A Gene Regulatory Network Cooperatively Controlled by Pdx1 and Sox9 Governs Lineage Allocation of Foregut Progenitor Cells

Abstract

The generation of pancreas, liver, and intestine from a common pool of progenitors in the foregut endoderm requires the establishment of organ boundaries. How dorsal foregut progenitors activate pancreatic genes and evade the intestinal lineage choice remains unclear. Here, we identify Pdx1 and Sox9 as cooperative inducers of a gene regulatory network that distinguishes the pancreatic from the intestinal lineage. Genetic studies demonstrate dual and cooperative functions for Pdx1 and Sox9 in pancreatic lineage induction and repression of the intestinal lineage choice. Pdx1 and Sox9 bind to regulatory sequences near pancreatic and intestinal differentiation genes and jointly regulate their expression, revealing direct cooperative roles for Pdx1 and Sox9 in gene activation and repression. Our study identifies Pdx1 and Sox9 as important regulators of a transcription factor network that initiates pancreatic fate and sheds light on the gene regulatory circuitry that governs the development of distinct organs from multi-lineage-competent foregut progenitors.

Figure 1. Principal component analysis for expression of transcription factors in endodermal cell populations

(A) Experimental strategy for principal component analysis of transcription factors in various endodermal cell populations. (B) Principal component (PC) analysis of the expression values (RPKM) characterizing the variance explained by transcription factors expressed in human embryonic stem cell (hESC)-derived populations and primary human cells. Each vector emanating from the origin represents an individual gene. Each dot represents a sample and each color represents the type of sample.

Figure 2. Pdx1 and Sox9 are coexpressed in the pancreatic domain in the foregut endoderm

(A–B″) Immunofluoresence staining for Sox2, Sox9 and Pdx1 (A–A″) and Cdx2, Sox9 and Pdx1 (B–B″) on embryonic sections at embryonic day (E) 8.75. The arrows in A′,A″ and B′,B″ indicate Pdx1⁺/Sox9⁺ cells co-expressing Sox2 and Cdx2, respectively. The dashed line in B–B″ demarcates the transition from the presumptive duodenal to the pre-pancreatic region. Fields demarcated by white dashed boxes in A′,A″,B′,B″ are shown at higher magnification in the same panels. Non-specific signal for Cdx2 is evident in the foregut lumen (B,B″, asterisks) due to antibody trapping. (C) Immunofluoresence staining for Cdx2, Sox2, and Pdx1 at E10.5. (D,E) Dams carrying R26^mT/mG embryos expressing CreER driven by either the Pdx1 or Sox9 regulatory sequences were injected with tamoxifen at E8.0, embryos sectioned at E10.5, and immunofluorescence staining performed for Cdx2, Pdx1 and GFP. Recombined, membrane-targeted GFP⁺ (mGFP⁺) cells trace to the pancreatic epithelium; scattered labeled cells are also detectable in the proximal duodenum in R26^mT/mG;Pdx1-CreER (D) and R26^mT/mG;Sox9-CreER (E) embryos. dp, dorsal pancreas; vp, ventral pancreas; duo, duodenum; stom, stomach. Scale bars = 50 μm (A–E).

Figure 3. Sox9 is dispensable for pancreas induction

(A,C) Confirmation of global Sox9 deletion by WMIF staining of Sox9 in E10.5 tail-tips. (B,D) 2D projections of 3D Imaris-reconstructed z-stacks through trunks of embryos after WMIF for Foxa2 and Pdx1. Although smaller, dorsal and ventral pancreatic buds are present in E10.5 Sox9-null embryos (D,D′). Fields demarcated by white dashed boxes in B,D are shown at higher magnification in B′,D′, respectively. Only single-channel Pdx1 signal is shown in B′ and D′. (E–H″) Immunofluorescence staining of sections through the pancreatic region of Sox9^fl/fl;Foxa3-Cre (Sox9^Δgut/Δgut) and control Sox9^fl/fl embryos at E9.5. Sox9 is efficiently deleted in dorsal (G′,G″) and ventral (H′,H″) pancreatic buds of Sox9^Δgut/Δgut embryos. Dashed line in G′ and H′ demarcates the Pdx1⁺ domain. (I–T) X-Gal staining for β-galactosidase expressed from the Pdx1^LacZko allele in embryonic day (E) 10.5 and E12.5 embryos carrying combinations of mutant alleles for Pdx1 and Sox9. With increasing loss of Sox9 dosage on either Pdx1-heterozygous (I–N) or Pdx1-null (O–T) backgrounds, dorsal and ventral pancreatic buds become increasingly hypoplastic. In Pdx1^−/−;Sox9^Δgut/Δgut embryos (S,T), pancreatic buds are not discernible. Note the reduced ventral pancreas in E12.5 compound heterozygous mutants (L). Asterisks denote absence of ventral pancreas. (U) With decreasing dosage of functional Pdx1 and Sox9 alleles, pancreatic morphogenesis becomes increasingly perturbed. dp, dorsal pancreas; vp, ventral pancreas; duo, duodenum; stom, stomach; li, liver; bd, bile duct. Scale bars = 50 μm (E–H″), 70 μm (B,B′,D, D′), 200 μm (A,C), 250 μm (I–T).

Figure 4. PDX1 and SOX9 co-occupy pancreatic and intestinal genes

(A) Genome-wide distribution of PDX1 and SOX9 binding peaks within the human genome from ChIP-seq analysis of human embryonic stem cell (hESC)-derived pancreatic progenitors. (B) Venn diagram of the overlap between PDX1 binding peaks and SOX9 binding peaks (minimum of 1 bp overlap). (C) Venn diagram of the overlap between genes bound by PDX1 and SOX9, showing 2,201 genes to be co-bound by PDX1 and SOX9 (hypergeometric analysis: p-value=4.3×10⁻⁹). (D) Gene ontology (GO) analysis of PDX1 and SOX9 co-bound genes (defined as PDX1 and SOX9 binding at enhancers and/or promoters within a 200 kb window). (E) Analysis of co-bound genes revealed that 82% of the co-bound genes are expressed and 18% are not expressed in hESC-derived pancreatic progenitors. (F) ChIP-seq binding profiles (reads per million) for PDX1, SOX9 and histone modifications (H3K4me1, H3K27ac, H3K4me3, H3K27me3) at the PTF1A and CDX2 loci in hESC-derived pancreatic progenitors. Enhancers were identified based on presence of H3K27ac and H3K4me1 and absence of H3K3me3. Black boxes indicate conserved regions in mice. kB, kilobases.

Figure 5. Pdx1 and Sox9 cooperatively silence genes encoding intestinal cell fate regulators

(A) Illustration of the experimental strategy for gene expression microarray analysis. The mRNA profiles of embryonic day (E) 12.5 pancreata (n=12 per genotype) from 1.) Pdx1^+/− versus Pdx1^+/−;Sox9^+/Δgut and 2.) Sox9^+/Δgut versus Pdx1^+/−;Sox9^+/Δgut littermates were compared. (B) 3,337 and 4,486 genes were differentially-expressed between 1.) and 2.), respectively. A total of 1,817 genes were common to both sets of significantly-regulated genes (FDR<0.05) with the same sign of change (i.e. up-regulated or down-regulated). (C) Pdx1- and Sox9-co-regulated genes were identified by cross-comparing mRNA profiles of E12.5 pancreata (n=12 per genotype) from 1.) Pdx1^+/− versus Pdx1^+/−;Sox9^+/Δgut and 2.) Sox9^+/Δgut versus Pdx1^+/−;Sox9^+/Δgut littermates. A total of 1,817 genes (denoted by red pixels) were common to both sets of significantly-regulated genes (FDR<0.05) with the same sign of change. (D) Gene ontology analysis of the 1,817 Pdx1- and Sox9-co-regulated genes. (E) The top twenty Pdx1- and Sox9-co-repressed genes with the highest fold-change.

Figure 6. Pdx1 and Sox9 are necessary and sufficient to repress the intestinal lineage choice

(A–G) Immunofluorescence analysis for Pdx1 and Cdx2 on E10.5 embryos carrying various combinations of Pdx1 and Sox9 mutant alleles. In compound Pdx1;Sox9 heterozygous mutant or Pdx1 or Sox9 single-homozygous mutant embryos, Cdx2 expression is restricted to duodenal precursors and excluded from the Pdx1^high dorsal pancreas (A–F). In Pdx1^−/−;Sox9^Δgut/Δgut embryos, a duodenal-pancreatic junction is not discernable and Pdx1 and Cdx2 are co-expressed in a broad domain (arrows in G″). (H–O) Immunofluorescence staining of sections from Sox9^GOF and control littermates shows repression of the intestinal markers Cdx2 (J,K) and Onecut-2 (Oc2; L,M) in mCherry⁺ duodenal precursors in Sox9^GOF mice. Pdx1 is upregulated (H,I) but Ptf1a is not induced (N,O) in duodenal precursors in Sox9^GOF embryos. Fields demarcated by dashed boxes in A–O are shown at higher magnification in A′–O″. (P) Summary of the phenotypes observed after combined Pdx1 and Sox9 deletion or Sox9 overexpression. (Q) Graphical model summary. Our data support a model whereby Pdx1 and Sox9 cooperatively specify the pancreatic lineage by inducing the pancreatic transcription factors Nkx6.1 and Ptf1a and repressing the duodenal transcription factor Cdx2. A positive regulatory loop between Pdx1 and Sox9 maintains the pancreatic fate choice. Repression of Sox9 by Cdx2 creates bistability of the fate choice (Gao et al. 2009). dp, dorsal pancreatic bud; vp, ventral pancreatic bud; duo, duodenum; stom, stomach. Scale bar = 50 μm.