This is an Accep ed Manusc ip o an a icle published by Wiley-Blackwell in Biology o he
Cell, 106 (3), 83 – 96 on Ma ch 2014, a ailable a : h ps://doi.o g/10.1111/boc.201300078 .
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Dab1 and eelin pa icipa e in a common signal
pa hway ha con ols in es inal c yp / illus uni
dynamics
Ma
´
ıa
D.
V
a
´
zquez-Ca e e
o,
Pablo
Ga
c
´
ıa-Mi anda,
Ma
´
ıa
L.
Calonge,
Ma
ı
´
a
J.
Pe al
1
and
Anunciacio
´
n
A.
Ilundain
Depa amen o de Fisiologı´a, Facul ad de Fa macia, Uni e sidad de Se illa,
Spain
Backg ound in o ma ion. The myo ib oblas s placed unde nea h
he epi helium o he oden small in es ine exp ess eelin, and
he eelin absence modi ies bo h he mo phology and he cell
enewal p ocesses o he c yp – illus uni . In he de eloping
cen al ne ous sys em, he eelin e ec s a e media ed by he
disabled-1 (Dab1) p o ein. The p esen wo k explo es whe he
Dab1 media es he eelin con ol o he c yp – illus uni
dynamics by examining in he mouse small in es ine he
consequences o he absence o (i) Dab1 (sc amble mu a ion)
on c yp – illus uni cell enewal p ocesses and mo phology and
(ii) eelin ( eele mu a ion) on he in es inal exp ession o Dab1.
Resul s. The e ec s o he sc amble mu a ion on he c yp –
illus uni enewal p ocesses a e ema kably simila o hose
caused by he lack o eelin. Thus, bo h mu a ions signi ican ly
educe epi helial cell p oli e a ion, mig a ion and apop osis, and
he numbe o Pane h cells; a ec he mo phology o he illus,
and expand he in e cellula space o he adhe ens junc ions
and desmosomes. The Wes e n blo assays e eal ha he
Dab1 iso o m p esen in he en e ocy es has a molecula weigh
o ~63 kDa and ha in he b ain o ~82 kDa. They also e eal
ha he absence o eelin inc eases Dab1 abundance in bo h
b ain and en e ocy es.
Conclusions. All oge he , he cu en indings link eelin wi h
Dab1 and sugges ha Dab1 unc ions downs eam o eelin
ac ion on he homeos asis o he c yp – illus uni .
In oduc ion
The homeos asis o he epi helium o he small in es ine is p ese ed
h ough he s ic egula- ion o cell p oli e a ion, g ow h a es ,
mig a ion/
di e en ia ion and apop osis. Epi helial cells o igi- na e om
mul ipo en s em/p ogeni o cells, loca ed
nea he bo om o each c yp o
Liebe ku¨ hn, and cell
cycle a es s when cell p ogeni o s each he c yp –
illus junc ion. As he p ogeny mig a e ou o he c yp owa ds he illus
ip, hey di e en ia e in
abso p i e en e ocy es, ho mone-sec e ing
en e oen-
doc ine cells, opioid-p oducing b ush cells, mic o old
cells and
mucus-p oducing Goble cells, and e en u-
ally shed in o he lumen wi hin
less han a week ( an de Flie and Cle e s, 2009). The an ibac e-
ial
pep ide-sec e ing Pane h cells also a ise om he
mul ipo en c yp s em
cells, bu hey mig a e o-
wa ds he bo om o he c yp , whe e hey su i e
o a ound 6–8 weeks be o e being elimina ed by phago- cy osis (Po e e al.,
2002). Spon aneous apop osis in he c yp s is a e and i may se e o
emo e de ec- i e/inju ed p ogeny cells and senescen Pane h cells
(Po en,
1997).
Epi helial cell u no e and mo phogenesis o he small in es ine a e
con olled by cell–cell and cell-unde lying basemen memb ane
in e ac ions (Gumbine , 1996). The na u e o cell–basemen memb ane
in e ac ions and hei in acellula
p ocessing emain la gely unde ined.
Benea h he epi helia a e he myo ib oblas s, which o ches a e
se e al
unc ions such as he con ol o epi helial
u no e , issue epai ,
in lamma ion and he immune esponse. They do so by sec e ing a ious
subs ances
o he ex acellula ma ix as well as exp essing ecep-
o s o
many o hem, allowing in o ma ion low o and om he in es inal epi helium
and he ex acel- lula ma ix (Andoh e al., 2007; Mi lin e al., 2011).
We
epo ed ha he myo ib oblas s placed unde nea h he epi helium o
he oden small in es-
ine exp ess eelin (Ga c´ıa-Mi anda e al., 2010)
and ha eelin absence ( eele mu a ion) modi ies bo h he mo phology and
he cell enewal p ocesses o
he c yp – illus uni (Ga c´ıa-Mi anda e al.,
2012, 2013). Wi hin he in es inal mucosa, eelin exp es-
sion is es ic ed
o he myo ib oblas s, bu bo h ep- i helial cells and myo ib oblas s exp ess
he eelin e ec o p o ein disabled-1 (Dab1) (Ga c´ıa-Mi anda e al., 2010).
In he de eloping cen al ne ous sys-
em, he binding o eelin o i s
ecep o s esul s in
y osine phospho yla ion o Dab1 and in ac i a ion o
mul iple downs eam signalling pa hway/s, esul - ing in cy oskele on
emodelling and p ecise neu onal
posi ioning (Howell e al., 1999).
Whe eas he in-
o ma ion on he cell signalling cascades ini ia ed by he
eelin/Dab1 signalling sys em in b ain is p o use,
he s udies on he ole o
eelin/Dab1 in non-neu al
issues a e sca ce. The pu pose o he cu en
wo k was o examine whe he Dab1 media es he obse ed eelin e ec s on
he c yp – illus uni dynamics. To
achie e his, we ha e examined in mice
small in-
es ine he consequences o (i) Dab1 gene de iciency (sc amble
mice) on he mo phology and on epi he- lial cell p oli e a ion, mig a ion,
di e en ia ion and apop osis and (ii) he absence o eelin ( eele mice)
on
Dab1 exp ession in he en e ocy es. We ha e used
sc amble mice because
hey exhibi a pheno ype in- dis inguishable om ha o he eele mice
(Swee
e al., 1996; Sheldon e al., 1997) e en hough he
sc amble mice
p oduce abou 5% o he no mal le el
o Dab1 p o ein (Sheldon e al., 1997).
A p elimina y epo o some o hese esul s was published as an abs ac
(Va´zquez-Ca e e o e al.,
2012).
Resul s
Weigh s o he body and small in es ine
Dab1-de icien mice (sc amble ) a e used in his s udy
o elucida e whe he Dab1
media es he eelin-induced e ec s on he c yp – illus uni homeos a
sis
p e iously epo ed (Ga cia-Mi anda e al., 2013).
The s udy was s a ed e alua ing
body and in es inal
weigh s, as well as in es inal leng h, o 15 and 60
day-old
sc amble and con ol mice. The da a sum-
ma ised in Figu e 1 e eal ha he
body weigh o
he sc amble mice is simila o ha o he con ol
li e ma es du ing
he suckling pe iod, bu smalle
in he 60 day-old mice. The sc amble mu a ion
also
dec eases bo h he weigh and leng h o he small in es ine a he wo ages
es ed.
In es inal mo phology
In o de o de e mine whe he he sc amble mu a ion
a ec s he
mo phology o he epi helium o he small
in es ine, he heigh and wid h
o he illi and he dep h and diame e o he c yp s we e measu ed in
he jejunum and ileum o 15 and 60 day-old con ol
and sc amble mice.
The esul s summa ised in Table 1 show ha he sc amble mu a ion
signi ican ly a ec s
he mo phology o he illi bu no ha o he c yp s. As
compa ed wi h con ol mice, he sc amble illi
a e sho e , mainly in he
jejunum, a he wo ages es ed, and hinne only in he 60 day-old
mice.
As he absence o Dab1 educes he illus leng h and simila ly in he
absence o eelin, he cell e-
newal p ocesses o he c yp – illus uni a e
modi ied
(Ga c´ıa-Mi anda e al., 2012, 2013) we decided o
examine he
e ec s o he sc amble mu a ion on ep- i helial cells o de e mine whe he
his mu a ion also a ec s epi helial cell p oli e a ion, mig a ion, di e -
en ia ion and apop osis.
Cell p oli e a ion and mig a ion a es in he epi helium o he small
in es ine o con ol and sc amble mice
To assess he e ec s o he sc amble mu a ion on ep-
i helial cell p oli e a ion and mig a ion a es, he in- co po a ion o B dU in o
DNA was measu ed in he
jejunum and ileum o 15 and 60 day-old con ol
and sc amble mice as desc ibed in he Ma e ials and
Me hods sec ion.
Cell p oli e a ion was de e mined by de ec ing B dU-ma ked cells 90
min a e he in ape i oneal injec ion o he ma ke . The esul s a e gi en
in
Figu e 2 and show ha , in bo h ypes o mice and a he wo ages es ed,
B dU is only obse ed in nuclei
o he c yp cells. The quan i ica ion o he

±
±
±
ma ked
nuclei e eals ha he cell p oli e a ion a e is g ea e
in he 60 day-
old mice han in he suckling mice in
bo h con ol and sc amble mice. The
mu a ion sig- ni ican ly educes he epi helial cell p oli e a ion a e
a he wo
ages and in es inal egions examined by 26 2%.
To e alua e he cell mig a ion a e, in es inal
B dU-ma ked cells we e
de ec ed 32 h a e injec ion o he ma ke and he esul s a e shown in
Figu e 3.
In bo h ypes o mice, he cell mig a ion a e along
he illi is
g ea e in he 60 day-old mice han in he
suckling mice and i is
signi ican ly educed by he
mu a ion in all he expe imen al condi ions
es ed by
34 ± 4%.
Cell apop osis in he epi helium o he small in es ine o con ol and
sc amble mice
Cell apop osis was e alua ed by immunological de ec ion o clea ed Caspase-3
in he jejunum and ileum
o 15 and 60 day-old con ol and sc amble mice. The
an i-
clea ed Caspase-3 an ibody de ec s on a Wes -
e n blo a band o 17 kDa ha
is indica i e o cell
apop osis (see Figu e 4A). The immunohis ochem- is y
assay e eals ha in bo h con ol and sc amble
mice he apop o ic cells a e
mainly obse ed a he illus (Figu e 4C). In con ol mice, bo h he den-
si y o
he 17-kDa band (Figu e 4A) and he numbe
o apop o ic cells (Figu e 4B) a e
highe in he 60
day-old han in he 15 day-old mice. The sc amble mu a ion
dec eases he densi y o he band (42 5%
dec ease) and he numbe o apop o ic
cells (59 4%
dec ease) in all he expe imen al condi ions es ed.
Cell di e en ia ion in he epi helium o he small in es ine o con ol
and sc amble mice
Epi helial cell di e en ia ion was e alua ed by measu ing he numbe o Goble
and Pane h cells in he jejunum and ileum o 15 and 60 day-old con ol and
sc amble mice.
Goble cells we e iden i ied by he pe iodic acid-
Shi (PAS)
±
s aining sys em, as desc ibed in Ma e ials
and Me hods sec ion, and Figu e 5
shows ha he
numbe o PAS posi i e cells inc eases wi h he age in bo h ypes o
mice. The sc amble mu a ion dec eases
he numbe o Goble cells, mainly in he
15 day-old jejunum. No signi ican di e ences a e obse ed in
he 60 day-old
jejunum.
Pane h cells we e quan i ied by immuno-de ec ion
o lysozyme and he
esul s a e summa ised in Figu e 6. They e eal ha age inc eases he num-
be o Pane h cells in bo h con ol and sc amble mice
and ha he mu a ion
educes hei numbe a he wo ages and in es inal egions examined by
29,3%.
Elec on mic oscopy s udies
The esul s discussed so a e eal ha he sc amble mu a ion educes epi helial
cell p oli e a ion, mig a- ion, di e en ia ion and apop osis. Because in e cellula
junc ions con ol hese p ocesses (Gumbine , 1996; He e´, 2009) and he eele
mu a ion a ec s hose junc ions in he in es inal epi helium (Ga c´ıa-
Mi anda e al.,
2013), he e ec s o he sc amble mu-
a ion on he cell- o-cell junc ions we e
examined a he elec on mic oscopy. In he in es inal epi helium,
he in e cellula
junc ions a e loca ed on he apical side o he la e al cell memb ane and o m he
api-
cal junc ional complex ha comp ises igh junc ions (TJ), adhe ens junc ions
(AJ) and desmosomes. The mic opho og aphs o Figu e 7 e eal ha he TJ ap-
pea no mal, bu he in e cellula space o bo h he AJ
and desmosomes is signi ican ly
wide in he sc amble
han in he con ol mice.
Immunolocalisa ion o E-cadhe in and
β
-ca enin in he epi helium o
he small in es ine o con ol and sc amble mice
Since he elec on mic oscopy s udies e ealed ha
he sc amble mice p esen AJ
wi h wide in e cel
lula space han he con ol mice, he cell loca ion o E-
cadhe in in he epi helium o he con ol and
sc amble small in es ine was de ec ed by immuno- his ochemis y (Figu e 8). β-
ca enin associa es wi h he cy osolic domain o he E-cadhe in and egula es
se e al
cell p ocesses. As i s cell memb ane loca ion in
pa depends on he amoun o E-
cadhe in p esen in
he cell memb ane, β-ca enin localisa ion was also in
es iga ed by
immunos aining in bo h ypes o mice
(Figu e 8). The speci ic signal p oduced by he
an i-E-
cadhe in an ibody is seen a he la e al memb ane o
he epi helial cells in bo h
con ol and sc amble mice.
β-Ca enin-speci ic s aining is also concen a ed a he
la e al cell memb ane. No signi ican di e ences be- ween he wo ypes o mice a e
obse ed, indica ing ha he mu a ion does no modi y he cell loca ion o ei he
p o ein. Immuno eac i e signal was no seen
in he absence o he p ima y
an ibodies (da a no shown).
Dab1 p o ein in en e ocy es isola ed om con ol, eele and sc amble mice
The las se o expe imen s was designed o (i) de e -
mine he Dab1
iso o m exp essed in mice en e ocy es,( ii)
compa e he in es inal iso o m
wi h ha exp essed
in b ain and (iii) de e mine whe he eelin modi-
ies he in es inal exp ession o Dab1. This was done by Wes e n blo
assays using an an i-Dab1 an ibody aised agains he C e minus o
Dab1 ha ecognises
all Dab1 iso o ms. The speci ici y o he an ibody was
e i ied using b ain and en e ocy es isola ed om he sc amble mice,
which should show d as ic educ ion in Dab1 exp ession on a Wes e n
blo . The esul s a e gi en in Figu e 9A. The bands de ec ed by he an i-
Dab1 an ibody ha is absen in he sc amble issues a e a polypep ide
band a ~63 kDa in he en e ocy es
and a polypep ide o ~82 kDa in
he b ain.
To es whe he eelin modi ies he exp ession o Dab1 i s abundance
was measu ed in en e ocy es isola ed om con ol and eele mice.
Figu e 9A shows
ha he eele mu a ion inc eases Dab1 abundance by
a
ac o o app oxima ely 2 ela i e o con ol in bo h
en e ocy es and
b ain.
The Wes e n blo does no p o ide e idence on he subcellula loca ion
o Dab1, which was in es iga ed by immunocy ochemis y. Figu e 9B
e eals ha he speci ic signal p oduced by he an i-Dab1 an ibody
is
seen h oughou he cy osol, and in some cells he signal is s onge
a he e minal web domain.
The speci ic labelling was absen om he
en e ocy es
isola ed om sc amble mice.
Discussion
The eelin-signalling sys em and i s ole in issues o he han he b ain
is poo ly unde s ood. We e- po ed ha in he oden small in es ine
eelin is
eleased by he myo ib oblas s placed unde nea h he
epi helium
and in ol ed in he homeos asis o he
c yp – illus uni (Ga c´ıa-
Mi anda e al., 2010, 2012,
2013). The cu en wo k ex ends he
knowledge o
he eelin-signalling sys em by e ealing ha in he
small
in es ine Dab1 also migh ansmi he eelin
signal o cy osolic
signalling pa hway/s, which ul ima ely migh a ec he cell enewal
p ocesses o he
c yp – illus uni . As compa ed wi h he b ain, mouse
en e ocy es
exp ess signi ican amoun s o Dab1 and a di e en Dab1
iso o m: he en e ocy e iso o m is ~63 kDa and ha in he b ain ~82 kDa.
Bo h he size o he bands
and he di e ences be ween b ain and
en e ocy es a e
consis en wi h he high di e si y obse ed in Dab1
exp ession. Dab1 polypep ide bands anging om 36 o 120 kDa ha e
been iden i ied in mouse emb yonic b ain, he 80-kDa Dab1 being he
p edominan o m
(Howell e al., 1997). I has also been epo ed ha
he Dab1 o ms esul ing om al e na i e splicing a e species speci ic,
ha e di e en issue exp ession p o- iles and some imes a e exp essed
in he same issue a he same s age o de elopmen o in di e en sub-
popula ions o cells depending on he s age o de elopmen (Ba e al.,
2003; Ka yal and Godbou , 2004;
Cos agli e al., 2006; Gao e al., 2010;
Long e al.,
2011; Gao e al., 2012). The physiological meaning o he
di e ing p esence o a Dab1 iso o m in he small in es ine om ha in
b ain is unclea a p esen . The molecula weigh o he in es inal iso o m
is close o
ha o he “Dab1 ea ly (Dab1-E) iso o m” exp essed
in he
p ogeni o cells o he human/chicken e ina and chicken emb yos gu
(Gao e al., 2010; Ka yal
e al., 2011). A “la e (Dab1-L) iso o m”
(commonly e e ed o as Dab1) has been isola ed om di e en
ia ing
e inal cells (Gao e al., 2010). The Dab1-E
is missing wo y osine
±
The esul s a e gi en as he numbe o posi i e lysozyme cells
pe c yp .
Elec on mic oscopy assays
Segmen s o small in es ine we e ixed in 4% glu a alde
hyde/0.1 mol/l sodium
cacodyla e, pH 7.4 a 4°C o 3 h. A e h ee inses in cacodyla e-bu e ed solu ion,
he issues we e
pos ixed in 1% OsO4 in 0.1 M phospha e bu e a 4°C o 1 h
and washed in cacodyla e-bu e ed solu ion con aining 7.5% suc ose. The
segmen s we e hen dehyd a ed in a g adua ed se ies o ace one (30%, 50%
and 70%), s ained wi h 2% u anyl ace a e and embedded in Spu ’s epoxy
esin. Ul a hin sec ions we e examined unde a Philips CM-10 ans-
mission
elec on mic oscope equipped wi h an Olympus Vele a.
The pho og aphs we e
p ocessed wi h iTEM so wa e and ImageJ p og am e sion 1.46 (Na ional
Ins i u es o Heal h, h p:// sb.in o.nih.go /ij/index.h ml).
S a is ical analysis
Da a a e p esen ed as mean SEM. The numbe o animals is
indica ed in he
legends. In Figu es 7 and 9 compa isons be ween di e en expe imen al g oups
we e e alua ed by he wo- ailed S uden ’s es . One-way ANOVA ollowed by he
Newman– Keuls’ es was used o mul iple compa isons (G aphPad P ism
p og am).
Di e ences we e se o be signi ican o P < 0.05.
Au ho con ibu ion
M.J.P. and A.A.I. concei ed and designed he expe imen s. M.D.V-C, P.G-M. and
M.L.C. pe o med he expe imen s. M.J.P., M.L.C. and A.A.I. analysed and
in e p e ed he da a. A.A.I. ca ied ou he discussion
and w o e he pape .
Acknowledgemen s
W
e
hank
D .
O.
Pin ado
(Cen o
de
P oducci
o
´
n
y
E
xpe imen aci
o
´
n
Animal,
U
ni e sidad
de
Se illa),
D . L. Collinson (Cance Resea ch Ins i u e o Lon-
don),
D . F. Rome o and D . N. W igh ’s labo a
o y (Cance Resea ch Ins i u e o

London) o hei echnical ad ice and R. Ca acuel o echnical sup- po The an i-
lysozyme and an i-β-ca enin an ibod
ies we e kindly gi ed by D . N. W igh .
Elec on-
mic oscopy images we e ob ained in he Cen o de In es igaci
o
´
n,
T
ecnolog
´
ıa
e
I
nno aci
o
´
n,
U
ni e sidad
de Se illa.
Funding
This wo k was suppo ed by a g an om he Jun a de Andalucia (CTS 884)
and by a ellowship om he
Spanish
Minis e io
de
Educaci
o
´
n
y
Ciencia
(AP2007-
04201) o M.D. Vazquez-Ca e e o.
Con lic o in e es s a emen
The au ho s ha e decla ed no con lic o in e es .
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Figu e 1 Body weigh and small in es inal weigh and leng h o con ol and sc amble mice
Da a a e p esen ed as he means ± SEM o i e di e -
en animals pe age. One-way ANOVA showed an e ec o age and mu a ion on body and
small in es ine weigh s (P < 0.001). Newman–Keuls’ es **P < 0.001 and *P < 0.05 sc amble
e sus con ol mice, aP < 0.01 e sus 15 day-old mice.
Figu e 2 Cell p oli e a ion a e in he in es inal epi helium o con ol and sc amble mice
The numbe o B dU-labelled cells was de e mined wi h a monoclonal an i-B dU an ibody
(1:300 dilu ion) in 30 well-o ien ed longi udinally c yp s pe mouse by ligh mic oscopy.
Ten mic ome e c yosec ions we e used. (A) Rep esen a i e sec ions o
in es inal c yp s. Scale ba = 25 and 10 µm in he inse . (B) Means ± SEM o he numbe o B dU-
labelled cells pe c yp . The
numbe o animals used pe age was h ee sc amble and h ee con ol mice. One-way
ANOVA showed an e ec o mu a ion and age on cell p oli e a ion (P < 0.001). Newman–
Keuls’ es : *P < 0.001 sc amble e sus con ol mice, aP < 0.001 e sus 15 day-old mice.
Table 1 Mo phome ic pa ame e s o small in es inal mucosa o sc amble and con ol mice
Villi C yp s
Heigh
Wid h
Dep h
Diame e
323* ± 11
162* ± 6
442*a ± 9
198*a ± 4
73* ± 2
Means ± SEM o in es inal mucosa measu emen s (in µm). Fi e sc amble and i e con ol mice pe age we e used. One-way ANOVA
Mice
Jejunum
Ileum
Jejunum Ileum
Jejunum
Ileum
Jejunum
Ileum
Age 15 days
Con ol:
Sc amble :
396 ± 9
199 ± 4
70
±
1 58
±
1
64
±
2
54
±
2
40
±
1
36
±
1
33
±
1
Age 60 days
Con ol:
Sc amble :
483
a
±
12
210
a
±
3
80
a
±
2 69
a
±
3
77
a
±
2
72
a
±
1
46
a
±
1
43
a
±
1
43
a
±
1
Figu e 3 Cell mig a ion a e in he in es inal epi helium o con ol and sc amble mice
The dis ance be ween he o emos B dU-labelled cells and he base o he illi was
measu ed and used o de e mine he cell mig a ion a e. Ten mic ome e c yosec ions we e
used. (A) Rep esen a i e sec ions o mice in es ine. Lines indica e he s a ing
and he on poin o labelled cells used o e alua e he en e ocy e mig a ion a e. Scale ba = 50
µm. (B) Means ± SEM
o he dis ance (in µm) mig a ed pe hou . The numbe o animals used pe age was
h ee sc amble and h ee con ol mice. One-way ANOVA showed an e ec o bo h,
mu a ion and age on cell mig a ion a e (P < 0.001). Newman–Keuls’ es :
*P < 0.001 sc amble e sus con ol mice, aP < 0.001 e sus 15 day-old mice.