GATA4 and GATA6 regulate pancreatic endoderm identity through inhibition of hedgehog signaling

Abstract

GATA4 and GATA6 are zinc finger transcription factors that have important functions in several mesodermal and endodermal organs, including heart, liver and pancreas. In humans, heterozygous mutations of either factor are associated with pancreatic agenesis; however, homozygous deletion of both Gata4 and Gata6 is necessary to disrupt pancreas development in mice. In this study, we demonstrate that arrested pancreatic development in Gata4(fl/fl); Gata6(fl/fl); Pdx1:Cre (pDKO) embryos is accompanied by the transition of ventral and dorsal pancreatic fates into intestinal or stomach lineages, respectively. These results indicate that GATA4 and GATA6 play essential roles in maintaining pancreas identity by regulating foregut endodermal fates. Remarkably, pancreatic anlagen derived from pDKO embryos also display a dramatic upregulation of hedgehog pathway components, which are normally absent from the presumptive pancreatic endoderm. Consistent with the erroneous activation of hedgehog signaling, we demonstrate that GATA4 and GATA6 are able to repress transcription through the sonic hedgehog (Shh) endoderm-specific enhancer MACS1 and that GATA-binding sites within this enhancer are necessary for this repressive activity. These studies establish the importance of GATA4/6-mediated inhibition of hedgehog signaling as a major mechanism regulating pancreatic endoderm specification during patterning of the gut tube.

Keywords: Foregut endoderm; GATA4; GATA6; Hedgehog; Mouse; Pancreas.

Fig. 1.

Transcriptome analysis of E12.5 pancreata revealed upregulation of hedgehog pathway in pDKO pancreatic anlage. (A) Representative brightfield images of E12.5 dissected visceral tissue from control and pDKO R26R:Tomato embryos. The fluorescent signal guided dissection of Pdx1-derived lineages. (B) Heatmap display of significant differentially expressed genes (P<0.0008). (C) Pie chart representation of 251 genes that are significantly altered in the pDKO embryos; 118 genes are downregulated and 133 genes are upregulated. (D) qRT-PCR confirmation of highly increased expression of several components in the hedgehog pathway. Error bars represent s.e.m. *P<0.05, ***P<0.001 (n=3). (E) Shh in situ hybridization of E10.5 pancreatic epithelium demonstrates increased Shh expression in pDKO pancreatic epithelium.

Fig. 2.

Pancreatic lineage cells in pDKO embryos switch cell fates. (A-H′) Representative sections of immunofluorescence-stained pDKO dorsal pancreatic lineage cells from E10.5 embryos. In control embryos, pancreatic lineage cells express Pdx1 (blue) (B) but do not express Sox2 (green) (C). Merged images (D,D′) show overlapping Tomato-expressing (red) cells derived from the Pdx1 lineage and Pdx1-expressing cells (blue). In pDKO embryos (E-H′), Pdx1 lineage cells (E) do not express Pdx1 (F), but instead express Sox2 (G). Merged images (H,H′) show overlapping Tomato- and Sox2-expressing cells, suggesting that pancreatic-derived lineages are converted to a stomach identity. (I-P″) Representative sections of E9.5 pDKO foregut endoderm showing that pancreatic lineage cells express intestinal markers. In control embryos, Tomato+ pancreatic-derived lineages (I) express Pdx1 (J), but not Cdx2 (K), confirming their pancreatic identity. Images from merged channels (L,L′) show overlapping expression of Tomato+ cells and Pdx1+ cells. In the pDKO ventral pancreatic domain, a few pancreatic lineage cells (M) have lost their expression of Pdx1 (N) and start to express the intestinal cell marker Cdx2 (O), suggesting that these cells are converting into intestinal cells. Merged images are shown in P-P″. (R-Y′) At E10.5, control pancreatic lineage cells (R) continue to express Pdx1 (S), but not Cdx2 (T). Merged images are shown in U and U′. In pDKO, increasing numbers of Pdx1-derived Tomato+ lineage cells (V) do not express Pdx1 (W) and instead express Cdx2 (X). Merged channels (Y,Y′) demonstrate overlapping expression of Tomato and Cdx2, suggesting that these cells are switching to an intestinal cell fate. Boxes indicate regions shown at higher magnification in D′, H, L′, P′, U′ and Y′.

Fig. 3.

GATA4 and GATA6 inhibit the activity of the Shh endoderm enhancer MACS1. (A) Schematic of the MACS1 enhancer element relative to the transcriptional start site of Shh. The MACS1 element is located ∼740 kb upstream of the Shh transcriptional start site. The MACS1 element is 806 base pairs, and is predicted to have four consensus GATA-binding sites (red text). Mutations in the GATA-binding sites are indicated by blue text. Four potential cryptic GATA-binding sites are designated with green text. (B) PCR analysis of E14.5 pancreata ChIP samples on four putative GATA-binding sites. The first two sites have strong binding for GATA4 and GATA6, whereas the third site has very weak binding. The fourth site has no detectable binding for GATA4 and GATA6. (C) Quantification of band intensities relative to their own inputs. Lane 1=Gata site 1 (GATA4 and GATA6 binding is about 25% and 63%, respectively); Lane 2=Gata site 2 (36% for GATA4 and 41% for GATA6); Lane 3=Gata site 3 (20% for GATA4 and 31% for GATA6); Lane 4=Gata site 4 (0.4% for GATA4 and 2.1% for GATA6); Lane 5=CPA1 site (no measurable binding). n=3. (D) Luciferase assay in α-TC6 cells. The pGL4.27-MACS1 plasmid has high luciferase activity in α-TC6 cells; this effect is suppressed by expression of GATA4 or GATA6. Co-transfection of GATA4 and GATA6 additively suppressed MACS1 expression. pGL4.27-MACS1mut refers to the MACS1 fragment that is mutated for the four GATA sites (red text in A). Error bars represent s.e.m. ***P<0.001. n=5. (E) Model summarizing the downstream consequences of a lack of GATA4 and GATA6 in the pancreatic epithelium. GATA4 and GATA6 expression in the presumptive pancreatic foregut endoderm represses hedgehog signaling to allow for the induction of Pdx1 expression (blue stripes) and initiation of pancreatic fates. In the absence of GATA4 and GATA6, hedgehog signaling is erroneously activated in the pre-pancreatic endoderm domain. As a result, the PDX1 domain is respecified into stomach fates expressing SOX2 (green stripes) and intestinal fates expressing CDX2 (yellow stripes). In the absence of GATA4 and GATA6, a small remnant of tissue co-expressing PDX1 and SOX2 or CDX2 can be detected. This domain transiently expresses pancreatic markers.