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. 2005 Feb 1;102(5):1490-5.
doi: 10.1073/pnas.0405776102. Epub 2005 Jan 24.

Lack of TCF2/vHNF1 in mice leads to pancreas agenesis

C Haumaitre  1 , E Barbacci, M Jenny, M O Ott, G Gradwohl, S Cereghini
Affiliations

Lack of TCF2/vHNF1 in mice leads to pancreas agenesis

C Haumaitre et al. Proc Natl Acad Sci U S A. .

"V体育官网入口" Abstract

Heterozygous mutations in the human POU-homeobox TCF2 (vHNF1, HNF1beta) gene are associated with maturity-onset diabetes of the young, type 5, and abnormal urogenital tract development VSports手机版. Recently, pancreas atrophies have been reported in several maturity-onset diabetes of the young type 5 patients, suggesting that TCF2 is required not only for adult pancreas function but also for its normal development. Tcf2-deficient mice die before gastrulation because of defective visceral endoderm formation. To investigate the role of this factor in pancreas development, we rescued this early lethality by tetraploid aggregation. We show that TCF2 has an essential function in the first steps of pancreas development, correlated with its expression domain that demarcates the entire pancreatic buds from the earliest stages. Lack of TCF2 results in pancreas agenesis by embryonic day 13. 5. At earlier stages, only a dorsal bud rudiment forms transiently and expresses the transcription factors Ipf1 and Hlxb9 but lacks the key transcription factor involved in the acquisition of a pancreatic fate, Ptf1a, as well as all endocrine precursor cells. Regional specification of the gut also is perturbed in Tcf2-/- embryos as manifested by ectopic expression of Shh and lack of Ihh and Ipf1 in the posterior stomach 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 into early molecular events controlling pancreas development and may contribute to the development of cell-replacement strategies for diabetes. .

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Figures

Fig. 2.
Fig. 2.
Early defective pancreas development in Tcf2-/- embryos. (A–H) Overall morphology in whole-mount β-gal-stained Tcf2 heterozygous and homozygous mutant embryos between E8.5 and E12.5. Tcf2-/- embryos exhibit, in addition to severe liver hypoplasia and abnormal ureteric branching (L. Lokmane, M. Pares-Fessy, C.H., and S.C., unpublished data), defective pancreas development. β-gal activity is more intensely observed in the neural tube (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 of the presence of two copies of the LacZ gene in mutant embryos and/or a negative autoregulation of Tcf2. The protruding dorsal pancreatic bud externally detected in heterozygous embryos between E9.5 and E12.5 is not observed in homozygous mutant embryos (black arrowhead, C–H). (I–X) Pancreatic bud morphogenesis in sagittal sections of control and Tcf2 homozygous mutant embryos between E9.5 and E13.5. (I–K) IPF1 immunostainings of sagittal sections of whole-mount β-gal-stained embryos reveal ventral and dorsal pancreatic buds in Tcf2+/- embryos, whereas the ventral pancreatic bud is totally absent and only a very reduced dorsal bud is observed in Tcf2-/- embryos at E9.5. vp, ventral pancreas; dp, dorsal pancreas. (L–T) At later stages, the dorsal pancreatic bud (white arrowhead), the duodenum (d), and the posterior stomach are stained by IPF1 in control embryos, but only a remnant pancreatic bud is stained in Tcf2-deficient embryos, which is abnormally close to the posterior stomachal epithelium. Whereas the pancreatic bud exhibits an important growth in control embryos particularly from E12.5, the remnant pancreatic bud in Tcf2-/- embryos regresses by E12.5 and is not further detected at E13.5 (pancreas agenesis) (U–X). (K, N, Q, and T) β-gal staining of the remnant pancreatic bud in sagittal sections of Tcf2 homozygous mutant embryos. β-gal and IPF1-stained sections are counterstained by safranin. β-gal+ mutant cells are detected in the rudiment of the pancreatic bud in Tcf2-/- embryos coexpressing IPF1 and display a broader expression domain including a thickness of the stomachal epithelium between E10.5 and E11.5. Note that the magnification in K, N, Q, and T is higher than in the corresponding IPF1-stained section in J, M, P, and S. (U–X) Stomachal epithelium morphology in E13.5 Tcf2 control and homozygous mutant embryos. Trichromic staining of the IPF1-stained sagittal sections. Arrows indicate the posterior stomachal epithelium, which is surrounded by the stomachal mesenchyme. Whereas the normal posterior stomach exhibits a columnar vacuolized epithelium, the posterior stomach of Tcf2-/- embryos appears squamous and nonvacuolized, as is normally the anterior stomach. In M, P, T, and X, an asterisk indicates thickening of the gastric epithelium.
Fig. 1.
Fig. 1.
Tcf2 expression in the embryonic pancreas. (A) Demarcation of the entire pancreatic buds at early stages by Tcf2 expression domain. Tcf2 and Ptf1a transcripts are visualized in the ventral and dorsal pancreatic buds in sagittal sections of E9.5 and E11.5 embryos by in situ hybridization (Left) with the corresponding IPF1 immunostaining on the same section (Middle). Merge images at lower and higher magnifications (Right) revealed that Tcf2 expression domain is correlated with Ptf1a expression domain and include Ipf1-expressing cells. Note also that Tcf2 and Ipf1 are coexpressed in the duodenum where Ptf1a is absent. vp, ventral pancreatic bud; dp, dorsal pancreatic bud, li, liver; g, gut; d, duodenum; p, pancreas. (B) Tcf2 expression in the mouse developing pancreas. At E11.5, Tcf2 transcripts are present in the pancreas (p) and duodenum (d) at lower levels than in the mesonephric tubules (mt). li, liver. At E13.5 the pancreatic epithelium is labeled by Tcf2 transcripts. β-gal staining of Tcf2 heterozygous embryos reveals an intense Tcf2 expression in ductal cells at E15.5 and E18.5. m, metanephros.
Fig. 3.
Fig. 3.
Decreased cell proliferation in pancreas of Tcf2-/- embryos. (A and B) The pancreatic bud in control and Tcf2-homozygous mutant embryos is defined by Ipf1 expression domain. (C and D) The mitosis marker antiphosphorylated histone H3 (P-H3) antibody detects proliferating cells in the pancreatic bud (circled by white dashed lines). (E and F) TUNEL experiments show cells in apoptosis in control and Tcf2-mutant pancreas (circled by white dashed lines). (G) The percentage of P-H3+ cells among IPF1+ cells of control and Tcf2-/- pancreatic buds reveals an important cell proliferation decrease in Tcf2-mutant pancreas. (H) The percentage of TUNEL+ cells among IPF1+ cells 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.
Fig. 4.
Fig. 4.
Impaired early pancreatic cell differentiation in Tcf2-/- embryos. (A–J) Immunohistochemical analysis of E11.5 control and Tcf2-/- dorsal pancreatic buds. Anti-Ipf1 (A and B), anti-Hlxb9 (C and D), anti-Isl1 (E and F), anti-Pax6 (G and H), and anti-glucagon (I and J) antibodies were used on sagittal sections of WT and Tcf2-mutant embryos. (A and B) Ipf1 was detected in the remnant dorsal pancreatic bud (marked by dashed lines), but not in the duodenum (circled by a white line), of Tcf2-/- embryos. (C and D) Hlxb9 also is expressed in 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 cell clusters, is still expressed in the mesenchyme. (K–V) In situ hybridization analysis of control and Tcf2-/- E11.5 dorsal pancreatic buds. In contrast to Ipf1, Ptf1a and Ngn3 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 expression is abolished in Tcf2-/- mutants (S and T), Shh is highly expressed in the anterior stomach with an expanded expression domain in posterior stomach and duodenum (Q and R) and induced Ptc expression (U and V).
Fig. 5.
Fig. 5.
Proposed model for Tcf2 function in the development of the pancreas and gut endoderm. (A) Expression domains of the transcription factors Tcf2, Ipf1, and Ptf1a, as well as the signaling molecules Shh and Ihh, in the fore-midgut area of Tcf2+/+ and Tcf2-/- embryos. Tcf2 deficiency leads to an extremely reduced pancreatic bud expressing Ipf1 but missing Ptf1a expression, associated with a perturbed gut regionalization reflected by an expansion of Shh expression domain and an absence of Ihh and Ipf1 expression in stomach and duodenum. +, expressed gene; -, nonexpressed gene. (B) Proposed model of the regulatory network that governs differentiation of pancreatic cells. The diagram illustrates the epistatic relations of genes required for pancreas differentiation, leading to endocrine and exocrine cells. Tcf2 is required early in pancreas development, activating Ptf1a, and regulating the Ipf1-Shh network, with Shh repression vs. Ihh activation. TCF2 also activates Ngn3 in endocrine precursor cells, in agreement with the hypothesis that TCF2-positive cells are the precursors of NGN3 positive cells (38).
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