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. 2008 Dec 15;22(24):3435-48.
doi: 10.1101/gad.1752608.

Dynamic regulation of Pdx1 enhancers by Foxa1 and Foxa2 is essential for pancreas development

Nan Gao  1 , John LeLay, Marko Z Vatamaniuk, Sebastian Rieck, Joshua R Friedman, Klaus H Kaestner
Affiliations

Dynamic regulation of Pdx1 enhancers by Foxa1 and Foxa2 is essential for pancreas development

Nan Gao et al. Genes Dev. .

Abstract

The onset of pancreas development in the foregut endoderm is marked by activation of the homeobox gene Pdx1 (IPF1). Pdx1 is essential for the expansion of the pancreatic primordium and the development of endocrine islets. The control of Pdx1 expression has been only partially elucidated. We demonstrate here that the winged-helix transcription factors Foxa1 and Foxa2 co-occupy multiple regulatory domains in the Pdx1 gene. Compound conditional ablation of both Foxa1 and Foxa2 in the pancreatic primordium results in complete loss of Pdx1 expression and severe pancreatic hypoplasia. Mutant mice exhibit hyperglycemia with severely disrupted acinar and islet development, and die shortly after birth. Assessment of developmental markers in the mutant pancreas revealed a failure in the expansion of the pancreatic anlage, a blockage of exocrine and endocrine cell differentiation, and an arrest at the primitive duct stage. Comparing their relative developmental activity, we find that Foxa2 is the major regulator in promoting pancreas development and cell differentiation. Using chromatin immunoprecipitations (ChIP) and ChIP sequencing (ChIPSeq) of fetal pancreas and islet chromatin, we demonstrate that Foxa1 and Foxa2 predominantly occupy a distal enhancer at -6.4 kb relative to the transcriptional start site in the Pdx1 gene. In addition, occupancy of the well-characterized proximal Pdx1 enhancer by Foxa1 and Foxa2 is developmental stage-dependent. Thus, the regulation of Pdx1 expression by Foxa1 and Foxa2 is a key early event controlling the expansion and differentiation of the pancreatic primordia.

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Figures

Figure 1.
Figure 1.
Conditional ablation of Foxa1 and Foxa2 in the pancreatic primordia causes pancreatic hypoplasia. (A) Control E18.5 pancreas, outlined by the dotted line, is located between the stomach and duodenum. (B–D) Foxa1 and Foxa2 compound mutant pancreata were severely hypoplastic, with variable penetrance of the affected pancreatic lobes. Asterisks indicate the complete absence of the ventral pancreas. (E–H) Adjacent sections of control and compound mutant pancreas (E18.5) were stained for Foxa1/2 and Pdx1, with all three proteins being absent in the mutant pancreas. (I,J) Foxa1/Foxa2 expression in duodenal epithelia of the compound mutants was unaffected.
Figure 2.
Figure 2.
Loss of Pdx1 expression in the pancreatic buds of compound mutant mice. (A–D) The pancreatic domain marked by the Pdx1CreE transgene was detected by β-galactosidase staining after introgression of the Rosa26R reporter allele into our Foxa1loxP/loxP,Foxa2loxP/loxP,Pdx1CreE mice. Both ventral and dorsal pancreatic buds (blue staining, arrows) were visible in compound mutant mice at E9.5. (E–H) These mutant buds expanded initially at E10.5. (I) As the pancreatic buds evaginated from the gut epithelium, Foxa1/2 expression was seen in a majority of control pancreatic precursor cells as well as in the gut epithelium. The arrow points to the pancreatic bud, which is positive for nuclear Foxa1/2. (J) Foxa1/2 expression was decreased in the pancreatic anlage of compound mutant mice at E9.5. (K) Higher magnification of I. (L) In compound mutant pancreatic buds, only scattered single cells remained Foxa1/a2-positive (arrow). (M–T) Confocal fluorescent staining demonstrates that Pdx1 expression is extinguished in the compound mutant pancreatic anlage on day E10.5. Control pancreatic epithelial cells strongly express Pdx1. E-cadherin staining indicates the extent of the pancreatic epithelium. Bars, 25 μm.
Figure 3.
Figure 3.
Pancreatic precursors fail to expand in Foxa1 and Foxa2 compound mutants. (A–F) Whole-mount gut tissues were dissected from E13.5 embryos of the indicated genotypes. Dorsal and ventral pancreata developed in Foxa2loxP/+,Foxa2loxP/+, Pdx1CreE and Foxa2loxP/loxP,Foxa2loxP/+, Pdx1CreE mice as indicated by β-galactosidase activity, but failed to grow in compound mutant mice (dotted lines). (G,H) E13.5 control pancreas formed a ductal epithelial tree with abundant exocrine structures visible at the ductal tips. Compound mutant pancreata consisted primarily of ducts. (I–R) Control and compound mutant sections were stained with glucagon (I,J), amylase (K,L), CPA1 (M,N), Ngn3 (O,P), and Ptf1a (Q,R) antibodies in red, and counterstained for E-cadherin (blue) or DNA (green). Bars, 25 μm.
Figure 4.
Figure 4.
Foxa1 and Foxa2 regulate pancreas growth with different potency. (A–F) Pancreas development in E14.5 mice with various combinations of Foxa1 and Foxa2 alleles, in mice with introgressed Rosa26R reporter allele. (A,B) One wild-type Foxa2 allele is sufficient to specify a nearly normal-size pancreas. Severe hypoplasia was seen in Foxa2loxP/+,Foxa2loxP/loxP,Pdx1CreE mice (C,D) and compound mutants (E,F), with the ventral pancreas being affected more severely (D,F). (G) The extent of the pancreas, as marked by β-galactosidase activity of the Rosa26R reporter present in all embryos, was quantified in mice with indicated Foxa1/Foxa2 genotypes.
Figure 5.
Figure 5.
Disrupted pancreatic differentiation in compound mutants. (A–R) Indirect immunofluorescent staining of E17.5 pancreas sections from mice with the indicated genotypes for amylase and glucagon (A–H), insulin (I–L), E-cadherin and insulin (M–P), DBA and insulin (Q–R), or vimentin (S–T) antibodies and visualized by confocal microscopy. Arrows in G and H point to apparently double-positive amylase+/glucagon+ cells, and the optical section depth in these micrographs is 0.7 μm. Arrow in L points to a single insulin+ cell in the compound mutant pancreas. Bars: A–D, 100 μm; E–H, 17.5 μm; I–T, 50 μm.
Figure 6.
Figure 6.
The impact of Foxa1 and Foxa2 ablation on the pancreatic transcription factor network. E18.5 pancreas sections from control and compound mutant mice were stained with Foxa1/2, Nkx6.1, and Pdx1 (A–H); Pax6, Nkx2.2, and Foxa1/2 (I–P); or Isl1 and Foxa1/2 (Q–T) antibodies by indirect immunofluorescence and visualized by confocal microscopy. Arrows, in compound mutant sections, point to remaining Foxa1/2-positive cells that coexpress additional pancreatic transcription factors. Asterisks in G, H, R, and S mark the ducts. H, P, and Q–T are bright-field images or merged images including bright-field images. Bars: R,T, 17.5 μm; all others, 30 μm.
Figure 7.
Figure 7.
Occupancy of Pdx1 enhancers by Foxa1/Foxa2 increases with developmental time. (A) Schematic diagram of the Pdx1 locus indicating the distal (Area IV), and proximal (Area I–II–III) enhancer. (B) Foxa2 ChipSeq demonstrates significant occupancy of Area IV in E14.5 mouse fetal pancreas, while increased occupancy of Area I–II–III is seen in pancreatic islets. Area IV (red box) is evolutionarily conserved as illustrated by multispecies sequence alignment. (C) ChIP experiments using chromatin from the developing pancreas or mature islets demonstrate preferential Foxa1/Foxa2 occupancy of Area IV. Error bars represent SEM. Each ChIP was performed on chromatin samples from two to six biological replicates, and enrichment of both enhancers in the ChIP samples compared with input chromatin was statistically significant (P < 0.05) for all time points and both antibodies analyzed, with the exception of binding of either Foxa1 or Foxa2 to Area I–II in mature islets, where the P-value was <0.06 (Student’s t-test).
Figure 8.
Figure 8.
Occupancy of two Pdx1 enhancers by Foxa1 and Foxa2 controls pancreas development. (A) Loss of Foxa1 and Foxa2 prevents the expansion and differentiation of pancreatic progenitor cells, leading to disrupted exocrine and endocrine development. (B) Foxa1 and Foxa2 are among the earliest factors required for the expansion of pancreatic progenitors, the differentiation of endocrine and exocrine compartments, and the maturation of islet cells. (C) Diagram illustrating the cis-regulatory elements of Pdx1, including the distal enhancer (Area IV; red box), and the proximal enhancer (Area I–II–III; green boxes). (a) In the wild-type embryo, both enhancers, together with key transactivators including Foxa1/Foxa2 (yellow diamonds) direct Pdx1 gene (blue box) expression and normal pancreas development. (b) Ablation of Pdx1 causes abrogation of pancreas development, with only a small dorsal ductule remaining (Jonsson et al. 1994; Offield et al. 1996). (c) A Pdx1 transgene directed by either a 6.2-kb fragment including Area I–II–III and half of Area IV, or a 4.5-kb fragment including only Area I–II–III is capable of rescuing the Pdx1−/− pancreas phenotype (Gannon et al. 2001; Boyer et al. 2006). (d) Targeted deletion of Area I–II–III leads to severe pancreatic hypoplasia, affecting in particular the endocrine compartment, while acinar tissue does form (Fujitani et al. 2006). (e) Removal of Foxa1 and Foxa2 in the early pancreatic buds, as shown in this study, causes elimination of Pdx1 expression and disrupted exocrine and endocrine pancreas development.
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