3);Sp8CreERT2/+!(B,C,E,G)!mutant!limb!buds.!Gain!of!function!mutation!of!
β
Fcatenin!resulted!in!ectopic!expression!of!Fgf8!(B),!Sp8!(C)!and!Bmp4!(E)!in!
the!dorsal!and!ventral!embryos,!while!En1!expression!from!the!ventral!limb!
ectoderm!was!lost!(G).!
!
Briefly,! our! experiments! are! consistent! with! previous! results! that!
demonstrate!or!even!suggested!that!Wnt!signalling!lies!upstream!of!both!Sp8!and!
Fgf8! expression! in! the! limb! ectoderm! (Bell! et% al.,! 2003;! Treichel! et% al.,! 2003;!
Barrow!et!al!2003;!Soshnikova!et%al.,!2003!Kawakami!et%al.,!2004;!Sahara!et%al.,!
Results!
!
143!
2007;!Lu!et%al.,!2008;!Talamillo!et%al.,!2010;!Lin!et%al.,!2013).!In!addition,!we!have!
demonstrated!that!Sp8!and!Fgf8!expression!patterns!are!identical!upon!Sp8CreERT2!
mediated!
β
Fcatenin!GOF.!These!results!fit!with!Sp8!acting!upstream!of!Fgf8!in!the!
limb!ectoderm,!in!a!Wnt/
β
Fcatenin!dependent!manner!(Lin%et%al.,!2013).!Finally,!
we!confirmed!the!ability!of!Wnt/
β
Fcatenin!to!upregulated!Bmp!signalling!in!the!
limb! ectoderm! as! assessed! by! the! ectopic! expression! of! Bmp4%(Barrow! et% al.,!
2003;! Soshnikova! et% al.,! 2003),! while! unexpectedly! En1!expression! was!
downregulated!in!the!ventral!limb!ectoderm!(Soshnikova!et%al.,!2003).!Thus,!it!is!
possible! that! the! GOF! of!
β
Fcatenin!did!not! rescue!the!DV! phenotype,!because!
there!was!not!enough!space!in!the!ventral!ectoderm!for!En1,!due!to!the!expanded!
expression!of!Fgf8.!!
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Discussion!
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147!
5.*Discussion*
The!use!of!conditional!and!knock!out!approaches!allows!us!to!determine!
the! requirement! and! redundancy! of!both!Sp6! and! Sp8! transcription! factors!in!
limb!development.!In!the!total!absence!of!Sp6%and!Sp8%or!even!when!only!a!single!
functional!allele!of!Sp6%remains,!in!the!absence!of!Sp8,!no!limbs!formed!leading!to!
a!tetramelic!phenotype.!In!addition,!the!disruption!of!one!of!the!Sp8!alleles!in!the!
absence! of! Sp6!resulted! in! a! phenotype! reminiscent! of! the! Split! hand/foot!
malformation!in!humans.!The!analysis!of!the!compound!mutants!demonstrates!
that! both! transcription! factors! are! conjointly! required! in! a! dose! dependent!
manner!and!that!Sp8!makes!a!more!substantial!contribution!to!limb!outgrowth!
than!Sp6,!probably,!due!to!a!higher!level!of!expression.!!
We!have!also!demonstrated!that!both!transcription!factors,!Sp6!and!Sp8,!
are!required!and!act!in!a!redundant!manner!mediating!the!induction!of!Fgf8!in!
the!AER.!!In!addition!they!are!required!to!ensure!proper!AER!morphology,!as!in!
their!total!absence!no!morphological!AER!is!observed.!Of!most!interest,!besides!
Bmp!expression!and!signalling!in!the!limb!ectoderm!at!early!stages,!as!assess!by!
the!expression!of!Bmp4!and!Msx2,!En1!expression!is!not!detected!in!the!ventral!
limb!ectoderm!and!double!dorsal!limb!are!developed.!
!
!5.1* Sp6*and* Sp8*are* conjointly* required* in* a* redundant* and* dose*
dependent*manner*for*limb*development!
The!limb!as!other!organs!is!generated!as!a!result!of!complex!interactions!
amongst!different!signalling!pathways!that!orchestrate!several!processes!such!as!
specification,! differentiation,! proliferation! or! apoptosis.! These! complex!
interactions!between! different! signaling! pathways!lead! to! the! generation! of!
intricate!networks!that!specifically!pattern!the!different!organs.!The!generation!
during!the!last!decades!of!different!mice!harboring!multiple!mutations!of!genes!
belonging! to! the! same! family! that! exhibited! similar! patterns! of! expression,!
highlighted!the!robustness!of!the!networks!provided!by!these!genes!acting!in!a!
redundant! manner! (Sun! et% al.,% 2000;! Boulet! et% al.,% 2004,! Mariani! et% al.,! 2008;!
Sheth!et%al.,%2013).!!
Discussion!
!
!
148!
In!the!case!of!the!Fgf,!Bmp!or!Hox!families,!it!is!well!documented!that!the!
members! of! these! families! play! redundant! roles! during! limb! development! and!
that!the!overall!final!dosage,!rather!than!particular!dosage,!is!the!most!important!
parameter!for!morphogenesis!(Sun!et%al.,%2000;!Boulet!et%al.,%2004,!Mariani!et%al.,!
2008;!Sheth!et%al.,%2013).!In!this!study,!through!the!generation!of!Sp6;Sp8!double!
knock! out! and! Sp8! conditional! mutant! in! absence! of! Sp6,!we! provide! further!
evidence! for! the!roles! of! Sp6!and! Sp8!transcription! factors! during! limb!
development.! Single! gene?targeting! analysis!related! their! role! with! AER!
maintenance!or!maturation!rather!than!induction!(Treichel!et%al.,!2003;!Bell!et%
al.,!2003;!Talamillo!et%al.,!2010).!!
The! analysis! of! the! Sp6;Sp8!double! mutants! performed! in! this! study!
demonstrated!that!these!transcription!factors!are!together!absolutely!required!
for! limb! development.! In! contrast! to! single! gene?inactivation! studies,! Sp6;Sp8!
double!mutants!or!even!mutants!bearing!a!single!functional!allele!of!Sp6!in!the!
absence!of!other!Sp!alleles!(Sp6!or!Sp8)!no!limbs!formed.!In!addition,!knock!out!
approaches!together!with!conditional! approaches!allowed!us!to! determine!not!
only! that! both! transcription! factors! act! in! a! redundant! manner! during! limb!
development,! but! also! in! a! dose! dependent! manner.! Despite! the! variability!
associated! to! each! genotype,! when! predominant! phenotypes! were! considered,!
the!phenotypes!displayed!a!strong!correlation!between!the!increase!in!severity!
and!the!decrease!in!the!level!of!Sp6!and/or!Sp8!gene!products,!with!Sp8!making!a!
more! substantial! contribution! to! limb! outgrowth! than! Sp6,%probably,! due! to! a!
higher!level!of!expression.!Regarding!the!forelimb,!progressive!decrease!of!Sp6!
and/or!Sp8!gene! products! resulted!in! an! increasing!severity! of! the! limb!
phenotype!leading!first!to!soft!tissue!syndactyly! of! digit!2?3!in!Sp6! KO! mutant!
animals,!then!Split!hand/foot!malformation!when!only!a!single!functional!allele!
of! Sp8! remains,! then! limb! truncations! in! the! absence! of! both! Sp8!alleles! and!
finally!amelic!phenotypes!in!the!absence!of!both,!Sp6!and!Sp8,!or!even!when!only!
a!functional!allele!of!Sp6!remains!(Fig.!42).!
The!increase!in!severity!observed!in!the!compound!mutants!as!the!level!of!
Sp6/Sp8!gene?products! decreased! showed! the! requirement! of! a! minimum!
threshold! level! of! Sp6/Sp8!gene?product! for! limb! development.! Lack! of! limbs!
Discussion!
*
!
149!
when! a! single! functional! allele! of! Sp6! still! remains,! in! the! absence! of! other! Sp!
alleles!(Sp6!and!Sp8),!indicate!that!the!level!of!Sp6!expression!provide!by!a!single!
functional! allele! of! Sp6!was! not! sufficient! to! support! limb! development! These!
level! of! expression! provided! by! a! single! functional! allele!is!below! a! minimum!
critical! threshold! required! for! limb! development.! In! addition,! the! more! severe!
limb! phenotype! of! the! Sp8! mutant! in! comparison! to! mutants! bearing! a! single!
functional!allele!of!Sp8,!in!the!absence!of!other!Sp!alleles!(Sp6!and!Sp8),!implies!
that!the!gene!dose!provided!by!two!alleles!of!Sp6!is!below!the!dose!provided!by!a!
single! allele! of! Sp8.! Moreover,! when! compared! with! the! phenotype! of! the! Sp8!
conditional! mutant! with! the! use! of! the! Msx2FCre%demonstrates! that! the! dose!
provided!by!two!alleles!of!Sp6!is%even!below!a!transient!expression!of!single!Sp8!
allele!(Fig.! 42).! Thus,! the! limb! phenotypes! of! compound! mutants! supports! a!
major!contribution!of!Sp8!to!limb!development!in!comparison!to!Sp6,%probably!
due!to!higher!level!of!expression.%
!
!
Figure* 42.* Scheme*illustrating* the* dose* dependent* requirement* of* Sp6*and* Sp8*
transcription*factors*for*proper*limb*development.*Progressive!decrease!of!Sp6!and/or!
Sp8!gene!products!resulted!in!an!increasing!severity!of!the!limb!phenotype!leading!first!to!
soft! tissue! syndactyly! of! digit! 2?3! in! Sp6!KO! mutant! animals,! then! Split! hand/foot!
malformation!when!only!a!single!functional!allele!of!Sp8!remained,!then!limb!truncations!in!
the!absence!of!both!Sp8!alleles!and!finally!amelic!phenotypes!the!in!absence!of!both,!Sp6!
and!Sp8,!or!even!when!only!a!functional!allele!of!Sp6!remained.!
Discussion!
!
!
150!
Sp9,!another!member!of!the!Sp!family!of!transcription!factors!that!is!also!
expressed!in!the!limb!ectoderm,!has!been!proposed!to!act!in!a!redundant!manner!
with!Sp8!during!limb!development!(Kawakami!et%al.,!2004).!However,!our!results!
show!that!in!the!absence!of!both!Sp6!and!Sp8,!Sp9!during!limb!development!is!
irrelevant!as!shown!by!the!total!absence!of!limbs!in!the!Sp6;!Sp8!double!mutant.!
*
5.2*Sp6*and*Sp8*are*required*for*Fgf8*induction*in*the*AER*
The!predominant!phenotypes!obtained!by!progressive!removal!of!the!Sp6!
and! Sp8!alleles! are! consistent! with! defects! involving! AER! development.!
Truncated! limbs! developed! in! the! absence! of! Sp8!result! from! a! premature!
regression!of!the!AER,!while!irregular!induction!of!Fgf8!leading!to!defects!in!AER!
maturation! are! responsible!for! the! SHFM! phenotype! developed! in! mutants!
bearing!a!single!functional!allele!of!Sp8,!in!the!absence!of!other!Sp!alleles!(Sp6!
and! Sp8).! Finally! the! total! absence! of! Sp6!and! Sp8!or! even! when! a! single!
functional!allele!of!Sp6!remains!in!the!absence!of!the!other!Sp!alleles!(Sp6!and!
Sp8)!lead!to!amelic!phenotypes!due!to!a!failure!in!Fgf8!induction!in!the!AER.!
The!induction!of!the!AER!relies!on!intricate!interactions!between!different!
signalling! pathways! that! lead! to! the! induction! of! Fgf8! in! the! limb! ectoderm!
(Barrow!et% al.,!2003;! Soshnikova! et% al.,!2003).! Big! efforts! have! been! made! to!
unveil!the! mechanisms!responsible!for! Fgf8! induction,! but! while! some! aspects!
have!been!unraveled!and!different!signalling!pathways!have!been!involved,!the!
crosstalk!between!them!is!not!fully!understood.!At!least!three!signalling!inputs!
have!been!demonstrated!to!be!critical!for!Fgf8!induction!in!the!limb!ectoderm.!
One! is! the! requirement! of! Fgf10!signalling! from! the! mesoderm! through! it!
receptor!Fgfr2,!expressed!in!the!limb!ectoderm!(Ohuchi!et%al.,%1997;!Min!et%al.,!
1998;!Sekine!et%al.,!1999;!Xu!et%al.,%1998;!Arman!et%al.,!1999;!Yu!et%al.,!2008;!Lu!et%
al.,!2008).! The! second!is!a! Wnt/β?catenin!activity!preferentially!signalling!into!
the!ventral!limb!ectoderm!where!AER!precursor!cells!are!induced!(Barrow!et%al.,!
2003;!Soshnikova!et%al.,!2003).!Finally,!the!third!involves!Bmp!signalling!in!the!
limb!ectoderm,!through!it!receptor!BmprIa%(Ahn!et%al.,!2001;!Pizette!et%al.,!2001;!
Soshnikova!et%al.,!2003;!Pajni?Underwood!et%al.,!2007).!The!disruption!of!any!of!
Discussion!
*
!
151!
the! mentioned! signalling!pathways! leads! to! amelic! phenotypes! due! to! the!
inability!to!induce!Fgf8!expression!in!the!limb!ectoderm.!
!Fgf10!signalling!from!the!mesoderm!through!its!Fgfr2!is!responsible!for!
Fgf8%induction,!in!a!Wnt/β?catenin!dependent!manner,!through!the!induction!of!
Wnt3a!and!Wnt3!in!chick!and!mouse!limb!ectoderm,!respectively.!Once!Fgf8!is!
induced,!its!signalling!to!the!mesoderm!is!required!to!maintain!Fg10!expression!
in!the!mesoderm!and!a!feedback!regulatory!loop!is!established.!Thus,!both!Fgf10!
and!Wnt/β?catenin!are! not!only! required!for! the!induction! of! Fgf8,!also!for! its!
maintenance%(Kawakami!et%al.,!2001).!Regarding!Bmp!signaling!but!disruption!of!
the!BmprIa!abolished!AER!induction!without!interfering!with!Fgf10!induction!in!
the!limb!mesoderm!(Ahn!et%al.,!2001;!Pajni?Underwood!et%al.,!2007;%Soshnikova!
et%al.,!2003).!However,!whether!Bmp!signalling!lies!upstream!or!downstream!to!
Wnt/β?catenin! signalling! remains! quite! controversial.! Nevertheless,! genetic!
evidence!favors! a! model! in! which!Wnt! /β?catenin!signalling! is! require!
downstream!of!Bmp!signalling!for!Fgf8!induction!in!the!AER!(Barrow!et%al.,!2003;%
Soshnikova!et%al.,!2003).!
We!have!demonstrated!here!that!the!combined!absence!of!Sp6!and!Sp8,!
results!in!absence!of!limbs!due!to!the!striking!inability!to!induce!Fgf8!expression!
in! the! AER,! while! normal! expression!of! Fgf10! in! the! limb! mesoderm! and! also!
Bmp!in!both!the!limb!ectoderm!and!mesoderm!is!achieved!at!early!stages!of!limb!
development,!when!the!AER!is!induced.!Because!both!transcription!factors!are!
expressed! in! a! temporal! and! spatial! manner! compatible! with! the! induction! of!
Fgf8%(Bell!et%al.,!2003;!Treichel!et%al.,!2003;!Talamillo!et%al.,!2010),!together!with!
compelling!evidence!positioning!them!downstream!of!Wnt/β?catenin!signalling!
in! the! limb! ectoderm! (Bell! et% al.,! 2003;! Treichel! et% al.,! 2003;! Kawakami! et% al.,!
2004;!Talamillo!et%al.,!2010)!and!the!binding!ability!of!Sp8!to!the!Fgf8!promoter!
(Sahara!et%al.,!2007),!our!results!are!consistent!with!a!model!in!which!Sp6!and!
Sp8!are!necessary!mediators!responsible!for!Wnt/β?catenin!dependent!induction!
of! Fgf8%(Fig.! 43),! acting! downstream! of! Fgf10!and! Bmp! signalling! in! AER!
induction.!In!addition,!the!phenotypes!obtained!and!the!gene!expression!analysis!
performed! suggest! that! both! factors!act! in! a! redundant! and! dose! dependent!
manner! in! the! induction! of! Fgf8.! However,! although!they! are! functionally!
Discussion!
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152!
redundant!they!do!not!contribute!in!the!same!manner!in!Fgf8!induction.!When!a!
single!functional!allele!of!Sp6%remains,!in!the!absence!of!other!Sp!alleles!(Sp6!and!
Sp8),!Fgf8!is!not!induced!implying!that!a!minimum!threshold!of!Sp!dose!that!is!
not! reached! in! this! mutants! is! required! to! ensure! the! induction! of! Fgf8.! In!
contrast,!the!dose!provided!by!a!single!functional!allele!of!Sp8,!in!the!absence!of!
other! Sp! alleles,! is! just! above! this! minimum! threshold! level! required! for! the!
induction! and! maintenance! of! Fg8!leading! to! the! development! of! all! three!
proximo?distal!skeletal!elements,!although!a!SHFM!phenotype!is!achieved.!
Of! most! interest,! disruption! of! either! Wnt/β?catenin%or! the! major!
contributors! of! the! Fgf! dose! provided! by! the! AER,! Fgf8!and! Fgf4,! resulted!in!
amelic!phenotypes!similar!to!the!total!absence!of!Sp6!and!Sp8!(Sun!et%al.,!2002;!
Boulet!et%al.,!2004).!However,!while!the!disruption!of!Wnt/β?catenin%in!the!limb!
ectoderm!prevents!Fgf8!induction!and!also!AER!morphogenesis!(Barrow!et%al.,!
2003;%Soshnikova!et%al.,!2003),!a!morphological!normal!or!even!hyperplastic!AER!
is!still!formed!in!the!double!Fgf4;Fgf8!conditional!mutant%implying!that!there!is!
not!a!simple!linear!relationship!between!Wnt/β?catenin!signalling!and!Fgf!in!the!
limb!ectoderm!(Sun!et%al.,!2002;!Boulet!et%al.,!2004).!Thus,!the!absence!of!Fgf8!
induction! together! with!the! ventral! ectodermal! thickening! present! in! double!
mutants! lacking! both! Sp6!and! Sp8,! implies! that! AER! morphogenesis! has! been!
initiated!but!the!process!is!arrested!and!cells!are!not!confined!to!DV!boundary.!
Hence,!our!results!favors!a!model!in!which!Wnt/β?catenin!accomplish!additional!
functions!during!AER!development!apart!from!the!induction!of!Fgf8,!related!with!
AER!morphogenesis!as!has!been!previously!suggested!(Barrow!et%al.,!2003).!We!
have!provided! further! evidence! that!support! that! the!role!of!Wnt/β?catenin!in!
the!limb!ectoderm!is!not!exclusive!to!the!Wnt/β?catenin?Sp6/Sp8?Fgf!regulatory!
loop.!In!addition,!the!early!expression!of!the!AER!markers!Msx2!and!Bmp4!in!the!
absence! of! Sp6!and! Sp8!(also! expressed! in! the! double! Fgf4;Fgf8)% that! are! lost!
when!Wnt/β?catenin!signalling!is!disrupted!(Sun!et%al.,!2002;!Barrow!et%al.,!2003;!
Soshnikova!et%al.,!2003),%demonstrated!that!Wnt/β?catenin!signalling!is!required!
for!their!induction!independents!of!the!Sp6/8!transcription!factors.!!
Based!on!Sp8%conditional!GOF!function!approaches,!it!has!been!recently!
reported!that!Sp8!partially!mediates!the!Wnt/β?catenin%dependent!induction!of!
Discussion!
*
!
153!
Fgf8,!function!that!we!have!demonstrated!here!that!is!fully!accomplished!by!Sp6!
and!Sp8.!Considering!the!dose!dependent!effect!of!the!Sp!factors,!the!inability!to!
rescue! the! loss! of! Wnt/β?catenin!with! forced! overexpression! of! Sp8%under!the!
Rosa26!locus,! may! rely! on! the! inability! to! reach! the! minimum! threshold! of! Sp!
level! required! for! the! induction! of! Fgf8.! In! support! of! this! notion,! it! has! been!
argued!that!the!incomplete!rescue!of!Wnt/β?catenin!LOF!after!overexpression!of!
Fgf8!under!the!Rosa26!locus!could!be!due!to!the!weaker!expression!level!of!Fgf8!
from!this!locus!compared!to!it!endogenous!level!of!expression!(Lin!et%al.,!2013).!!
*
5.3*Role*of*Sp6*and*Sp8*in*Dorso[Ventral*pattern*establishment*
The! AER! is! a! highly! dynamic! and! transitory! structure! subject! to!
morphogenetic!movements!during!its!development.!In!mouse,!the!pre?AER!cells!
are!induced!in!the!ventral!limb!ectoderm!by!the!time!limb!ectoderm!is!polarized!
(Kimmel!et%al.,!2000).!As!the!limb!bud!grows,!morphogenetic!movements!confine!
the!pre?AER!cells!to!the!DV!boundary!of!the!limb,!that!separates!En1!expressing!
and! non?expressing! cells.! The! fact! that! AER! induction! and! DV! pattern!
establishment!occurs!concomitantly!together!with!the!positioning!of!the!mature!
AER!at!the!DV!interface,!reflects!a!link!between!these!two!processes.!
Several! experiments! in! chick! as! well! as! gene?targeting! analysis! in! mice!
together!with!spontaneous!mutations!in!chick!suggested!that!the!establishment!
of!a!DV!boundary!is!an!indispensable!pre?requisite!for!AER!induction.!Grafting!
experiments!in!chick!in!which!confrontation!of!ventral!and!dorsal!ectoderm!led!
to!the! development!of!ectopic!AERs!at!the!junction! of!the!manipulated!ventral!
and!dorsal!ectoderms!(Laufer!et%al.,!1997;!Tanaka!et%al.,!1997),!together!with!Fgf?
soaked! bead! induced! ectopic! limb! buds! developing!at! the! right! DV! boundary!
disregarding!the!position!of!the!bead!supported!this!linkage!(Altabef!and!Tickle,!
2002).! In! addition,! overexpression! experiments! in! chick! suggested! that! a!
boundary!of!En1!expressing!and!non?expressing!cells!was!an!indispensable!pre?
requisite! for! AER! induction! (Pizette! et% al.,!2001).! Consistent! with! this,! the!
inability!to!induce!an!AER!in!the!chick!mutant!limbless!associates!on!the!absence!
of! En1! expression! in! the! ventral! limb! ectoderm! and! the! subsequent! extended!
Discussion!
!
!
154!
expression! of! Wnt7a!in! the! ventral! ectoderm! (Fallon,! 1983;! Carringthon! and!
Fallon,!1988;!Ros!et%al.,!1996;!Grieshammer!et%al.,!1996;!Noramly!et%al.,!1996).!!
In! contrast,! the! chick! mutant! eudiplopodia!is! characterized! by! the!
development!of!an!ectopic!AER!in!the!dorsal!ectoderm!leading!to!digits!with!a!
double!dorsal!phenotype!(Goetinck,!1964).!In!addition,!experiments!in!chick!in!
which!prospective!limb!mesoderm!was!grafted!between!two!rows!of!somites!led!
to!the!development!of!ectopic!double!dorsal!limbs!due!to!the!dorsalizing!effects!
of! signals! emanating! from! the! somites,! are! against! the! requirement! of! En1!
expressing! and! non?expressing! boundary! for! AER! formation! (Michaud! et% al.,!
1997).!Moreover,!En1!mutant!mice!and!also!the!double!mutant!En1;Wnt7a!that!
failed!to!express!En1!in!the!ventral!ectoderm!developed!an!AER!and!in!the!case!
of!the!double!mutant!was!morphologically!normal!(Cygan!et%al.,!1997;!Loomis!et%
al.,!1996;!Loomis!et%al.,!1998;!Parr!and!McMahon,!1995).!Based!on!the!ventrally!
extended!AER!of!En1!mutants,!it!is!currently!accepted!that!the!role!of!En1!is!also!
involved! with! AER! maturation! (Loomis! et% al.,! 1996;! Loomis! et% al.,!1998).!
Therefore,!the!requirement!of!a!DV!pattern!for!the!induction!of!the!AER!remains!
controversial.!
We!have!demonstrated!that!a!progressive!decrease!in!the!Sp6/Sp8!dose!
lead!to!DV!pattern!establishment!defects.!As!shown!in!the!total!absence!of!Sp6!
and!Sp8!or!even!when!a!single!functional!allele!of!Sp6!remains!in!the!absence!of!
other!Sp!alleles!(Sp6!and!Sp8),!the!limb!buds!emerges!but!rapidly!regresses.!Gene!
expression! analysis! of! these! limb! buds! showed! that! En1!was! absent! from! the!
ventral!limb!ectoderm!leading!to!ectopic!expression!of!Wnt7a!in!the!ventral!limb!
ectoderm! what! confers! a! double! dorsal! phenotype! to! the! emerging! limb! bud.!
Interestingly,!lack!of!En1!induction!in!the!ventral!ectoderm!in!the!absence!of!Sp6!
and!Sp8!occurs!disregarding!normal!expression!of!Bmp!ligands!and!signalling!as!
confirmed!by!normal!expression!of!Msx2,!indicating!the!involvement!of!Sp6!and!
Sp8!in!the! induction!of! En1! in! the! limb! ventral! ectoderm.! Since! the! ability! of!
members! of! the! Sp! family! to! interact! with! other! transcription! factor!has! been!
demonstrated,!including!SMADs,!we!hypothesize!that!Sp6/Sp8!could!be!required!
for! the! induction! of! En1! acting! downstream! of! Bmp! signalling,! through! the!
Discussion!
*
!
155!
cooperation! with! SMADs! (Fig.! 43)(Pardali! et% al.,!2000;! Sakaguchi! et% al.,!2005;!
Kim!et%al.,!2006).!!
On!the!other!hand,!when!a!single!functional!allele!of!Sp8!remains!in!the!
absence!of!other!Sp!alleles!(Sp6!and!Sp8),!irregular!activation!of!Fgf8!is!achieved!
and!as!the!AER!develops!maturation!defects!with!gaps!of!Fgf8!expression!along!
its! AP! axis! are! developed! together! with! expression! of! Fgf8! in! the! ventral! limb!
ectoderm.! These! AER! defects! could! account! for! the! SHFM! phenotype! in! these!
mutants.! Remarkably,! ventrally! abnormal! extended! expression! of! Wnt7a!at!
variable!degree!was!always!in!correlation!with!the!restriction!of!En1!to!a!more!
proximal!domain.!This!defect!in!DV!pattern!establishment!can!explain!the!double!
dorsal!digits!of!mutants!bearing!a!single!functional!allele!of!Sp8!in!the!absence!of!
the! other! Sp! alleles! (Sp6!and! Sp8).! Therefore,! it!is! consistent! with! previous!
reports!indicating!the!continuous!requirement!of!Wnt/β?catenin%for!the!correct!
DV!pattern!establishment!(Barrow!et%al.,!2003).!The!ventrally!extended!AER!is!
also!consistent!with!phenotypes!in!which! AER! maturation! defects!arise!due!to!
lack!of!En1%expression!in!the!limb!ectoderm!(Loomis!et%al.,!1998).!!
Finally,!the!limb!phenotype!of!the!Sp6;Sp8!double!mutant!resembles!that!
of! the! chicken! mutant! limbless.! Limbless!is! a! Mendelian! autosomal! recessive!
mutation! in! chick! affecting! the! ectodermal! component! of! the! limb! that! is!
characterized!by!the!total! absence!of! limbs!(Prahlad! et%al.,! 1979).! However,!in!
contrast!to!double!Sp6;Sp8!mutants! limbless!defects!are!restricted!to!the!limbs!
(Carrington!and!Fallon,!1984b;!Lanser!and!Fallon,!1984).!In!limbless!mutants,!the!
limb!bud!is!initiated!but!at!early!stages!(20!HH)!the!mesoderm!undergoes!cell?
death! as! a! consequence! of! the! inability!of!these! mutants! to! activate!Fgf8!
expression! in! the! limb! ectoderm! and! the! limb! bud! consequently! regresses!
(Fallon!et%al.,! 1983;!Ros!et%al.,!1996).!In!addition,!limbless!limb!buds! lacks! En1!
expression! in! the! ventral! ectoderm! with! the! subsequent! Wnt7a!expression! in!
both!dorsal!and!ventral!ectoderm,!leading!to!the!development!of!bidorsal!limb!
buds.! Interestingly,! the! genetic! cause! of! the! chicken! limbless! mutation! has! not!
been! identified! yet.! Therefore,! the! similarity! between! limbless!and! Sp6;Sp8!
double! mutants! limb! phenotype,! together! with! the! fact! that! Sp6! seems! to! be!
absent! in! the! chick,! lead!us! to! consider! the! possibility!that! Sp8!could! be! the!
Discussion!
!
!
156!
candidate! gene! responsible! of! the! limbless!phenotype,! as! has! been! previously!
proposed!(Robb!et%al.,%2011).!However,!as!limbless!defects!are!restricted!to!the!
limbs!and!mutants!do!not!exhibit!any!neural!defect!as!occurs!in!Sp8!mutants,!if!
Sp8!is! the! responsible!gene! for! the! limbless! mutation! in! chick,! this! mutation!
needs! to! be! in! a! limb! specific! regulatory! element! rather! than! in! the! gene.!
Nevertheless,!another!possibility!could!be!that!a!possible!redundancy!between!
Sp8!and!Sp9%in!chick!could!account!for!the!differences!between!limbless!and!the!
double!Sp6;Sp8!mutant.!However,!the!absence!of!a!morphological!AER!in!limbless!
does!not!support!Sp8!as!responsible!gene.!
*
5.4*Sp6/Sp8*and*Split*hand/foot*malformation*
SHFM! is! a! genetically! heterogeneous! congenital! human! malformation!
characterized!a!central!cleft!in!upper!and!lower!limbs!where!central!digits!may!
be!absent!and!the!remaining!posterior!digits!appear!normally!fused!(Temtamy!
and!McKusick,!1978;!Sifakis!et%al.,!2001).!The!phenotype!is!highly!variable!even!
within! the! same! individual! and! ranges! from! mild! central! syndactyly! to! severe!
loss!of!elements! with! oligodactyly!and! it! may!include!defects!in! the!zeugopod.!
The! incidence! of! SHFM! is! about! 1:18,000! live! births! and! can! appear! as! an!
isolated! entity! or! as! part! of! a! syndrome.! In! human,! at! least! 6! loci! have! been!
associated! to! this! SHFM! phenotype! (Scherer! et% al.! 1994;! Johnson! et% al.,!1995;!
Mills!et%al.,!1999;!Yang!et%al.,!1999;!Lo!Iacono!et%al.,!2008).!It!is!currently!accepted!
that! these! limb! defects! are! the! result! of! a! failure! in! the! maintenance! of! the!
central! region! of! the! AER! that! losses!Fgf8! expression.! We! have! demonstrated!
that!the!dose!provided!by!a!single!functional!allele!of!Sp8!in!the!absence!of!other!
Sp!alleles! (Sp6!or! Sp8)! is! just! above! the! minimum! threshold!of! the! Sp! dose!
required!for!Fgf8!induction!leading!to!its!irregular!activation!that!results!in!the!
formation!of!an!irregular!AER!presenting!gaps!along!its!AP!length.!We!show!that!
these!irregularities!in!the!AER!from!early!stages!also!result!in!SHFM!phenotype.!
Discussion!
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157!
Although!6!loci!has!been!associated!to!the!different!types!of!SHFM,!only!
Tp63!(SHFM!type!IV)!and!Dlx5!and!Dlx6!(SHFM!type!I)!have!been!unequivocally!
involved!in!this!malformation.!Both!of!them!have!been!shown!to!be!components!
of!the!same!regulatory!network.!Regarding!the!epistatic!relation!between!Tp63!
and!Dlx!genes!in!this!network,!it!has!been!shown!that!Tp63!lies!upstream!of!Dlx!
genes!and!it!is!required!for!its!expression!(Lo!Iacono!et%al.,!2008;!Kouwenhoven!
et% al.,! 2010).! Since! similar! phenotypes! are! frequently! caused! by! disruption! of!
components!of!a!regulatory!network,!we!have!considered!the!possibility!that!Sp6!
and!Sp8!genes!might!be!part!of!the!Tp63!network.!Indeed,!the!phenotypes!of!our!
mutants!are!identical,!including!the!dorso?ventral!component!to!those!recently!
reported!in!a!new!identified!mutation!in!Dlx5!(Shamseldin!et%al.,!2012).!However,!
the!fact!that!Tp63!and!Dlx5!and!Dlx6!are!expressed!virtually!normal!in!Sp6/Sp8!
mutants!indicate!that,!if!Sp6/Sp8!transcription!factors!would!act!downstream!of!
Tp63?Dlx!factors!if!they!are!in!the!same!network.!
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Figure* 43.* Schematic* representation* of* the* major* signalling* pathways*
leading*to*Fgf8*induction*and*DV*pattern*establishment*and*the*relative*
position*of*Sp6*and*Sp8.!Sp6!and!Sp8!act!in!a!redundant!manner!in!the!Wnt/β?
catenin! dependent! induction! of! Fgf8!in! the! limb! ectoderm.! In! addition! both!
transcription!factors!are!required!for!the!induction!of!En1!in!the!ventral!limb!
ectoderm!maybe!through!cooperation!with!the!Bmp!signalling!pathway.!Finally!
if!Sp6!and!Sp8!are!involved!in!the!Tp63!network!leading!to!SHFM!they!must!act!
downstream!the!Dlx!genes.!Arrows!indicated!induction!and!bars!repression.!
!
Discussion!
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158!
Briefly,! we! provide! strong!evidence!that! support! the! functional!
redundancy! and! a! dose! dependent! requirement! of! Sp6!and! Sp8! in! both! AER!
induction!and!DV!pattern!establishment.!We!demonstrate!that!they!are!required!
and!act! in! a! redundant! and! dose! dependent! manner! downstream! of! Wnt/β?
catenin%in!the!induction!of!Fgf8!in!the!limb!ectoderm,!as!Fgf8!is!not!induced!in!
the!absence!of!both!transcription!factors!and!limbs!are!not!formed.!In!addition,!
both! transcription! factors! are! also! required! for! the! induction! of! En1!in! the!
ventral!limb!ectoderm!and!subsequent!ventral!identity!specification.!Since,!Sp1!
is! known! to! interact! with! Smad,! the! Bmp! signalling! transducers,! it! is! possible!
that!cooperation!between!Sp6/8!and!Smad!are!required!for!the!induction!of!En1.!
Therefore,!we!propose!a!model!in!which!Sp6!and!Sp8!are!absolutely!required!for!
Fgf8!induction!and!DV!pattern!establishment.!
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6.CONCLUSIONS*
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6.*Conclusions*
1[ Sp6!and! Sp8! are! conjointly! required! for! limb! development! as! in! the!
complete! absence! of! both! transcription! factors,! no! limbs! form.! The!
progressive!reduction!in!the!dose!of!Sp6!and!Sp8!gene!products!leads!first!
to! split! hand/foot! malformation,! then! oligodactyly,! then! truncation! and!
finally! amelia.! Furthermore,! the! digits! that! form! with! reduced! Sp6/Sp8!
gene!dosage!exhibit!bi?dorsal!features.!
!
2[ In!the!absence!of!others!Sp!alleles!(either!Sp6!or!Sp8),!a!single!functional!
allele! of! Sp6!is! not! sufficient! to! support! any! limb! development,! while! a!
single!functional!allele!of!Sp8%permits!development!of!all!three!proximo?
distal! segments,! although! exhibits!an! split! hand/foot! malformation!
phenotype.! This! functional! difference! likely! relies! on! the! much! higher!
level!of!expression!of!Sp8!in!the!limb!ectoderm,!compared!to!Sp6!.!
!
3[ The!molecular!characterization!of!the!mutant!limb!buds!shows!that,!when!
the! dosage! of! Sp6/Sp8!is! significantly! reduced,! Fgf8!and! En1!are! not!
activated!although!initiation!of!AER!morphology!occurs.!!
!
4[ The!limb!phenotype!of!the!embryos!that!develop!with!a!single!copy!of!Sp8!
mimicked! the! human! split! hand/foot! malformation.! The! cause! of! the!
phenotype! is! an! irregular! Fgf8!activation! associated! with! a! constant!
abnormal!extension!of!Wnt7a!in!the!ventral!ectoderm!in!correlation!with!
a! corresponding! restriction! of! En1.! Tp63,! Dlx5!and! Dlx6! are! normally!
expressed!in!these!limbs!indicating!that!if!Sp6/Sp8!are!components!of!the!
Tp63!network,!they!act!downstream!of!Dlx5!and!Dlx6.!
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!
Conclusions!
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162!
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5[ Our!results!are!consistent!with!Sp6!and!Sp8!being!functionally!equivalent!
and!working!in!concert!during!limb!development!as!necessary!mediators!
of! the! Wnt/β?catenin! induction! of! Fgf8!and! also! as! cooperators! of! the!
Bmp!signalling!in!the!induction!of!En1.!
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!7.CONCLUSIONES*
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Conclusiones!
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165!
7.*Conclusiones*
!
1[ Sp6!y! Sp8! son! conjuntamente! requeridos! para! el! desarrollo! de! la!
extremidad,! dado! que! en! la! completa! ausencia! de! ambos! factores! de!
transcripción!las!extremidades!no!se!desarrollan.!La!reducción!progresiva!
en! la! dosis! de! Sp6!y! Sp8!da! lugar! primero! a! malformaciones! de! mano!
hendida/pie! hendido,! luego! oligodactilia,! después! truncamientos! y!
finalmente!amelia.!Además,!los!dedos!que!se!forman!con!dosis!reducidas!
de!Sp6/Sp8!muestran!características!bi?dorsales.!
!
2[ En! ausencia! de! otros! alelos! de! Sp! (tanto! Sp6! como! Sp8),! un! solo! alelo!
funcional! de! Sp6!no! es! suficiente! para! el! desarrollo! de! la! extremidad,!
mientras! que! un! solo! alelo! de! Sp8! permite! el! desarrollo! de! los! tres!
segmentos!próximo?distales,!aunque!presentando!un!fenotipo!de!de!mano!
hendida.! Esta! diferencia! funcional! probablemente! reside! en! un! mayor!
nivel! de! expresión! de! Sp8! en! el! ectodermo! de! la! extremidad,! en!
comparación!con!Sp6.!
!
3[ La! caracterización! molecular! de! los! esbozos! de! extremidad! de! los!
mutantes! muestra! que,! cuando! la! dosis! de! Sp6/Sp8! se! reduce!
significativamente,!Fgf8!y!En1!no!se!activadan!a!pesar!de!que!se!produce!
la!inducción!de!una!AER!morfológica.!
!
4[ El!fenotipo!de!la!extremidad!de!los!embriones!que!se!desarrollan!con!una!
única! copia! de! Sp8! reproducen! la! malformación! en! humanos! de! mano!
hendida.! La! causa! del! fenotipo! es! una! activación!irregular! de! Fgf8!
asociada!a!una!anormal!y!constante!extensión!de!Wnt7a!en!el!ectodermo!
ventral,! en! correlación! con!la! correspondiente! restricción! de! En1.! La!
normal!expresión!de!Tp63,!Dlx5!y!Dlx6!en!estas!extremidades!indica!que!
si!Sp6/Sp8! son!componentes! de!la! vía!de! Tp63,! éstos! estarían!actuando!
por!debajo!de!Dlx5!y!Dlx6.!
!
Conclusiones!
!
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166!
!
!
5[ Nuestros! resultados! muestran! que! Sp6!y! Sp8! son! funcionalmente!
equivalentes! y! actúan! de! manera! conjunta! durante! el! desarrollo! de! la!
extremidad.! Estos! resultados! apoyan! un! modelo! en! el! que! Sp6!y! Sp8!
estarían!actuando!como!mediadores!necesarios!para!la!inducción!de!Fgf8!
mediada! por! Wnt/β?Catenin,! además! de! cooperar! con! la! vía!de!
señalización!de!Bmp!para!la!inducción!de!En1.!
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8.REFERENCES!
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References!
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169!
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AGRADECIMIENTOS*
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Agradecimientos!
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Hay! tantas! personas! a! las! que! debo! estar! agradecido! por! su! apoyo! a! lo!
largo! de! estos! cuatro! años,! que! me! resultaría! muy! difícil! saber! por! donde!
empezar.! Aun! así,! intentando! ser! breve,! esta! tesis! no! puede! terminar! sin! unas!
palabras!de!agradecimiento!hacia!las!personas!que!han!marcado!mi!estancia!en!
Santander.!!
En!primer!lugar,!quiero!agradecer!a!Marian!y!a!Federica!todo!lo!que!me!
han!enseñado!sobre!biología!del!desarrollo!y!sobre!ciencia!en!general.!Me!siento!
muy! agradecido! de! poder! decir! que! he! tenido! como! directoras! no! solo! a! unas!
grandes!investigadoras,! sino! también! unas! personas! cercanas! y! con! las! que!
poder!discutir.!Del!mismo!modo,!me!gustaría!agradecerles!todos!los!consejos!que!
durante!este!tiempo!me!han!dado.!!!
No!me!puedo!olvidar!de!todos!los!compañeros!de!laboratorio:!Marisa,!Mª!
Félix,!Marta,!Patricia,!Paula,!Lucia,!Carlos,!Rushi,!Marian!T.,!Antonio,!Sara,!Irene,!
Mamen!y!Mar.!Muchas!gracias!por!haber!sido!además!de!compañeros,!grandes!
amigos.!Me!considero!afortunado!por!haber!compartido!estos!años!con!vosotros,!
todos!en!mayor!o!menor!medida,!habéis!sido!muy!importantes!y!habéis!marcado!
de!forma!especial!mi!estancia!aquí.!!
También!me!gustaría!agradecer!en!general!el!ambiente!de!compañerismo!
que! existe! entre! los! diferentes! departamentos! de! la! facultad! y! del! recién!
inaugurado!IBBTEC,!a!todos!y!cada!uno!de!los!becarios!(que!son!muchos!y!me!
seria! imposible! agradecerles! a! todos! sin! dejarme! seguramente! alguno! por! el!
camino)! siempre! dispuestos! a! escuchar! y! a! ayudar! en! lo! que! estuviese! en! su!
mano.!Gracias!a!todos,!aquí!me!tendréis!siempre!para!lo!que!necesitéis.!!
Por!último,!pero!no!por!ello!menos!especial!quiero!agradecer!a!mi!familia!
y!amigos!en!general!,!y!a!mis!padres!y!a!mi!amama!en!particular,!su!paciencia!e!
incondicional!apoyo!durante!estos!cuatro!años,!porque!sin!ellos!esto!no!hubiese!
sido!posible.!Laura,!por!supuesto!que!no!me!olvido!de!ti,!siempre!has!estado!ahí!
ayudándome!a!conseguir!lo!que!me!proponía!y!esta!última!vez!no!ha!sido!menos,!
siempre!te!estaré!agradecido!por!tu!comprensión!y!alegría,!solo!puedo!decirte:!!
?!UBI TU - IBI EGO?!
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