GATA4 and GATA6 control mouse pancreas organogenesis

Abstract

Recently, heterozygous mutations in GATA6 have been found in neonatal diabetic patients with failed pancreatic organogenesis. To investigate the roles of GATA4 and GATA6 in mouse pancreas organogenesis, we conditionally inactivated these genes within the pancreas. Single inactivation of either gene did not have a major impact on pancreas formation, indicating functional redundancy. However, double Gata4/Gata6 mutant mice failed to develop pancreata, died shortly after birth, and displayed hyperglycemia. Morphological defects in Gata4/Gata6 mutant pancreata were apparent during embryonic development, and the epithelium failed to expand as a result of defects in cell proliferation and differentiation. The number of multipotent pancreatic progenitors, including PDX1+ cells, was reduced in the Gata4/Gata6 mutant pancreatic epithelium. Remarkably, deletion of only 1 Gata6 allele on a Gata4 conditional knockout background severely reduced pancreatic mass. In contrast, a single WT allele of Gata4 in Gata6 conditional knockout mice was sufficient for normal pancreatic development, indicating differential contributions of GATA factors to pancreas formation. Our results place GATA factors at the top of the transcriptional network hierarchy controlling pancreas organogenesis.

Figure 1. Single inactivation of Gata4 and Gata6 does not affect pancreas formation.

(A–D) Immunohistochemical analyses show strong expression of GATA4 in acinar cells (arrowheads in A) and GATA6 in endocrine cells (arrows in C) in pancreatic sections of control mice at P1. Loss of GATA4 (B, arrowheads) and GATA6 (D, arrows) in newborn conditional mutant mice is confirmed by immunohistochemical analysis. (E–G) Gross morphology of neonatal WT and conditional mutant guts. H&E-stained sections of newborn control (H), Gata4 (I), and Gata6 (J) conditional knockout pancreata does not reveal major defects in pancreas architecture. Mature acinar (amylase), ductal (mucin) (K–M), and islet (insulin and glucagon) markers (N–P) are normally expressed in single Gata4 and Gata6 conditional knockout mice. Insets in H–M show higher magnification of acinar cells. Mild ductal dilation in acinar cells of Gata4^flox/flox;Pdx1-Cre is observed (I and L, insets). Nuclei are counterstained with DAPI in K–P. Scale bars: 50 μm; 25 μm (insets).

Figure 2. Pancreatic agenesis in Gata4/Gata6 double mutant.

Gross appearance of neonatal WT and conditional mutant guts (A–D) and pancreatic sections stained with H&E (E–H) reveal the abnormal morphology of double-mutant pancreata at P1. Gata4^flox/flox;Gata6^flox/+;Pdx1-Cre mice show pancreatic hypoplasia with scarcity of acinar cells (B and F). Gata4^flox/+;Gata6^flox/flox;Pdx1-Cre mice display normal pancreatic mass and architecture (C and G). Immunohistochemical analysis shows reduced expression of the acinar marker, amylase, in Gata4^flox/flox;Gata6^flox/+;Pdx1-Cre pancreatic sections (J) compared with Gata4^flox/+;Gata6^flox/flox;Pdx1-Cre (K) and control littermates (I). The double-mutant pancreatic remnant displays cystic structures surrounded by abundant stroma (H, L, P, and Q). The cystic structures express mucin (L) and cytokeratin 19 (S) and react with DBA lectin (T), which are markers of differentiated ductal cells. Immunostaining for E-cadherin confirms the epithelial nature of the cysts (R). Insulin and glucagon staining reveals normal differentiation of the endocrine lineage in Gata4^flox/flox;Gata6^flox/+;Pdx1-Cre (N) and Gata4^flox/+;Gata6^flox/flox;Pdx1-Cre (O) mutant mice in comparison with control mice (M). In contrast, Gata4/Gata6 double-mutant mice lack endocrine cells (P). Counterstaining with DAPI was performed to reveal nuclei. Scale bars: 50 μm.

Figure 3. Pancreatic epithelial expansion is impaired in the absence of GATA4 and GATA6 activity.

The pancreatic epithelia of control, Gata4^flox/flox;Gata6^flox/+;Pdx1-Cre, and Gata4^flox/+;Gata6^flox/flox;Pdx1-Cre embryos at E13.5 display normal morphology (A–C), whereas double-mutant pancreatic epithelium appears disorganized and reduced in epithelial area (D). Immunohistochemistry analysis of the mitotic marker phospho-histone H3 (PHH3) reveals a significant reduction in proliferating pancreatic epithelial cells in the double-mutant (H) compared with littermate embryos at E13.5 (E–G). Immunostaining with another proliferation marker, Ki67, confirms the reduction in proliferation of E13.5 pancreatic epithelial cells in the double mutant compared with littermates (I–L). Counterstaining with DAPI was performed to reveal nuclei. Quantification of proliferating cells, measured as the number of PHH3 and Ki67-positive cells (M and N, respectively) per E-cadherin–positive cells. *P < 0.05. Scale bars: 50 μm.

Figure 4. Endocrine and acinar differentiation are compromised in Gata4/Gata6 double-mutant embryos.

The enzyme Carboxipeptidase A1 (Cpa1) is expressed in the multipotent progenitor cell population located at the tip of the E13.5 branching epithelium in control, Gata4^flox/flox;Gata6^flox/+;Pdx1-Cre, and Gata4^flox/+;Gata6^flox/flox;Pdx1-Cre mice (A–C, arrowheads). In stark contrast, Cpa1⁺ cells were not detected in the double-mutant pancreatic epithelium (D). The proendocrine markers Ngn3 (E–G) and Nk2.2 (I–K) were mainly expressed in the epithelial trunk of control, Gata4^flox/flox;Gata6^flox/+;Pdx1-Cre, and Gata4^flox/+;Gata6^flox/flox;Pdx1-Cre embryos at E13.5. On the contrary, endocrine differentiation is disrupted in the double-mutant embryos as the number of cells expressing Ngn3 (H) and Nkx2.2 (L) are reduced compared with littermate embryos. The pancreatic epithelium is outlined in white in H and L. Counterstaining with DAPI was performed to reveal nuclei. Scale bars: 50 μm.

Figure 5. Reduced number of MPCs in Gata4/Gata6 double-mutant mice.

Control embryos show strong expression of all multipotent pancreatic progenitor markers, Pdx1, Ptf1a, Sox9, and Nkx6.1 at E13.5 (A, E, I, and M, respectively). Similarly, Gata4^flox/+;Gata6^flox/flox;Pdx1-Cre embryos display normal distribution and expression levels (C, G, K, and O). However, Gata4^flox/flox;Gata6^flox/+;Pdx1-Cre mice show a significant decrease of cell numbers expressing Pdx1, Ptf1, and Nkx6.1 (B, F, and N) and a moderate reduction in the number of Sox9⁺ cells (J). The reduced number of cells expressing all pancreatic progenitor markers is even more dramatic in the double mutant (D, H, L, and P). (Q) qPCR analysis of multipotent pancreatic progenitor markers in E13.5 pancreata. *P < 0.02; **P < 0.001. Scale bars: 50 μm.

Figure 6. GATA4 and GATA6 bind to the Pdx1 conserved area III in vitro and in pancreatic cell line.

(A) Highly conserved region in the cis-regulatory area III of Pdx1. Two conserved GATA sites, as revealed by bioinformatics analysis, are shown in blue boxes. Numbers indicate the position of the GATA sites relative to the Pdx1 translational start site. Point mutations introduced into GATA sites, G1m and G2m, are indicated in red lowercase. Asterisks denote nucleotides that have been perfectly conserved between mouse and human. (B) Recombinant GATA4 and GATA6 proteins are able to bind to G1 and G2 GATA sites of the Pdx1 enhancer as shown by EMSA. Competition experiments were performed by adding excess unlabeled probes of G1, G2, or control (denoted as c in competitor row) GATA sites, and the corresponding mutant versions (G1m, G2m, or cm) to the binding reaction. (C) ChIP experiments performed in mouse pancreatic ductal cells (mPAC cells) using specific GATA4 and GATA6 antibodies (lanes 2, 3, respectively) and nonspecific anti-IgG (lane 4) show that anti-GATA4 and anti-GATA6 antibodies are able to immunoprecipitate the GATA sites of the Pdx1 conserved area III, but not nonspecific genomic regions. Lane 1 contains PCR products from input DNA (Inp) amplified prior to immunoprecipitation. Sizes of the PCR products in bp are shown on the right. (D) A WT Pdx1 promoter-luciferase construct (pGL3-Pdx1-WT) is significantly activated by endogenous factors present in mPAC cells compared with the activity of the empty reporter pGL3 vector. Mutations in the GATA sites of Pdx1 (pGL3-Pdx1-mut) significantly attenuate the luciferase activity. *P = 0.002; **P = 0.005.

Figure 7. Conserved GATA sites in area III are required for Pdx1 enhancer activation in vivo.

Whole-mount (A, C, E, and G) and transversal sections (B, D, F, and H) of representative Pdx1-WT-lacZ and Pdx1-mut-lacZ transgenic embryos stained with X-gal. β-gal activity in both dorsal and ventral pancreatic buds is first observed in Pdx1-WT-lacZ embryos at E9.5 (A and B). In contrast, no X-gal staining is observed in the pancreatic buds of Pdx1-mut-lacZ embryos (E and F, asterisk). By E10.0, β-gal activity in the Pdx1-mut-lacZ embryos (square in G, arrowhead in H) is dramatically reduced compared with Pdx1-WT-lacZ embryos (C and D). At E13.5, all the pancreatic epithelial cells in Pdx1-WT-lacZ embryos show homogeneous β-gal activity (I). Similarly, strong X-gal staining is observed in most of the pancreatic cells of Pdx1-WT-lacZ embryos at E17.5 (M). In contrast, lacZ expression is markedly reduced in the pancreatic epithelium of Pdx1-mut-lacZ embryos both at E13.5 (arrows in N) and at E17.5 (R). Immunofluorescence staining at E13.5 reveals a complete overlapping expression pattern of Pdx1 and β-gal in Pdx1-WT-lacZ pancreas (J–L), while only a fraction of Pdx1⁺ cells express β-gal in Pdx1-mut-lacZ embryos (O–Q). dp, dorsal pancreas; vp, ventral pancreas. Scale bars: 50 μm.