PDX:PBX complexes are required for normal proliferation of pancreatic cells during development

Abstract

The homeobox factor PDX-1 is a key regulator of pancreatic morphogenesis and glucose homeostasis; targeted disruption of the PDX-1 gene leads to pancreatic agenesis in pdx-1(-/-) homozygotes. Pdx-1 heterozygotes develop normally, but they display glucose intolerance in adulthood. Like certain other homeobox proteins, PDX-1 contains a consensus FPWMK motif that promotes heterodimer formation with the ubiquitous homeodomain protein PBX. To evaluate the importance of PDX-1:PBX complexes in pancreatic morphogenesis and glucose homeostasis, we expressed either wild-type or PBX interaction defective PDX-1 transgenes under control of the PDX-1 promoter. Both wild-type and mutant PDX-1 transgenes corrected glucose intolerance in pdx-1 heterozygotes. The wild-type PDX-1 transgene rescued the development of all pancreatic lineages in pdx-1(-/-) animals, and these mice survived to adulthood. In contrast, pancreata from pdx-1(-/-) mice expressing the mutant PDX-1 transgene were hypoplastic, and these mice died within 3 weeks of birth from pancreatic insufficiency. All pancreatic cell types were observed in pdx-1(-/-) mice expressing the mutant PDX-1 transgene; but the islets were smaller, and increased numbers of islet hormone-positive cells were noted within the ductal epithelium. These results indicate that PDX-1:PBX complexes are dispensable for glucose homeostasis and for differentiation of stem cells into ductal, endocrine, and acinar lineages; but they are essential for expansion of these populations during development.

Figure 1

Expression of wild-type or PBX interaction defective PDX-1 transgenes rescues glucose homeostasis in pdx-1 (+/−) mice. (A) Transient transfection assay of wild-type and PBX interaction defective (Pdxμ) PDX-1 constructs in 293T cells, with a TSEII luciferase reporter containing two PDX:PBX heterodimer recognition sites. Cells were transfected with wild-type or mutant PDX plus E2A-PBX expression vector where indicated. The first bar on the left represents control transfection with empty vector alone plus TSEII reporter. Luciferase activity shown after normalizing to β-galactosidase activity from cotransfected RSV-βgal plasmid. (B) Genotypic analysis of mice expressing PDX-1 transgene. (Left) Inheritance of PDX-1 transgene was evaluated by PCR assay with primers that are selective for rat PDX-1. (Right) Mice heterozygous or homozygous for targeted disruption of the murine PDX-1 gene were identified by genomic Southern blot analysis with a ³²P-labeled PDX-1 cDNA probe. Insertion of the inactivating β-galactosidase gene is indicated by a 3.8-kb vs. a 3.0-kb EcoRI fragment. (C) Wild-type (Lane 1) and mutant (Lane 2) PDX-1 transgenes are comparably expressed in transgenic mice. (Left) RNase protection assay of total RNA from whole pancreas of adult mice, with the use of ³²P-labeled wild-type PDX-1 antisense RNA probe. The shorter fragment in mutant mice results from RNase digestion in sequences encoding mutant FPWMK/AAGGQ motif. Lane 3, control nontransgenic littermate RNA; the rat PDX-1 probe does not recognize murine PDX-1. Lane 4, position of undigested PDX-1 antisense probe. Lane 5, molecular weight marker. (Right) Western blot assay of whole pancreas extract, showing levels of PDX-1 protein in wild-type, pdx-1(+/−), pdx-1(+/−);mTg, and pdx-1(−/−);wtTg mice. (D) Line graph of blood glucose levels in wild-type (—■—), pdx-1(+/−) heterozygous (– –⧫– –), pdx-1(+/−); wtTg, and pdx-1 (+/−);mTg mice (⋅⋅⋅●⋅⋅⋅) after i.p. glucose injection. Results for pdx-1(+/−); wtTg and pdx-1(+/−);mTg mice were indistinguishable.

Figure 2

Islet hypoplasia in pdx-1(−/−);mTg neonates. (A) Comparison of pdx-1(−/−);mTg (smaller) and wild-type (larger) littermates 16 days after birth. (B) Whole mount of pdx-1(−/−); wtTg and pdx-1(−/−); mTg mice. Ventral (Left) and dorsal (Right) views are included. p, pancreas; d, duodenum; sp, spleen; s, stomach. (C) Immunocytochemical analysis of insulin (red, arrows) and glucagon + somatostatin (green, asterisks) cells in sections from pdx-1(−/−); wtTg (Upper) and pdx-1(−/−):mTg (Lower). (D) Whole mount showing smaller developing islets (dark blue; arrowheads) in pdx-1(−/−) mTg compared with pdx-1(+/−) mTg^t mice. Blue color indicates expression of β-galactosidase from the endogenous pdx-1 allele pdx-1^lacZKO (1).

Figure 3

Pancreatic islets of neonatal pdx-1(−/−);mTg mice are smaller and have disrupted architecture. (A–D) Sections of pdx-1(+/−); mTg islets. (E–H) Sections of pdx-1(−/−); mTg islets. pdx-1(−/−); mTg islets are much smaller and are located in close proximity to the ducts. Immunohistochemistry (brown) shows insulin-positive (F), glucagon-positive (G), and somatostatin-positive (H) cells in pdx-1(−/−); mTg islets. Glucagon- and somatostatin-positive cells (G and H) are not localized at the islet periphery as in wild-type islets (compare with C and D).

Figure 4

Pancreata from pdx-1 (−/−);mTg^t mice show a defect in proliferation and contain increased numbers of hormone-positive cells in the ductal epithelium. (A) Immunocytochemical staining of whole-pancreas sections from pdx-1 (−/−);wtTg and pdx-1 (−/−);mTg neonates, with K_i-67 antiserum as the marker of cell proliferation. (B) Immunocytochemistry of pancreas sections from neonatal wild-type mice, showing the highest levels of PBX-1 (green) in ductal cells (D), followed by islet cells. Acinar cells stain weakly for PBX-1. Insulin immunostaining (red) is overlaid to indicate the islet. (C) Immunohistochemical detection of insulin (Left), glucagon (Middle), and somatostatin (Right) cells (arrowheads) within the pancreatic ductal epithelium of neonatal pdx-1 (−/−);mTg mice. Mice of all other genotypes did not show hormone-positive cells within ducts in any sections examined.