基因缺失造成鼠胰腺發(fā)育不全

Lack of TCF2vHNF1 in mice leads to
pancreas agenesis
C. Haumaitre*, E. Barbacci*, M. Jenny?, M. O. Ott*, G. Gradwohl?,and S. Cereghini*?
*Biologie du De′ veloppement, Unite′ Mixte de Recherche 7622,Centre National de la Recherche Scientifique, Universite′ Pierre etMarie Curie, 9 Quai St.
Bernard Ba t C, 75005 Paris, France; and ?Institut National de laSante′ et de la Recherche Me′ dicale U381, 3 Avenue Molie` re,67200 Strasbourg, France
Edited by Kathryn V. Anderson, Sloan–Kettering Institute, New York,NY, and approved December 20, 2004 (received for review August 6,2004)
Heterozygous mutations in the human POU-homeobox TCF2
(vHNF1, HNF1) gene are associated with maturity-onset diabetes
of the young, type 5, and abnormal urogenital tractdevelopment.
Recently, pancreas atrophies have been reported in severalmaturity-
onset diabetes of the young type 5 patients, suggesting that
TCF2 is required not only for ***** pancreas function but alsofor
its normal development. Tcf2-deficient mice die beforegastrulation
because of defective visceral endoderm formation. Toinvestigate
the role of this factor in pancreas development, we rescued
this early lethality by tetraploid aggregation. We show thatTCF2
has an essential function in the first steps of pancreasdevelopment,
correlated with its expression domain that demarcates the
entire pancreatic buds from the earliest stages. Lack of TCF2results
in pancreas agenesis by embryonic day 13.5. At earlier stages,only
a dorsal bud rudiment forms transiently and expresses thetranscription
factors Ipf1 and Hlxb9 but lacks the key transcription
factor involved in the acquisition of a pancreatic fate, Ptf1a, aswell
as all endocrine precursor cells. Regional specification of thegut
also is perturbed in Tcf2/ embryos as manifested by ectopic
expression of Shh and lack of Ihh and Ipf1 in the posteriorstomach
and duodenum. Our results highlight the requirement of Tcf2 for
ensuring both accurate expression of key regulator molecules in
the stomach–duodenal epithelium and proper acquisition of the
pancreatic fate. This study provides further insights intoearly
molecular events controlling pancreas development and maycontribute
to the development of cell-replacement strategies for
diabetes.
diabetes MODY5 homeodomain transcription factor pancreas
development gut regionalization tetraploid aggregation
In mammals, the pancreas emerges as ventral and dorsal
evaginations from the foregut–midgut junction that subsequently
fused to form a complex organ. The signaling molecule
Sonic Hedgehog (SHH) demarcates a molecular boundary between
the prepancreatic endoderm and adjacent stomach and
duodenal anlagen and exerts an inhibitory action on pancreas
development (1–3). Genetic studies in mice have identified a
hierarchical regulatory network involved in pancreasmorphogenesis,
with significant and sequential differences between
ventral and dorsal pancreas. In the mouse, the dorsal bud
appears at embryonic day 9.5 (E9.5) concomitantly with thefirst
differentiated glucagon-producing cells. The homeobox gene
Ipf1(Pdx1) is expressed before and during this budding, and all
pancreatic cell types derive from IPF1 progenitors (4, 5).
However, in Ipf1-deficient mice, pancreas development isarrested
after budding (6, 7), implying that other factors promote
pancreas specification. Recently, the transcription factorPtf1a
(P48) has been shown to be essential for the acquisition of a
pancreatic fate by undifferentiated ventral foregut endoderm,
being required for the specification of the ventral pancreasand
robust outgrowth of the dorsal bud. In its absence, ventral
pancreas progenitors differentiate into duodenal cells bydefault
(8). By contrast, the homeobox gene Hlxb9 is required only
dorsally, for specifying the gut epithelium to a pancreatic fate(9,
10). A key regulator of endocrine development is the basic
helix–loop–helix protein Neurogenin3 (Ngn3), which isabsolutely
required to promote islet cell development (11). The Isl1
gene, which encodes a LIM-homeodomain protein, performs
two functions in the developing pancreas. It is initiallyrequired
in the dorsal mesenchyme for proper exocrine differentiation
and later in the pancreatic epithelium for islet survival (12).
Downstream of them, other transcription factors are essentialfor
proper pancreatic endocrine differentiation such as Nkx2.2 and
Pax6 (13–15). However, the initial stages of pancreaticdevelopment
occur early in mammalian embryogenesis, and molecular
mechanisms governing these first steps remain to be
elucidated.
In humans, mutations in the POU-homeobox TCF2 gene are
associated with the human disease maturity-onset diabetes ofthe
young type 5, a form of dominantly inherited type II diabetes
mellitus characterized by pancreatic beta cell dysfunction atthe
age of 25 years or younger, nondiabetic early onset renaldisease,
liver dysfunction, and abnormal urogenital tract development
(16–18). In addition to these phenotypes, variable levels of
pancreas atrophies have recently been associated with different
TCF2 mutations (19, 20). Remarkably, we have recentlyidentified
a severe pancreas hypoplasia in two fetuses carrying
previously undescribed mutations in the TCF2 gene (A. L.
Delezoide, C.H., and S.C., unpublished results). These data,
together with the observation that vHnf1(Tcf2)-mutant embryos
show underdevelopment of the pancreas in zebrafish (21),
strongly suggest a critical function of Tcf2 in pancreasdevelopment.
However, the molecular bases of these pancreatic phenotypes
are poorly understood, as are the Tcf2 target genes
involved. In mice, the precise implication of Tcf2 during early
organogenesis remains essentially unknown, because Tcf2-
deficient embryos die before gastrulation due to defective
visceral endoderm formation (22, 23).
In this study, we rescued this early lethality by tetraploid
aggregation, by using Tcf2/ embryonic stem (ES) cells. We
observed in these rescued Tcf2-null embryos an absence of the
ventral pancreatic bud and an extremely reduced and transient
dorsal bud that leads to pancreas agenesis by E13.5. Ourresults
uncover the requirement of Tcf2 for the specification of the
ventral pancreas and for proper morphogenesis anddifferentiation
of the dorsal pancreas. They further suggest that Tcf2 also
is required for both accurate regionalization of the primitivegut
through Hedgehog (Hh) signaling and proper acquisition of the
pancreatic fate by regulating Ptf1a expression, thus placingthis
transcription factor at one of the highest positions in thegenetic
network that controls pancreas development.
Materials and Methods
Diploid and Tetraploid Chimera. Because chimeric embryosgenerated
with our previously isolated Tcf2/ and Tcf2/ ES cells
This paper was submitted directly (Track II) to the PNASoffice.
Abbreviations: En, embryonic day n; ES, embryonic stem; Hh,hedgehog; vHnf1, variant
Hepatocyte nuclear factor 1; Shh, Sonic Hedgehog; Ihh, IndianHedgehog; Ngn3, Neurogenin3;
Isl1, Islet-1; Hnf6, Hepatocyte nuclear factor 6.
?To whom correspondence should be addressed. E-mail: silvia.cereghini@snv.jussieu.fr.
? 2005 by The National Academy of Sciences of the USA
1490–1495 PNAS February 1, 2005 vol. 102 no. 5 www.pnas.orgcgidoi10.1073pnas.0405776102
presented neural tube defects that were inherent to theparental
cell line (22), we isolated seven previously undescribed EScell
lines (four Tcf2/ and three Tcf2/) from blastocysts obtained
after crossing Tcf2-heterozygous mice (129sv background), as
described in ref. 24. Tetraploid embryos were generated by
electrofusion on a cell fusion instrument (CF-150, BLS Ltd.,
Budapest; voltage, 80 V; duration, 80 ms; 1 pulse) at thetwo-cell
stage. Tetraploid or diploid chimeric embryos were generated as
described in ref. 25. Two four-cell stage CD1 tetraploidembryos
or a wild-type (WT) CD1 morula were aggregated with a single
loose clump of 15–20 Tcf2-deficient ES cells, cultured in M16
medium (Sigma) up to the blastocyst stage, and implanted into
pseudopregnant females. We first confirmed that tetraploid or
diploid embryos generated with our Tcf2/ ES cells were similar
to WT or heterozygous Tcf2 embryos, indicating that thephenotype
of Tcf2/ ES cells-derived embryos is specifically due to
the lack of TCF2. We used as control embryos in a given litter
blastocysts obtained from cultured morulae not aggregated with
ES cells and implanted together with ES-cell-aggregatedembryos.
Because the yield of tetraploid embryos was low, we
established optimal conditions to obtain diploid chimera with
maximal ES-cell contribution, and we verified that these very
highly diploid chimeric embryos displayed the same phenotype
as tetraploid embryos. Because we disrupted the Tcf2 gene by
inserting the LacZ gene, the relative contribution of WT and
mutant cells in ES-cell-derived embryos was evaluated bywholemount
X-Gal staining (22). We analyzed here only very highly
chimeric and tetraploid embryos, characterized by the presence
of exclusively -gal mutant cells in the Tcf2-expressing tissues
(defined as Tcf2/ embryos).
Immunohistochemistry, in Situ Hybridization, and TUNEL. Mouse
embryos were fixed in 4% paraformaldehyde and embedded in
paraffin. Then, 5-msagittal sections were dewaxed, rehydrated,
and subjected to microwave antigen retrieval in 10 mM citrate.
For immunostaining, we used rabbit anti-Ipf1 (M. German,
Hormone Research Institute, San Francisco), mouse antiglucagon
(Sigma), rabbit anti-Hlxb9 (10), mouse anti-Islet-1
(39.4D5 and 40.2D6), mouse anti-Pax6 (Developmental Studies
Hybridoma Bank, Iowa City, IA), and rabbit anti-phosphohistone
H3 (Upstate Biotechnology, Lake Placid, NY) as primary
antibodies, and FITC- and Cyanine3-conjugated (The Jackson
Laboratory) as secondary antibodies. For in situ hybridization,
we prepared frozen sections from timed embryos, as described
in ref. 11. The following cRNA probes were used: Ptf1a (P.
Wellauer, Swiss Institute for Experimental Cancer Research,
Epalinges, Switzerland); Hnf6 (F. Lemaigre, Universite′
Catholique de Louvain, Brussels); Ngn3 (11); Shh and Ihh (A. P.
McMahon, Harvard University, Cambridge, MA); and Ptc (M.
Scott, Howard Hughes Medical Institute, Stanford, CA).
TUNEL was performed by using the fluorescein cell death
detection kit (Roche).
Results and Discussion
Tcf2 Is Expressed in the Developing Pancreas from Its EarlyStages.
Tcf2 heterozygous embryos for a null allele with the LacZ gene
under the control of regulatory regions of the Tcf2 locusexhibit
at E8–E8.5 high -gal expression in the neural tube and in the
entire gut from the foregut–midgut region and by E9.5 in the
hepatic, ventral, and dorsal pancreatic primordia (22) (seealso
Fig. 2 A and C). As the ventral and dorsal pancreatic budsstarted
to form, we observed Tcf2 transcripts at high levels in theentire
epithelial cells of the pancreatic buds (Fig. 1A).Interestingly,
Tcf2 expression domain included that of Ptf1a and Ipf1, two of
the earliest markers of the pancreatic bud (6–8) (Fig. 1A), as
well as early glucagon-expressing cells (data not shown). At
E13.5, Tcf2 transcripts were detected in the branchedpancreatic
epithelium. As the buds grew and fused, Tcf2 appeared more
intensely expressed in exocrine ducts, as shown by X-Galstaining
of Tcf2/ embryos at E15.5 and E18.5 (Fig. 1B).
Thus, in the ventral and dorsal pancreatic anlagen, Tcf2,Ptf1a,
and Ipf1 are expressed concurrently, suggesting that TCF2 might
control early steps of pancreas differentiation.
Lack of TCF2 Disrupts Early Pancreas Development.Tcf2-deficient
mice die before gastrulation due to defective extra-embryonic
visceral endoderm formation (22, 23). Therefore, to examine the
role of Tcf2 in pancreas development, we generated diploid and
tetraploid chimeric mouse embryos by aggregation with -gal
Tcf2-deficient ES cells. In tetraploid embryos, 4n cellscontribute
to extra-embryonic lineages, whereas the resulting fetusesderive
exclusively from ES cells. We set up conditions by which very
highly chimeric embryos generated by diploid aggregationexhibited
the same phenotype as embryos generated by tetraploid
aggregation. In this study, we focused on the severe pancreatic
phenotype of these two equivalent types of embryos, further
defined as Tcf2/ embryos. In both cases, we confirmed that
these embryos essentially were derived from Tcf2-deficient ES
cells, as manifested by -gal staining of Tcf2-expressingtissues
Fig. 1. Tcf2 expression in the embryonic pancreas. (A) Demarcationof the
entire pancreatic buds at early stages by Tcf2 expression domain.Tcf2 and
Ptf1a transcripts are visualized in the ventral and dorsalpancreatic buds in
sagittal sections of E9.5 and E11.5 embryos by in situhybridization (Left) with
the corresponding IPF1 immunostaining on the same section (Middle).Merge
images at lower and higher magnifications (Right) revealed thatTcf2 expression
domain is correlated with Ptf1a expression domain and includeIpf1-
expressing cells. Note also that Tcf2 and Ipf1 are coexpressed inthe duodenum
where Ptf1a is absent. vp, ventral pancreatic bud; dp, dorsalpancreatic bud,
li, liver; g, gut; d, duodenum; p, pancreas. (B) Tcf2 expression inthe mouse
developing pancreas. At E11.5, Tcf2 transcripts are present in thepancreas (p)
and duodenum (d) at lower levels than in the mesonephric tubules(mt). li,
liver. At E13.5 the pancreatic epithelium is labeled by Tcf2transcripts. -gal
staining of Tcf2 heterozygous embryos reveals an intense Tcf2expression in
ductal cells at E15.5 and E18.5. m, metanephros.
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DEVELOPMENTAL
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(Fig. 2 B, D, F, and H). Tetraploid chimeric embryos generated
by aggregation of Tcf2-heterozygous ES cells werephenotypically
normal at the stages examined here (data not shown).
Pancreatic bud formation in Tcf2/ and control embryos (see
Materials and Methods) was analyzed from E9.5 to E13.5, by
scoring the expression of Ipf1 (Fig. 2 I, J, L, M, O, P, R, S, U,and
V). We verified that all Ipf1 cells in Tcf2/ embryos were
mutant -gal cells (Fig. 2 K, N, Q, and T). At E9.5, we observed
a severely reduced dorsal pancreatic bud (an 60% reduction)
in Tcf2 mutants, whereas the ventral bud was undetectable (Fig.
2 D and J). The dorsal pancreatic bud rudiment did not grow
further, remaining close to the stomach lumen (Fig. 2 M, P, and
S), in contrast to control embryos that displayed an important
branching phase between E10.5 and E12.5 (Fig. 2 L, O, and R).
The pancreatic bud in Tcf2/ embryos was extremely reduced
at E12.5 (Fig. 2S) and became absent at E13.5 (Fig. 2V). In
accordance with these observations, by using the mitosis marker
phosphorylated histone-H3 (26, 27), we found a lowerproliferating
rate in the mutant pancreatic bud from E9.5, whereas the
number of proliferating cells in the surrounding mesenchyme
was not affected (Fig. 3G). TUNEL experiments showed that
cells of this remnant dorsal bud did not undergo apoptosis
because the percentage of apoptotic cells of Tcf2/ vs. control
pancreatic bud was not significantly different (Fig. 3H),suggesting
that these cells were either eliminated by necrosis orrecruited
to another tissue.
We subsequently examined the expression of early pancreatic
markers (28) by immunohistochemical (Fig. 4 A–J) and in situ
hybridization (Fig. 4 K–V) analyses in E11.5 sagittal sections.We
observed that the Ipf1 dorsally reduced expression domain in
Fig. 2. Early defective pancreas development in Tcf2/ embryos.(A–H) Overall morphology in whole-mount-gal-stained Tcf2heterozygous and homozygous
mutant embryos between E8.5 and E12.5. Tcf2/ embryos exhibit, inaddition to severe liver hypoplasia and abnormal ureteric branching(L. Lokmane, M.
Pares-Fessy, C.H., and S.C., unpublished data), defective pancreasdevelopment. -gal activity is more intensely observed in the neuraltube (nt) of E8.5 Tcf2/
than Tcf2/ embryos (A and B), as well as in mesonephric ducts (md)and primitive gut (g) at E9.5 (C and D), probably as a result ofthe presence of two copies
of the LacZ gene in mutant embryos andor a negative autoregulationof Tcf2. The protruding dorsal pancreatic bud externally detectedin heterozygous
embryos between E9.5 and E12.5 is not observed in homozygous mutantembryos (black arrowhead, C–H). (I–X) Pancreatic bud morphogenesisin sagittal sections
of control and Tcf2 homozygous mutant embryos between E9.5 andE13.5. (I–K) IPF1 immunostainings of sagittal sections ofwhole-mount-gal-stained embryos
reveal ventral and dorsal pancreatic buds in Tcf2/ embryos, whereasthe ventral pancreatic bud is totally absent and only a veryreduced dorsal bud is observed
in Tcf2/ embryos at E9.5. vp, ventral pancreas; dp, dorsalpancreas. (L–T) At later stages, the dorsal pancreatic bud (whitearrowhead), the duodenum (d), and
the posterior stomach are stained by IPF1 in control embryos, butonly a remnant pancreatic bud is stained in Tcf2-deficient embryos,which is abnormally close
to the posterior stomachal epithelium. Whereas the pancreatic budexhibits an important growth in control embryos particularly fromE12.5, the remnant
pancreatic bud in Tcf2/ embryos regresses by E12.5 and is notfurther detected at E13.5 (pancreas agenesis) (U–X). (K, N,Q, andT) -gal staining of the remnant
pancreatic bud in sagittal sections of Tcf2 homozygous mutantembryos. -gal and IPF1-stained sections are counterstained bysafranin. -gal mutant cells are
detected in the rudiment of the pancreatic bud in Tcf2/ embryoscoexpressing IPF1 and display a broader expression domain includinga thickness of the
stomachal epithelium between E10.5 and E11.5. Note that themagnification in K, N, Q, and T is higher than in the correspondingIPF1-stained section in J, M,
P, and S. (U–X) Stomachal epithelium morphology in E13.5 Tcf2control and homozygous mutant embryos. Trichromic staining of theIPF1-stained sagittal
sections. Arrows indicate the posterior stomachal epithelium, whichis surrounded by the stomachal mesenchyme. Whereas the normalposterior stomach exhibits
a columnar vacuolized epithelium, the posterior stomach of Tcf2/embryos appears squamous and nonvacuolized, as is normally theanterior stomach. In M,
P, T, and X, an asterisk indicates thickening of the gastricepithelium.
1492 www.pnas.orgcgidoi10.1073pnas.0405776102Haumaitre et al.
mutant embryos also expressed the early pancreatic marker
Hlxb9 (Fig. 4 B and D). By contrast, both Ipf1 and Hlxb9
expression were not detected in the presumptive ventralpancreatic
bud area. Remarkably, we found no expression of the key
transcription factor Ptf1a (8) (Fig. 4L). Moreover, Tcf2/
mutants displayed a very reduced expression of Hnf6 and no
Ngn3 expression in the remnant dorsal pancreatic bud, two
factors required for endocrine fate acquisition (11, 29) (Fig. 4N
and P). Consistent with this absence of Ngn3, the earliestknown
marker of endocrine precursors (11), expressions of Isl1, Pax6,
and glucagon were lost (Fig. 4 F, H, and J). Isl1 remained,
however, expressed in the mesenchyme (7) (Fig. 4F), a tissue
where Tcf2 was not expressed. Thus, endocrine precursors are
totally absent in Tcf2/ pancreatic epithelium.
Taken together, our results show that Tcf2 controls initial
specification of the ventral pancreas and is required forproper
proliferation and differentiation of the dorsal pancreas.
Lack of TCF2 Perturbs Regionalization of the Primitive Gut.Because
Hh signaling was shown to exert an inhibitory action onpancreatic
development (1, 30), we further investigated whether the
impairment of pancreatic development in Tcf2/ embryos
could result from a modified expression of either Sonichedgehog
(Shh) or Indian hedgehog (Ihh). Whereas Ihh was expressed
at E12 in the caudal epithelium of the stomach and duodenum
in WT embryos (Fig. 4S), no Ihh transcripts were detected in
Tcf2/ embryos (Fig. 4T). We propose that Ihh could be a direct
target gene of Tcf2, because we found that Ihh is also absentin
Tcf2/ embryoid bodies (31). By contrast, Shh, normally not
expressed in the caudal stomach and in the pancreatic bud (Fig.
4Q), was highly ectopically expressed in the stomachalepithelium
and duodenum with a rostrocaudal gradient of expression,
but remained excluded from the mutant pancreatic bud (Fig.
4R). As expected, the Hh receptor Patched (Ptc) was intensively
expressed along the Shh expression domain with thecorresponding
rostro-caudal expression gradient (Fig. 4V), suggesting that
the Hh pathway is active. Interestingly, in E13.5 Tcf2/embryos,
the posterior stomach exhibited characteristics of the
anterior stomach and appeared essentially squamous andnonvacuolized,
instead of exhibiting a columnar epithelium with
mucin-negative vacuoles as posterior stomach of control embryos
(Fig. 2 W and X). A similar anteriorly directed transformation
of the posterior stomach resulting from ectopic Shh
expression also has been observed in mice carrying mutations of
activin receptors ActRIIA and ActRIIB (32). However, Tcf2/
embryos present a more severe pancreatic phenotype than that
of ActRIIA/ ActRIIB/ double-mutant embryos, suggesting
an additional role of TCF2 in early pancreas development.
We also observed a thickening of the posterior stomach
epithelium by E10.5 (Fig. 2 M and P). Subsequently, part ofthis
multilayered stomachal epithelium appeared to be delaminated
by E12.5 and remained as clusters of tissue in the lumen of a
distended stomach (Fig. 2 T and X). This phenomenon might be
caused by the loss of Ihh expression, because a multilayer
epithelium is also observed in the colon of Ihh/ embryos (33).
These results show that loss of Tcf2 function results in a
perturbed anteroposterior regionalization of the primitive gut,
through a deregulation of Hh signaling.
TCF2 Is a Critical Regulator in the Transcriptional NetworkThat
Governs Pancreas Morphogenesis. Our data strongly suggest a
critical role for Tcf2 in the orchestratred network oftranscription
factors and secretory molecules (34) controlling the expansionof
endodermal progenitors and their differentiation intopancreatic
primordia.
Remarkably, Tcf2-deficient embryos exhibit a very close
phenotype to that caused by Ptf1a deficiency, in regard to the
absence of a ventral pancreas and a reduced dorsal pancreas.
Intriguingly, we identified a TCF12-DNA consensus-binding
site (ATTAATGTTTAAC) in the Ptf1a promoter at 5,092
bp from the initiation site, within a domain highly conserved
between mouse and human, which specifically binds TCF12
proteins (data not shown). This finding suggests that TCF2
could regulate Ptf1a expression directly. However, thephenotype
of Tcf2/ embryos is more severe than that of Ptf1a/
embryos, because in the absence of Ptf1a expression the dorsal
pancreas is maintained and relatively developed, with endocrine
cells still present (8, 35), indicating that other regulatory
factors may contribute to Tcf2 mutant phenotype. In this
context, Ipf1 and Ngn3 were previously identified as direct
target genes of TCF2 (36, 37). Yet, Ipf1 was still detectablein
the Tcf2/ pancreatic bud rudiment (Fig. 2), implying that
TCF2 is not required for Ipf1 initial induction. By contrast,the
expression of Ngn3, a gene whose expression was transiently
reduced in Hnf6/ embryos (29, 38), was completely abolished
in Tcf2/ embryos. Because Hnf6 expression also was
Fig. 3. Decreased cell proliferation in pancreas of Tcf2/ embryos.(A and B)
The pancreatic bud in control and Tcf2-homozygous mutant embryos isdefined
by Ipf1 expression domain. (C and D) The mitosis markerantiphosphorylated
histone H3 (P-H3) antibody detects proliferating cells in thepancreatic
bud (circled by white dashed lines). (E and F) TUNEL experimentsshow cells in
apoptosis in control and Tcf2-mutant pancreas (circled by whitedashed lines).
(G) The percentage of P-H3 cells among IPF1 cells of control andTcf2/
pancreatic buds reveals an important cell proliferation decrease inTcf2-
mutant pancreas. (H) The percentage of TUNEL cells among IPF1cells
reveals no significant difference between control and Tcf2/pancreatic buds.
A total of 13 sections from control (n 5) and 7 sections from Tcf2/(n 4)
of E9.5 and E10.5 embryos were evaluated.
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DEVELOPMENTAL
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severely reduced in Tcf2/ dorsal pancreatic bud, activation
of Ngn3 may require the concurrent action of Tcf2 and Hnf6.
Recent studies have reported a transient reduction in TCF2
levels in the pancreatic duct cells in Hnf6-deficient embryos
from E13.5 to E15.5, leading to the suggestion that Hnf6 is
upstream of Tcf2 (38). Nevertheless, the more severe pancreatic
phenotype of Tcf2-mutant embryos, compared with that of
Hnf6/ embryos (29), indicates that this transcriptional hi-
Fig. 5. Proposed model for Tcf2 function in the development of thepancreas and gut endoderm. (A) Expression domains of thetranscription factors Tcf2, Ipf1,
and Ptf1a, as well as the signaling molecules Shh and Ihh, in thefore-midgut area of Tcf2/ and Tcf2/ embryos. Tcf2 deficiency leadsto an extremely reduced
pancreatic bud expressing Ipf1 but missing Ptf1a expression,associated with a perturbed gut regionalization reflected by anexpansion of Shh expression domain
and an absence of Ihh and Ipf1 expression in stomach and duodenum.,expressed gene;, nonexpressed gene. (B) Proposed model of theregulatory network
that governs differentiation of pancreatic cells. The diagramillustrates the epistatic relations of genes required for pancreasdifferentiation, leading to endocrine
and exocrine cells. Tcf2 is required early in pancreas development,activating Ptf1a, and regulating the Ipf1-Shh network, with Shhrepression vs. Ihh activation.
TCF2 also activates Ngn3 in endocrine precursor cells, in agreementwith the hypothesis that TCF2-positive cells are the precursors ofNGN3 positive cells (38).
Fig. 4. Impaired early pancreatic cell differentiation in Tcf2/embryos. (A–J) Immunohistochemical analysis of E11.5 control andTcf2/ dorsal pancreatic
buds. Anti-Ipf1 (A and B), anti-Hlxb9 (C and D), anti-Isl1 (E andF), anti-Pax6 (G and H), and anti-glucagon (I and J) antibodieswere used on sagittal sections of
WT and Tcf2-mutant embryos. (A and B) Ipf1 was detected in theremnant dorsal pancreatic bud (marked by dashed lines), but not inthe duodenum (circled by
a white line), of Tcf2/ embryos. (C and D) Hlxb9 also is expressedin this bud. (E–J) None of the early endocrine pancreatic markers,Isl1, Pax6, and glucagon,
were detected in the dorsal pancreatic epithelium of Tcf2 mutants.(E and F) Isl1, although absent in pancreatic epithelial cellclusters, is still expressed in the
mesenchyme. (K–V) In situ hybridization analysis of control andTcf2/ E11.5 dorsal pancreatic buds. In contrast to Ipf1, Ptf1a andNgn3 are not expressed in
the remnant dorsal pancreatic bud of Tcf2 mutants (K, L, O, and P),whereas Hnf6 is severely reduced (M and N). Whereas Ihh expressionis abolished in Tcf2/
mutants (S and T), Shh is highly expressed in the anterior stomachwith an expanded expression domain in posterior stomach andduodenum (Q and R) and
induced Ptc expression (U and V).
1494 www.pnas.orgcgidoi10.1073pnas.0405776102Haumaitre et al.
erarchy most likely is not involved at earlier stages ofpancreas
development.
Regulatory circuits also are involved in regional specification
of the gut endoderm. One important aspect of thisregionalization
is the restricted expression of Ipf1 and Shh, essential to
permit pancreas development (12). Several studies havesuggested
that Shh and Ipf1 mutually repress their expression
through a regulatory loop in the gut endoderm (1, 21, 30). In
correlation with this finding, we found in Tcf2/ embryos an
expanded domain of Shh expression in the posterior stomach and
duodenum, whereas the Ipf1 expression domain remainedrestricted
to the rudimentary dorsal pancreatic bud but was absent
in posterior stomach and duodenum (Fig. 4). Thus, Tcf2 appears
to regulate regional specification of the gut endoderm through
the Shh-Ipf1 network.
Taken together, these findings allow us to propose a model
highlighting the critical role played by Tcf2 in the control of
pancreas development, in relation with the regionalization ofthe
primitive gut (Fig. 5). We propose that in the absence of TCF2,
Ptf1a expression is not induced, leading to defectivespecification
of the ventral pancreas and a reduced dorsal pancreas, which is
subsequently not maintained because of an alteredregionalization
of the gut through deregulation of Hh signaling.
This study provides further insight into the early molecular
events controlling pancreas development in mice and thefunction
of the transcription factor TCF2 in this process. Our
observation of pancreas hypoplasia in two fetuses carryingnovel
TCF2 mutations (A. L. Delezoide, C.H, and S.C, unpublished
data) suggests that decreased levels of TCF2 also perturbnormal
pancreas growth and function in humans. The role played by Tcf2
in pancreas development thus appears to be conserved during
evolution. Then, understanding how Tcf2 together with other
regulatory molecules direct early pancreas development in mice
may help to elaborate cell-replacement strategies for diabetes
mellitus.
We thank B. Thorens (Institute of Physiology of Lausanne,Lausanne,
Switzerland), M. German, P. Wellauer, F. Lemaigre, A. P. McMahon,M.
Scott, and S. Schneider-Maunoury (Unite′ Mixte de Recherche7622,
Centre National de la Recherche Scientifique, Universite′ Pierre etMarie
Curie) for reagents, and J. F. Colas, J. L. Duband, and S.Schneider-
Maunoury for comments on the manuscript. This work was supportedby
Association pour la Recherche sur le Cancer Contracts 58243231,
Institut National de la Sante′ et de la Recherche Me′dicale,Centre
National de la Recherche Scientifique, and Universite′ Pierre etMarie
Curie. C.H. is a recipient of Ph.D. student fellowships fromMiniste`re de
la Recherche et de la Technologie and Association pour la Recherchesur
le Cancer.
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