*Fo co espondence: chapuli@
uma.es
Compe ing in e es s: The
au ho s decla e ha no
compe ing in e es s exis .
Funding: See page 14
Recei ed: 15 Ma ch 2016
Accep ed: 08 Sep embe 2016
Published: 19 Sep embe 2016
Re iewing edi o : Ma ga e
Buckingham, Ins i u Pas eu ,
F ance
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a icle is dis ibu ed unde he
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edis ibu ion p o ided ha he
o iginal au ho and sou ce a e
c edi ed.
Condi ional dele ion o WT1 in he
sep um ans e sum mesenchyme causes
congeni al diaph agma ic he nia in mice
Ri a Ca mona
1,2
, Ana Can
˜e e
1,2
, Elena Cano
3
, Lau a A iza
1,2
, Anabel Rojas
4,5
,
Ramon Mun
˜oz-Cha
´puli
1,2
*
1
Depa men o Animal Biology, Uni e si y o Ma´laga, Ma´laga, Spain;
2
Andalusian
Cen e o Nanomedicine and Bio echnology (BIONAND), Ma´laga, Spain;
3
Max
Delb u€ck Cen e o Molecula Medicine, Be lin, Ge many;
4
Andalusian Cen e o
Molecula Biology and Regene a i e Medicine (CABIMER), Se illa, Spain;
5
Cen o de
In es igacio´ n Biome´ dica en Red de Diabe es y En e medades Me abo´ licas
Asociadas (CIBERDEM), Se illa, Spain
Abs ac Congeni al diaph agma ic he nia (CDH) is a se e e bi h de ec . W 1-null mouse
emb yos de elop CDH bu he mechanisms egula ed by WT1 a e unknown. We ha e gene a ed a
mu ine model wi h condi ional dele ion o WT1 in he la e al pla e mesode m, using he G2
enhance o he Ga a4 gene as a d i e . 80% o G2-Ga a4
C e
;W 1
l/ l
emb yos de eloped ypical
Bochdalek- ype CDH. We show ha he pos hepa ic mesenchymal pla e coelomic epi helium gi es
ise o a mesenchyme ha popula es he pleu ope i oneal olds isola ing he pleu al ca i ies be o e
he mig a ion o he somi ic myoblas s. This p ocess ails when W 1 is dele ed om his a ea.
Mu an emb yos show Raldh2 down egula ion in he la e al mesode m, bu no in he in e media e
mesode m. The mu an pheno ype was pa ially escued by e inoic acid ea men o he p egnan
emales. Replacemen o in e media e by la e al mesode m ecapi ula es he e olu iona y o igin o
he diaph agm in mammals. CDH migh hus be iewed as an e olu iona y a a ism.
DOI: 10.7554/eLi e.16009.001
In oduc ion
Congeni al diaph agma ic he nia (CDH) is a se e e bi h de ec , cha ac e ized by incomple e o ma-
ion o muscula iza ion o he diaph agm and, as a consequence, he nia ion o he s omach, in es-
ines, li e o spleen in o he pulmona y ca i ies, leading o pulmona y hypoplasia. CDH occu s
app oxima ely in 1 ou o 3000 bi hs, accoun ing o abou 8% o all se e e congeni al anomalies.
80–90% o all he cases a e pos e ola e al he nias, also known as Bochdalek- ype CDH, cha ac e ized
by a de ec in he pos e o (do sal in mice) la e al a ea o he diaph agm. In mos cases (>85%), his
de ec is loca ed a he le side (Pobe e al., 2010). The classical hypo hesis is ha Bochdalek CDH
is due o a ailu e o he usion o pleu ope i oneal olds (PPFs) wi h he sep um ans e sum (ST).
PPFs a e la e al ims o issue connec ing caudally wi h he neph ic idges and an e io ly wi h he ST
(Maye e al., 2011). Some au ho s desc ibe ha his usion a he occu s wi h he pos hepa ic mes-
enchymal pla e (PHMP), an accumula ion o mesenchymal cells de i ed om he ST and loca ed in
he pos e odo sal ma gin o he li e lobes (I i ani e al., 1984). Al hough all hese issues o m an
ana omical con inuum, we will e e o he pos e io , do sola e al a eas o he li e as PHMP, and
PPFs o he issue olds loca ed in he do sal pa o he coelomic ca i y, ma king he limi be ween
he pe i oneal and he pleu al ca i ies.
The clinical aspec s o CDH a e well s udied bu i s e iology is s ill poo ly known
(Klaassens e al., 2006). Animal models a e he e o e e y aluable in o de o in es iga e he
Ca mona e al. eLi e 2016;5:e16009. DOI: 10.7554/eLi e.16009 1 o 17
RESEARCH ARTICLE
cellula and molecula p ocesses leading o CDH in humans. Recen ly, i has been desc ibed in mice
ha dele ion o GATA4 in he PPFs mesenchyme using a P x1
C e
d i e leads o he de elopmen o
amuscula , weak a eas in he diaph agm and CDH (Me ell e al., 2015). This pape assumes ha
P x1 is exp essed in he mesenchyme o he pleu ope i oneal olds (PPFs) bu no in he ST, and in
ac , i sugges s ha he ST con ibu es minimally o he de ini i e diaph agm. Howe e , his epo
does no dis inguish be ween PPFs and PHMP and he pheno ype does no display diaph agma ic
discon inui ies, nei he does i show a p e alence on he le side, which a e common ea u es in
human CDH.
The e inoic acid (RA) signaling pa hway has also been in ol ed in diaph agma ic de elopmen . A
classical animal model o CDH consis s in he ea men o p egnan a s wi h ni o en, a subs ance
ha inhibi s he syn hesis o RA. I has been shown ha ni o en ea men leads o educed size o
he PPFs/PHMP, especially in he le side, dec easing cell p oli e a ion in his a ea (Clugs on and
G een, 2007;Clugs on e al., 2010) and also dec easing exp ession o WT1 and GATA4
(Dingeman e al., 2011,2013). RA ea men in his model pa ially escues he pulmona y hypopla-
sia de ec (Mon edonico e al., 2008;Sugimo o e al., 2008). The pheno ype o his animal model
is hus, qui e simila o he human one.
To da e, only a ew examples o CDH in human ha e been associa ed o mu a ions in WT1 locus,
such as he Denys-D ash, Meacham and WAGR synd omes (Sco e al., 2005;Su i e al., 2007;
An onius e al., 2008). WT1 is exp essed in he coelomic epi helium o he sep um ans e sum o
mouse emb yos by he s age E9.0. Loss o unc ion o he Wilms’ umo supp esso gene W 1 in
mice leads o de ec i e diaph agms (K eidbe g e al., 1993;Clugs on e al., 2006). Howe e , he
mechanism by which WT1 con ibu es o diaph agm de elopmen is s ill unknown.
To s udy he ole o WT1 in CDH we pe o med loss-o - unc ion expe imen s by condi ionally
inac i a ing he W 1 gene in he ST/PHMP/PPFs mesenchyme. The use o WT1 condi ional knockou
o e comes he ea ly emb yonic dea h caused by sys emic de iciency o WT1. We used a d i e based
on he G2 enhance o he Ga a4 gene (Rojas e al., 2005). This enhance d i es exp ession o
Ga a4 in he la e al pla e mesode m om he s age E7.5, and by he s age E9.5 is ac i e in he sep-
um ans e sum and p oepica dium, ceasing i s ac i i y by E12.5 The ac i i y o his enhance is
comple ely absen in he in e media e mesode m. Ou indings indica e ha WT1 is in ol ed in he
gene a ion o he mesenchyme o he ST/PHMP/PPFs con inuum h ough epi helial-mesenchymal
ansi ion and hey p o ide a no el pe spec i e on he genesis o he Bochdalek he nia and he e o-
lu iona y o igin o he diaph agm.
Resul s
The sep um ans e sum, pos hepa ic mesenchymal pla e and
pleu ope i oneal olds con ain he e ogeneous mesenchymal
popula ions
In no mal E10.5 emb yos, he pos e io and do sal ma gin o he li e shows an accumula ion o mes-
enchymal issue, which ex ends om he do sal mesen e ium o he li e o he la e al ips o he
lobes (Figu e 1). This mesenchymal laye is he PHMP desc ibed by I i ani (1984). The PPFs, by his
de elopmen al s age, appea as a pai o ou g ow hs o he body wall loca ed a bo h sides o he
lung buds. (Figu e 1A,B). They a e also cons i u ed o mesenchymal cells lined by he coelomic epi-
helium. The G2-Ga a4 enhance di ec ed LacZ epo e exp ession in bo h PPF and PHMP a E10.5
and E11.5 in he mouse emb yo (Figu e 1A). Howe e , no be a galac osidase ac i i y was obse ed
in he pos e io and medial pa o he sep um ans e sum, whe e he li e is connec ed wi h he
diges i e ac (as e isk in Figu e 1A), indica ing ha he G2 enhance is no ac i e in his speci ic
domain.
PHMP and PPFs a e no ana omically independen en i ies. The la e al ips o he PHMP appea
con inuous wi h he PPFs a he cephalic le el while hey a e sepa a ed mo e caudally (Figu e 1B).
The con inuous pa o he PHMP/PPFs ensemble appa en ly o ms i sel by he an e io g ow h o
he pe i oneal ecess, which sepa a es he li e sides om he body wall. Thus, he mesenchymal
popula ion o he ST o igina es he PHMP and also he mos cephalic po ion o he PPFs. The la e al
closu e o he pleu al ca i ies occu s be ween E11.5 and E12.5 by pos e io g ow h o he c escen -
Ca mona e al. eLi e 2016;5:e16009. DOI: 10.7554/eLi e.16009 2 o 17
Resea ch a icle De elopmen al Biology and S em Cells Human Biology and Medicine
Figu e 1. Cell lineages in he sep um ans e sum (ST), pos hepa ic mesenchymal pla e (PHMP) and pleu ope i oneal olds (PPF). (A) G2-LacZ emb yo
a he s age E11.5. The G2 enhance is ac i e in he li e (LI) meso helium and pos hepa ic mesenchymal pla e (PHMP). Howe e , he cen al a ea o he
sep um ans e sum shows no ac i i y o his enhance (as e isk). (B)W 1
C e
;R26R
LacZ
emb yo, E10.5. Cells om he WT1 lineage appea in blue. Se ies
o ans e se sec ions om cephalic (le ) o caudal ( igh ) le els. The an e io pe i oneal ecess (PR) is spli ing he li e om he body wall. The PHMP is
con inuous wi h he PPF a cephalic le els. No e he abundance o mesenchymal cells in he an e io PHMP and how his mesenchyme is less abundan
in pos e io le els. The PPF is con inuous wi h he enal idge (RR). The mesenchymal cells o all hese s uc u es belong o a WT1-exp essing cell
lineage. (C)W 1
LacZ
emb yo, E11.5. Ac i a ion o he W 1 epo e can be seen in he li e meso helium. PHMP, PPF and RR. The RRs appea a he le el
o he s omach (STO). OE: oesophagus. (D)W 1
C e
;R26R
YFP
emb yo, E11.5. Cells om he WT1-exp essing cell lineage (g een) a e mo e abundan a
Figu e 1 con inued on nex page
Ca mona e al. eLi e 2016;5:e16009. DOI: 10.7554/eLi e.16009 3 o 17
Resea ch a icle De elopmen al Biology and S em Cells Human Biology and Medicine
like con inuum o med by he PHMP and he PPFs, a p ocess which is pa allel o he as g ow h o
he li e lobes du ing hese s ages (Figu e 1B,C).
As seen in he W 1
C e
labeled cells, he PPFs con inue pos e io ly as he neph ic idges, he p o-
geni o issue o he mesoneph os (Figu e 1B and C). A loose mesenchymal issue appea s in he
PPF be ween he mo e compac a angemen o he cells in he PHMP and in he enal idges, bu
he commissu al a ea o he PPF is always cons i u ed by compac mesenchyme simila o ha om
he PHMP (a ow in Figu e 2C). All hese mesenchymal popula ions a e de i ed om a WT1-
exp essing cell lineage, as shown by wo models, he W 1
C e
;R26R
LacZ
(Figu e 1B) and he W 1
C e
;
R26R
YFP
(Figu e 1D). Exp ession o he W 1 gene is also e ealed in he PPF and PHMP mesen-
chyme by he WT1-LacZ epo e and by immunohis ochemis y (Figu es 1C and 3A, espec i ely).
Epi helial and mesenchymal cells o he PHMP and mos an e io PPFs de i e om a cell lineage
in which Ga a4 exp ession is d i en by he G2 enhance . This is demons a ed by he exp ession o
YFP in G2-Ga a4
C e
;R26R
YFP
emb yos, and suppo s he exis ence o a p ocess o cell mig a ion om
PHMP o PPFs (Figu e 1E,F). Howe e , he neph ic idges and he mesoneph os de i ed om hem
show no YFP+ cells in his model, indica ing ha he p ogeni o s o hese issues de i ed om a di -
e en cell lineage. Thus, G2-Ga a4 lineage acing allows o es ablishing a well-de ined limi
be ween wo la e al mesode mal e i o ies ha we will call he ein he G2
+
and he G2
-
domains
(see igu e 3B,C). In e es ingly, he gonad and he ad enal s oma we e YFP+ (Figu e 1G), and his
can be ela ed wi h he ea ly exp ession o GATA4 in he coelomic epi helium, which will co e he
geni al bu no he enal idges (Figu e 1H). Inciden ally, he GATA4 posi i e cells om he cen al
pa o he ST do no exp ess YFP in G2-Ga a4
C e
;R26R
YFP
emb yos (Figu e 1F), an obse a ion con-
sis en wi h he lack o epo e ac i i y in he G2- Ga a4
LacZ
emb yos (Figu e 1A). These da a e eal
a signi ican he e ogenei y be ween cen al and la e al mesenchymal popula ions in he ST.
A clea asymme y was obse ed in he dis ibu ion o PHMP mesenchyme in emb yos o E10.5-
E12.5 (Figu e 1D,E). Mesenchymal cells we e always mo e abundan in he igh PHMP han in he
le , which migh a o he he nia ion in he le side. We es ima ed, by image analysis, he olume
aken up by he YFP+ cells in he igh and le PHMP o ou G2-Ga a4
C e
; R26R
YFP
emb yos. The R/
L a io o hese olumes was 1,21 and 1,45 in wo E11.5 emb yos, and 1,53 and 1,61 in wo E12,5
emb yos (mean = 1,45; S.E.M = 0,086). The - es o one sample compa ed wi h an expec ed alue
= 1 (symme ical dis ibu ion) ga e a esul o 5,21 (p- alue=0,0069 o h ee d.o. ). Thus, he igh
PHMP con ains abou 50% mo e cells om he G2-Ga a4 lineage han he le one.
WT1 condi ional knockou mice display Bochdalek’s he nia
This is a basically desc ip i e s udy o he pheno ype o mouse emb yos wi h condi ional dele ion o
W 1 in la e al mesode m. We ha e included in he s udy 36 ou 104 mu an emb yos de ec ed by
geno yping. The es o hem had been p e iously used o a s udy o he ca diac pheno ype
(Cano e al., 2016).
To unde s and he ole o WT1 posi i e cells in he de elopmen o diaph agm, we condi ionally
inac i a ed W 1 gene using he desc ibed G2-Ga a4
C e
line. Dele ion o WT1 in he PHMP/PPFs con-
inuum, i.e. in he G2
+
domain, causes a pheno ype cha ac e ized by de ec s in he in low ac o
he hea , co ona y essels (Cano e al., 2016) and, impo an ly, Bochdalek’s he nia, mos equen ly
Figu e 1 con inued
he igh PHMP (R) as compa ed wi h he le one (L). No e he lack o YFP+ cells in he cen al a ea o he ST. AO: ao a; LU: lung. (E) G2-Ga a4
C e
;
R26R
YFP
emb yo, E11.5. Cells om he lineage exp essing GATA4 unde he con ol o he G2 enhance a e s ained in g een, and RALDH2 is s ained in
ed. Mos cells in he PHMP and PPFs belong o he G2-Ga a4 lineage, bu hey a e e y sca ce in he cen al a ea o he ST. Abundan YFP+ cells a e
p esen do sally o he ao a, and a ound he no ocho d (NC). (F) G2-Ga a4
C e
;R26R
YFP
emb yo, E11.5. GATA4 immunos aining in ed. Colocaliza ion o
GATA4 and YFP is obse ed in he PHMP and PPF, bu he cen al a ea o he ST shows GATA4 exp ession no d i en by he G2 enhance . (G) G2-
Ga a4
C e
; R26R
YFP
emb yo, E11.5. Smoo h muscle alpha-ac in is s ained in ed. Gonads (GON) and ad enals (AD) con ain a la ge numbe o G2-Ga a4
lineage cells, bu hey a e sca ce in o he mesoneph os (MN). Da a eused, wi h pe mission, om Figu e 4A, Mun
˜oz-Cha
´puli e al. De elopmen al
Dynamics, Special Issue: Mechanisms o Mo phogenesis, 245:307–322 (2016). Ó2015 Wiley Pe iodicals, Inc. (H) Immunolocaliza ion o GATA4 ( ed) in an
E10.5 G2-Ga a4
C e
; R26R
YFP
emb yo. The G2 lineage is shown in g een. The e is a sha p bounda y (a ow) be ween he meso helial cells exp essing
GATA4 o he geni al idge (GR), close o he do sal mesen e y (DM), and he meso helial cells o he enal idges (RR), which a e GATA4-. AO: ao a;
OE: oesophagus.
DOI: 10.7554/eLi e.16009.002
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Resea ch a icle De elopmen al Biology and S em Cells Human Biology and Medicine
loca ed in he le side (Figu e 2). The geno ypes o he emb yos ob ained a di e en ages show
he expec ed pe cen age o mu an s a all emb yonic s ages (abou 25%) excep o he s age
E15.5, when we only ob ained ou mu an s among 60 emb yos (Table 1). The numbe o emb yos
s udied in olde s ages (42 emb yos, nine mu an s, 21,4%), was no enough o con i m i he mu a-
ion causes some le hali y in la e ges a ion.
We analyzed WT1 mu an emb yos a di e en s ages o de elopmen . De ec in he PHMP in
G2-Ga a4
C e
;W 1
l/ l
mu an emb yos could be obse ed as ea ly as E10.5, since less mesenchymal
cells a e p esen be ween he coelomic epi helium and he hepa oblas s when compa ed wi h con-
ol li e ma es (Figu e 2A–D). A la e s ages, he closu e o he pleu al ca i y by g ow h o he ST/
PPFs c escen is delayed wi h espec o he con ols, and he mesenchymal cells o he PHMP a e
Figu e 2. Pheno ype o he G2-Ga a4
C e
; W 1
l/ l
emb yos. (A–D) Wild ype (A,C) and mu an (B,D), E10.5 li e ma es. The la ge amoun o mesenchymal
cells in he pos hepa ic mesenchymal pla e (PHMP) is e iden in he wild ype, especially in he igh side. Se ial sec ions co esponding o he
connec ion be ween he PHMP and he pleu ope i oneal olds (PPF) a e shown in C. No e he p esence o compac mesenchyme in he PHMP and also
in he closes pa o he PPF (a ow in C). A co esponding sec ion o he mu an in shown in D. No e he lack o PHMP, he coelomic epi helium lying
di ec ly on he hepa ic issue (a ows in D) and he limi o he sep um ans e sum (ST) mesenchymal cells (a owhead in D), which do no ex end
la e ally. (E) Se ial sec ions o a G2-Ga a4
C e
; W 1
l/ l
E11.5 emb yo a le els equi alen o hose shown in Figu e 1C. Despi e he p esence o no mal
PHMP in he an e io pa , he pos e io a eas o he li e lack o la e al mesenchymal cells (a ow). The mesenchyme is es ic ed o he cen al ST.
Renal idges (RR) appea a a le el co esponding o he en ance o he oesophagus (OE) in o he ST. MG: mesogas ium. (F) Wild ype E13.5 emb yo
showing comple e isola ion o he pleu al ca i ies by he pleu ope i oneal memb anes ha cons i u e he main pa o he diaph agm (D). (G) G2-
Ga a4
C e
; W 1
l/ l
E14.5 emb yo a eigh di e en le els showing le diaph agma ic de ec wi h he nia ion o he le li e lobe (LI) in o he pleu al ca i y
and se e e hypoplasia o he le lung (LL). Ec opic muscle appea in he medias inum (MM). Ad enals (AD), mesoneph os (MN), gonads (GON) and
spleen (SP) appea no mal. LU: lungs; H: hea ; RL: igh lung; STO: s omach.
DOI: 10.7554/eLi e.16009.003
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Resea ch a icle De elopmen al Biology and S em Cells Human Biology and Medicine

loca ed mo e medially, no in he la e al ips o he li e lobes. This is mo e e iden a he le side
(a ow in Figu e 2E). Thus, hese cells canno mig a e owa ds he PPFs p ecluding caudal g ow h o
he la e al commissu es o he pleu ope i oneal sep a and closu e o he pleu al ca i ies.
Fi e E12.5 mu an emb yos s udied al eady showed s ong educ ion o he PHMP size as com-
pa ed wi h con ol li e ma es, bu he diaph agma ic de ec o he WT1 condi ional knockou
emb yos became clea ly appa en by E13.5, when he pleu al ca i ies a e almos comple ely isola ed
in wild ype emb yos (Figu e 2F). O 23 mu an emb yos analyzed by he s ages E13.5 o olde , only
ou o hem showed no mal de elopmen o he diaph agm, while he es o emb yos showed di -
e en deg ees o diaph agma ic de ec s (Table 1). Th ee o he emb yos showed hin, memb anous
diaph agm in he le side and 16 showed a wide de ec in he diaph agm, equen ly wi h he nia ion
o he li e in o he le pleu al ca i y and hypoplasia o he le lung (Figu e 2G). One o hem
(E13.5) showed he de ec only a he igh side and he CDH was p esen in he le side in he es
o hem. Thus, abou 80% o he E13.5 o olde mu an emb yos showed abno mal diaph agma ic
de elopmen .
Renal idges appea well de eloped in mu an E11.5 emb yos a a le el co esponding o he
en ance o he oesophagus in o he ST (Figu e 2E). The p esence o in e media e mesode m in
hese ho acic enal idges was con i med by ISH o Pax2 (Figu e 4). In wild ype emb yos o he
same age ho acic enal idges can also be seen, bu loca ed mo e caudally, a he le el o he s om-
ach (Figu e 1C).
Figu e 3. WT1, GATA4 and RALDH2 exp ession in G2-Ga a4
C e
; W 1
l/ l
emb yos. (A,B) Immunolocaliza ion o WT1 (A) and GATA4 (B) in wild ype E11.5
emb yos. The pos hepa ic mesenchymal pla e (PHMP) and pleu ope i oneal olds (PPF) show abundan posi i e cells. Howe e , he WT1
immuno eac i e cells a e p esen in he base o he PPF and he adjacen body wall, whe e GATA4+ cells a e absen . This di e ence de ines he G2+
and he G2- domains. Da a in A eused, wi h pe mission, om Figu e 3E, Mun
˜oz-Cha
´puli e al. De elopmen al Dynamics, Special Issue: Mechanisms o
Mo phogenesis, 245:307–322 (2016). Ó2015 Wiley Pe iodicals, Inc. (C–D) Immunolocaliza ion o WT1 (C) and GATA4 (D) in G2-Ga a4
C e
; W 1
l/ l
E11.5
emb yos. The PHMP and he G2+ domain o he PPF show GATA4+ cells bu no WT1+ cells due o he condi ional dele ion o his gene in he G2+
domain. WT1 exp ession emains in he G2- domain o he base o he PPF and body wall. GATA4 exp ession is no mal in mos cephalic PPF, bu
immuno eac i i y disappea s in mo e caudal a eas o PPF, as shown in D’ and D’’. (E–H) Immunolocaliza ion o RALDH2 in E10.5 (E,F) and E11.5 (G,H)
wild ype and G2-Ga a4
C e
; W 1
l/ l
mu an emb yos. RALDH2 immuno eac i i y is high in PPF and he adjacen body walls. No e s ong immuno eac i i y
in he enal idges (RR) o he E11.5 mu an emb yo. Howe e , RALDH2 immuno eac i i y is educed o absen in he PHMP and li e meso helium o
he mu an emb yos, as compa ed wi h he con ols. CV: ca dinal eins; LU: lungs.
DOI: 10.7554/eLi e.16009.004
Ca mona e al. eLi e 2016;5:e16009. DOI: 10.7554/eLi e.16009 6 o 17
Resea ch a icle De elopmen al Biology and S em Cells Human Biology and Medicine
The sha p bounda y be ween he abo e explained G2
+
and G2

domains was clea ly obse ed
by he immunolocaliza ion o WT1 and GATA4 p o ein in G2-Ga a4
C e
;W 1
l/ l
mu an emb yos (Fig-
u e 3). WT1 immuno eac i i y is s ong in he PHMP and PPF o he wild ype emb yos and localizes
in he coelomic epi helium and, wi h a lowe in ensi y, in mesenchymal cells (Figu e 3A). Howe e ,
WT1 is clea ly down egula ed in he G2
+
domain o he mu an emb yos indica ing an e icien
Table 1. Numbe o emb yos s udied and % o G2-Ga a4
C e
;W 1
l/ l
ound. The i e E12.5 emb yos showed abno mal pos hepa ic
mesenchymal pla es a he le side, bu hey we e no compu ed o he diagnosis o he diaph agma ic he nia, ha was pe o med
only on he 23 emb yos a he s ages E13.5 o olde .
S age
Numbe o emb yos
s udied
Numbe and% G2C e+;
W 1 l/ l
Emb yos analyzed o
pheno ype
No
de ec
Mild
de ec
Diaph agma ic
he nia
E10–10.5 42 11 (26,2%) 3
E11.5 89 21 (23,6%) 5
E12.5 76 21 (27,6%) 5 (5)
E13,5 65 19 (27,1%) 13 1 1 11*
E14,5 53 17 (32,1%) 4 2 2
E15,5 60 4 (6,7%) 4 2 2
E16,5 21 4 (19,0%) 1 1
E17,5–
18,5
21 5 (23,4%) 1 1
To al 427 102 (23,6%) 36 4 (17,4%) 3 (13,0%) 16 (69,6%)
*We ha e included se en emb yos om emales ed wi h con ol die in he RA- escue expe imen . One emb yo showed CDH only a he igh side.
DOI: 10.7554/eLi e.16009.005
Figu e 4. Localiza ion o Pax2 exp ession by in si u hyb idiza ion. (A) Con ol E11.5 emb yo. Pax2 is exp essed in he neu al ube (NT). The
pleu ope i oneal olds (PPF) lack o Pax2 exp ession. (B) G2-Ga a4
C e
; W 1
l/ l
E11.5 emb yo. Pax2 is exp essed in he PPF a he le el o he lung buds
(LU). The exp ession is s onge in he ubula s uc u es ha a e de eloping in he le PPF (inse ).
DOI: 10.7554/eLi e.16009.006
Ca mona e al. eLi e 2016;5:e16009. DOI: 10.7554/eLi e.16009 7 o 17
Resea ch a icle De elopmen al Biology and S em Cells Human Biology and Medicine
excision o he WT1 loxed allele (Figu e 3C). Immunolocaliza ion o GATA4 p o ein ma ked he bo -
de be ween bo h G2 domains, con i ming he absence o endogenous GATA4 p o ein and he inac-
i i y o he G2 enhance in he G2
-
domain. (Figu e 3B,D). The p esence o GATA4+ cells in he
mo e cephalic pa o he PPF suppo s a mig a ion o PHMP cells owa ds he PPFs.
Since WT1 is in ol ed in epi helial-mesenchymal ansi ion (EMT) o he epica dium by ep ession
o E-cadhe in and ac i a ion o Snail1 (Ma ı´nez-Es ada e al., 2010), we checked he exp ession o
E-cadhe in in he de eloping PHMP o G2-Ga a4
C e
;W 1
l/ l
mu an emb yos. Con ol E11.5 emb yos
show an E-cadhe in nega i e coelomic epi helium o e a laye o mesenchymal cells in he de elop-
ing PHMP (Figu e 5A–D). Howe e , mu an emb yos show exp ession o E-cadhe in in he epi he-
lium o he co esponding a ea and lack o E-cadhe in nega i e mesenchymal cells (Figu e 5E–H).
Sys emic WT1 loss o unc ion leads o comple e lack o PHMP
We ha e compa ed he G2-Ga a4
C e
;W 1
l/ l
CDH pheno ype wi h ha displayed by he sys emic
WT1 knockou mouse emb yos, which do no su i e beyond E13.5. Despi e he ea ly emb yonic
dea h o hese models, we ha e obse ed a mo e se e e de ec , wi h a comple e lack o PHMP and
displaying, in some cases, pe sis en ho acic neph ic idges in E12.5 and E13.5 emb yos (Figu e 6).
In hese emb yos only he cen al pa o he ST is conse ed, i.e. he a ea whe e he G2 enhance is
no ac i e, as shown in Figu e 1E.
Re inoic acid pa ially escues he de ec s in he diaph agm o WT1
condi ional knockou emb yos
The e inoic acid (RA) signaling pa hway has been in ol ed in he o ma ion o diaph agm. To es
whe he RA signaling migh be dis u bed in ou CDH model, we checked he exp ession o RALDH2
by immuno luo escence in G2-Ga a4
C e
; W 1
l/ l
mu an and con ol emb yos. RALDH2 exp ession is
ound in he PHMP and PPF o wild ype E10.5-E11.5 emb yos (Figu e 3E,G). The PPF o he G2-
Figu e 5. Immunolocaliza ion o E-cadhe in in he le pos hepa ic mesenchymal pla e (PHMP) o ou E11.5 con ol emb yos (A–D) and ou E11.5 G2-
Ga a4
C e
; W 1
l/ l
emb yos (E–H). E-cadhe in appea s up egula ed in he coelomic epi helium o he PHMP o he mu an emb yos (a ows in E–H). In
wild ype emb yos he coelomic epi helium o he PHMP lacks o E-cadhe in exp ession (a ows in A–D) and co e s a laye o E-cadhe in-nega i e
mesenchymal cells (as e isks).
DOI: 10.7554/eLi e.16009.007
Ca mona e al. eLi e 2016;5:e16009. DOI: 10.7554/eLi e.16009 8 o 17
Resea ch a icle De elopmen al Biology and S em Cells Human Biology and Medicine
Ga a4
C e
; W 1
l/ l
mu an emb yos show an inc eased immuno eac i i y o RALDH2 bu we ound a
educed o absen exp ession o his enzyme in he PHMP (Figu e 3F,H). This p obably e lec s, as
discussed below, he pe sis ence o he in e media e mesode m o he neph ic idges in o he PPF.
In ac , RALDH2 is no mally exp essed a high le els in he in e media e mesode m e en in sys emic
WT1 knockou emb yos, which also show dec eased RALDH2 immuno eac i i y in he li e meso-
helium (Figu e 6C,D).
RA ea men pa ially escues he pulmona y pheno ype and p omo es al eologenesis in he
ni o en model o induced CDH in a s (Mon edonico e al., 2008;Sugimo o e al., 2008). In o de
o check he e ec s o a RA ea men on ou mu an pheno ype, in a i s expe imen we ed p eg-
nan emales wi h a RA-supplemen ed die . Fo e hical easons we decided o limi he s udy o ou
li e s in which we de ec ed eigh G2-Ga a4
C e
; W 1
l/ l
mu an emb yos. Fi e o hem (62,5%) dis-
played a comple e sep a ion o he pleu al ca i y by E15.5, i.e. abou h ee imes he p opo ion o
no mal pheno ypes ound in he non- ea ed mu an s (Z es = 2,09, wo- ailed p- alue= 0,037). Con-
ol li e ma es we e no a ec ed by he RA- ea men .
Figu e 6. Diaph agma ic pheno ype in sys emic WT1 loss o unc ion. (A,B) WT1-/- E13.5 emb yos. The comple e lack o pos hepa ic mesenchymal
pla e is e iden , mesenchymal cells a e absen om he la e al ends o he li e lobes and emain es ic ed o he cen al a ea o he sep um
ans e sum (ST), close o he oesophagus (OE). The pleu al ca i ies a e con inuous wi h he pe i oneal ca i y. No e he enal idges (RR) pe sis ing a
ho acic le el, and he abno mal adhesion o he igh li e lobe wi h he body wall (a ow in B). AO: ao a; LI: li e ; LU: lungs. (C,D) Immunolocaliza ion
o RALDH2 in WT1-/- emb yos, E11.5 (A) and E12.5 (B). RALDH2 immuno eac i i y is s ong in he enal idges (RR), bu weak o absen in he li e (LI)
meso helium (MES). AO: ao a; LU: lung.
DOI: 10.7554/eLi e.16009.008
Ca mona e al. eLi e 2016;5:e16009. DOI: 10.7554/eLi e.16009 9 o 17
Resea ch a icle De elopmen al Biology and S em Cells Human Biology and Medicine
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