Decreased proliferation of early pancreatic precursors in Ptf1a hypomorphic mutant mice. A–C: Sections of pancreata stained with X-gal and phosphorylated histone H3 (pHH3), a mitosis maker, at e12.5 (A–C). D and E: The ratios of pHH3-positive cells/Ptf1a lineage-labeled pancreatic epithelial cells at e10.5 (D) and e12.5 (E). Data are expressed as means ± SE (n = 4). *P < 0.05 compared with Ptf1a^cre/+ mice. (Please see http://dx.doi.org/10.2337/db07-1558 for a high-quality digital representation of this figure.)

Impaired glucose tolerance with insufficient insulin secretion in Ptf1a hypomorphic mutant mice. A: Blood glucose levels during intraperitoneal glucose tolerance test (data from 21- to 30-day-old wild-type, n = 4; Ptf1a^cre/+, n = 16; Ptf1a^cbll/cbll, n = 33; and Ptf1a^cre/cbll mice, n = 6). Note that glucose intolerance is observed in a Ptf1a dosage–dependent manner. B: Serum insulin levels 15 min after intraperitoneal glucose injection measured by enzyme-linked immunosorbent assay (data from 23- to 30-day-old wild-type, n = 4; Ptf1a^cbll/cbll, n = 9; and Ptf1a^cre/cbll mice, n = 5). Serum insulin levels are inappropriately lower in hypomorphic mutants compared with wild-type mice. Data are expressed as means ± SE. *P < 0.05 compared with wild-type mice.

Reduction of Ptf1a dosage results in small body size and pancreatic hypoplasia. A: Quantification of Ptf1a mRNA in pancreata by real-time RT-PCR. The relative expression levels of Ptf1a mRNA in pancreata from Ptf1a hypomorphic mice are shown compared with wild-type mice at e12.5 and P1. Data are expressed as means ± SE (n = 4). *P < 0.05, **P < 0.01. B and C: Gross appearance of Ptf1a^cre/cbll mice compared with Ptf1a^cre/+ littermates (B) and Ptf1a^cbll/cbll mice compared with wild-type littermate (C) at P23. D: Body weights of Ptf1a^cre/+, Ptf1a^cbll/cbll, and Ptf1a^cre/cbll mice during postnatal periods. Data are expressed as means ± SE. *P < 0.05 compared with Ptf1a^cre/+ mice. E–G: Macroscopic views of pancreata of Ptf1a^cre/+;R26r (E), Ptf1a^cbll/cbll (F), and Ptf1a^cre/cbll;R26r mice (G) at P7 stained with X-gal. Broken lines delineate pancreata in F, H, and I. At P30, pancreatic hypoplasia is observed in Ptf1a^cbll/cbll (I) and Ptf1a^cre/cbll;R26r mice (J) compared with Ptf1a^cre/+ mice (H). (Please see http://dx.doi.org/10.2337/db07-1558 for a high-quality digital representation of this figure.)

Pdx1 expression levels are reduced in early pancreatic precursors in Ptf1a hypomorphic mutant mice. A–E: Sections of pancreata stained with X-gal (blue) and Pdx1 (red). F and G: Sections of pancreata stained for hematoxylin and eosin (upper panels) and Pdx1 protein (lower panels). Signal intensity is similar in the distal stomach epithelium, so that the Pdx1 expression level in pancreatic precursors can be judged compared with it. Note that Pdx1 expression is reduced in pancreatic precursors at e10.5, e11.5, and e12.5 in Ptf1a^cre/cbll mice compared with Ptf1a^cre/+ and wild-type mice. G: Dashed line shows pancreatic epithelium. In Ptf1a null mice, Pdx1 expression is also reduced at e12.5 (E). H: Quantification of Pdx1 mRNA in pancreatic rudiments by real-time RT-PCR. The relative expression levels of Pdx1 in pancreatic rudiments from Ptf1a hypomorphic mice are shown compared with wild-type mice at e12.5. Data are represented as means ± SE (n = 4). *P < 0.05 compared with wild-type mice. Du, duodenum; St, distal stomach; P, pancreas. (Please see http://dx.doi.org/10.2337/db07-1558 for a high-quality digital representation of this figure.)

Pdx1 expression persists in the postnatal pancreatic duck-like epithelial cells in Ptf1a hypomorphic mutant mice. Sections of pancreata stained for X-gal (blue) and Pdx1 (red) (A–G and K) and stained for Pdx1 (H and I). The expression level of Pdx1 protein is similar among all genotypes at e14.5 and e17.5. At P1 and P30, strong Pdx1 expression is observed in pancreatic β-cells in Ptf1a^cre/+ mice (arrowhead in E), and its lower expression is maintained in pancreatic exocrine cells, whereas Pdx1 expression is not observed in duct cells (arrows in E). Note that a moderate level of Pdx1 expression persists in the pancreatic duck-like epithelial cells in Ptf1a^cre/cbll mice at P1 (arrows in F) and P30 (G) and in Ptf1a^cbll/cbll mice at P7 (arrows in I). The arrowhead in F shows strong Pdx1 expression in β-cells in Ptf1a^cre/cbll mice. J: Quantification of Pdx1 mRNA in pancreatic rudiments by real-time RT-PCR. The relative expression levels of Pdx1 in pancreatic rudiments of Ptf1a^cre/cbll mice are shown compared with those of wild-type mice at e14.5. Data are expressed as means ± SE (n = 4). Du, duodenum; St, distal stomach; P, pancreas. (Please see http://dx.doi.org/10.2337/db07-1558 for a high-quality digital representation of this figure.)

Endocrine pancreas development in Ptf1a hypomorphic mutant mice. A–L: Sections of pancreata costained for insulin (red) and glucagon (green). Normal islet structures are observed in Ptf1a^cre/+ mice at P1 (D), whereas they are not observed in Ptf1a^cbll/cbll (H) and Ptf1a^cre/cbll mice (L). M: Ratios of endocrine cell types at e18.5 in Ptf1a^cre/+ (red bars), Ptf1a^cbll/cbll (green bars), and Ptf1a^cre/cbll mice (purple bars). N and O: Relative total β-cell number at e14.5 (N) and e18.5 (O) in each genotype. P–R: Representative sections of pancreatic islets colabeled for insulin (Ins, red) and glucagon (Glu, green), somatostatin (Som, green), or pancreatic polypeptide (PP, green) at P30. Note that pancreatic α-, ɛ-, and δ-cells are increased in number and α-cells are intermingled with β-cells in Ptf1a^cbll/cbll and Ptf1a^cre/cbll mice. S–X: Colabeling for Pdx1 (red) and insulin (green) and staining for Glut2 (green) in islets. Pancreatic β-cells express Pdx1 and Glut2 in all genotypes at P30, suggesting their maturation. Data are expressed as means ± SE (n = 3). *P < 0.05 compared with Ptf1a^cre/+ mice. (Please see http://dx.doi.org/10.2337/db07-1558 for a high-quality digital representation of this figure.)

Cell fate conversion of pancreatic precursors into the CBD and duodenal cells in hypomorphic Ptf1a^cre/cbll mice. A–L: Macroscopic views of pancreatic regions stained with X-gal. Hypomorphic pancreas is observed from the beginning of pancreatic bud formation at e10.5 (B) and becomes more apparent as development proceeds to e14.5 (D) and e17.5 (F and H) in Ptf1a^cre/cbll;R26r mice. Broken lines in (I) delineate moderate hypoplastic pancreas in Ptf1a^cbll/cbll mice at e17.5. Note that substantial proportions of the Ptf1a lineage-labeled cells are ectopically observed in the CBD (red arrows in H) and the duodenum (Du) (outlined boxes in H and L) in Ptf1a^cre/cbll;R26r mice at e17.5 (H) and P7 (L). Endogenous β-gal activity is observed in the duodenum as background. The cell fate conversion to the CBD is observed in Ptf1a^cre/cbll;R26r mice as early as e14.5 (red arrows in D). Ectopic Ptf1a lineage-labeled areas are also observed in the CBD and duodenum in Ptf1a^cre/cre;R26r mice at e17.5 (J and K). M–Q: Histological analyses of Ptf1a^cre/cbll;R26r mice at P7. Substantial proportions of Ptf1a lineage-labeled cells are misspecified and located to the CBD epithelium (arrows in M and N [M and N are the same sections]), including the peribiliary glands (arrows in O) and duodenal epithelial cells (P) stained with hematoxylin and eosin, which are positive for cytokeratin (red; arrow in N) and the intestinal marker Cdx2 (brown; Q), respectively, in Ptf1a^cre/cbll;R26r mice. (Please see http://dx.doi.org/10.2337/db07-1558 for a high-quality digital representation of this figure.)

Exocrine cytodifferentiation is reduced during development with delayed branching morphogenesis in hypomorphic mutant mice. A–T: Sections of pancreata stained with X-gal (blue) and amylase (Amy, green). (Left panels in D, G, J, M, P, and R: hematoxylin and eosin [HE] staining.) Extensive branching morphogenesis of the pancreatic epithelial tree is observed at both e12.5 and e14.5 in Ptf1a^cre/+;R26r mice (A and C), whereas almost no branching morphogenesis is observed in Ptf1a^cre/cbll;R26r mice at those stages (B and E). At e17.5, branching of the pancreatic epithelium could be observed in Ptf1a^cre/cbll;R26r mice (H). At e14.5, amylase-positive cells are observed in Ptf1a^cre/+ mice (C), whereas they are not seen at all in Ptf1a^cre/cbll (E) and Ptf1a^cbll/cbll mice (D). Amylase-positive cells are first apparent at P1 in Ptf1a^cre/cbll mice (K), but they are premature in that the nucleus-to-cytoplasm ratio in pancreatic exocrine cells of Ptf1a^cre/cbll mice at P1 is as large as that of Ptf1a^cre/+ mice at e14.5 (outlined boxes in K and C). In some areas, normal pancreatic acinar structures are observed at P7 in Ptf1a^cre/cbll mice (Q), but the nucleus-to-cytoplasm ratio of the acinar cells in Ptf1a^cre/cbll mice at P7 is as large as that of Ptf1a^cre/+ mice at e17.5 (outlined boxes in Q and F). At P30, in the ventral pancreas (v.p.) and the proximal dorsal pancreas (d.p.) in Ptf1a^cre/cbll mice, amylase-positive acinar structures are observed (arrows and outlined box in R and S), whereas, in their dorsal pancreas, the duct-like structures still exist (arrowheads and dashed-line box in R) and rarely express amylase (T). In Ptf1a^cbll/cbll mice, immature duct-like epithelial cells (arrowheads and dashed-lined boxes in J and M) are observed at P1 and P7, but acinar tissues are fully developed at P30 (P). U: Stool smears of Ptf1a^cre/+, Ptf1a^cbll/cbll, and Ptf1a^cre/cbll mice stained with Oil Red O. Large numbers of undigested lipid droplets are observed in stool smears of Ptf1a^cre/cbll mice, indicative of maldigestion. Very rarely, a small amount of lipid droplets is also observed in stool smears of Ptf1a^cbll/cbll mice. (Please see http://dx.doi.org/10.2337/db07-1558 for a high-quality digital representation of this figure.)