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1.
Figure 7

Figure 7. Pdx1 participates in a cross-regulatory network with Sox9, Hnf6, Foxa2, and Hnf1β.. From: The diabetes gene Pdx1 regulates the transcriptional network of pancreatic endocrine progenitor cells in mice .

(A) mRNA levels in total pancreas from Pdx1+/+, Pdx1+/ΔC, and Pdx1ΔC/ΔC pancreata at E13.5 (n = 7–9 per genotype; *P < 0.05). (B and C) ChIP with Pdx1 antisera of Min6 mouse insulinoma cells (B) or wild-type E13.5 pancreata (C). Two peaks of enrichment confirmed at +1 and +550 relative to the transcriptional start site of the Foxa2 gene and 1 peak at –31,087 relative to the transcriptional start site of the Hnf1b gene (n = 3; *P < 0.05). (D) Model depicting the role of Pdx1 in the cross-regulatory transcription factor network governing early pancreas development.

Jennifer M. Oliver-Krasinski, et al. J Clin Invest. 2009 July 1;119(7):1888-1898.
2.
Figure 6

Figure 6. Pdx1 cooperates with Hnf6 to regulate Ngn3. . From: The diabetes gene Pdx1 regulates the transcriptional network of pancreatic endocrine progenitor cells in mice .

HepG2 cells transfected with enhancer reporter Ngn3(–3379 to –3227)-tkluc and expression vectors for Pdx1, Hnf6, Sox9, Foxa2 (A) or Pdx1, Pdx1ΔC, and Hnf6 (B). *P < 0.05 versus empty vector; P < 0.01 versus Pdx1 plus Hnf6; P < 0.01 versus Pdx1; P < 0.05 versus Pdx1 plus Hnf6. n = 4. (C and D) Pdx1 physically interacts with Hnf6. 293T cells were transfected with expression vectors for Pdx1 or Pdx1ΔC and Hnf6 (C) or Pdx1 and Sox9, Hnf1β, or Foxa2 (D). Cell lysates were immunoprecipitated with mouse monoclonal Pdx1 antisera or mouse IgG control. Western blots (WBs) using polyclonal antisera for Pdx1, Hnf6, Sox9, Hnf1β, and Foxa2 are shown. A representative example of 3 independent experiments is shown. (E) Model depicting the role of Pdx1 in the transcriptional regulation of Ngn3. The question mark denotes the absence of in vivo evidence to support the in vitro studies, suggesting Foxa2-mediated regulation of Ngn3.

Jennifer M. Oliver-Krasinski, et al. J Clin Invest. 2009 July 1;119(7):1888-1898.
3.
Figure 2

Figure 2. Dose-dependent regulation of pancreas organogenesis.. From: The diabetes gene Pdx1 regulates the transcriptional network of pancreatic endocrine progenitor cells in mice .

(AE) Normal pancreas organogenesis in Pdx1ΔC/ΔC mice. (A) Foregut and accessory organs were dissected from newborn animals. The pancreas is highlighted by a black dotted line. (B) Body weights and (C) pancreatic weights were also measured at P1 (n = 6–15 per group). (D and E) Development of overt diabetes in Pdx1ΔC/ΔC mice. (D) Random blood glucose levels were measured at birth and (E) followed for 3 weeks in Pdx1ΔC/ΔC (triangles), Pdx1+/ΔC (squares), and Pdx1+/+ (circles) littermates (n = 6–21 per group; *P < 0.003 compared with both other groups). (FI) Pancreatic hypoplasia in Pdx1ΔC/– mice. (F) Foregut and accessory organs were dissected from newborn animals. The pancreata from wild-type (left panel) and Pdx1ΔC/– (right panel) mice are highlighted by the black dotted line. (G) Body weights, (H) pancreatic weights, and (I) random blood glucose levels were measured in wild-type and Pdx1ΔC/– littermates at P1 (n = 5 per group; *P < 0.003).

Jennifer M. Oliver-Krasinski, et al. J Clin Invest. 2009 July 1;119(7):1888-1898.
4.
Figure 3

Figure 3. Impaired glucose tolerance and insulin secretion in Pdx1ΔC mice. . From: The diabetes gene Pdx1 regulates the transcriptional network of pancreatic endocrine progenitor cells in mice .

(A and B) Glucose tolerance in male (A) and female (B) mice. Intraperitoneal glucose tolerance tests using 2 g glucose/kg body weight performed on 3- to 4-week-old mice (n = 4–7 per group; ANOVA; male Pdx1+/+ versus Pdx1+/ΔC, P = 0.0037; male Pdx1+/+ versus Pdx1ΔC/ΔC, P < 0.0001; male Pdx1+/ΔC versus Pdx1ΔC/ΔC, P < 0.0001; female Pdx1+/+ versus Pdx1+/ΔC, P = NS; female Pdx1+/+ versus Pdx1ΔC/ΔC, P <0.0001; female Pdx1+/ΔC versus Pdx1ΔC/ΔC, P < 0.0001). Glucose levels in Pdx1ΔC/ΔC mice rose above 500 mg/dl, the limit of detection of the handheld glucometer. (C and D) Blunted acute insulin release in Pdx1ΔC/ΔC animals. Insulin levels were measured from whole blood collected at 0 and 5 minutes after intraperitoneal glucose injection (2 g glucose/kg body weight). Both male (C) and female (D) mice were 3–4 weeks of age (n = 4–7 animals per group). *P < 0.05 compared with level at 0 minutes after injection of same genotype; P < 0.05 compared with level at 0 minutes after injection of wild-type mice.

Jennifer M. Oliver-Krasinski, et al. J Clin Invest. 2009 July 1;119(7):1888-1898.
5.
Figure 5

Figure 5. Pdx1 regulates Ngn3, at least in part, via its direct occupancy of a conserved upstream enhancer of Ngn3. . From: The diabetes gene Pdx1 regulates the transcriptional network of pancreatic endocrine progenitor cells in mice .

(A) Ngn3 mRNA levels in total pancreas of E13.5 animals (n = 5; **P = 0.005). (B) Expression of Ngn3 in PDCs transduced with pBABE or pBABE-Pdx1 retroviruses. Pdx1 and Ngn3 mRNA levels are expressed compared with empty vector (n = 6; P < 0.007). (C) Colocalization of Pdx1 and Ngn3 during pancreas development. Double immunofluorescence for Ngn3 (green) and Pdx1 (red) in wild-type pancreata. Arrows indicate double-positive cells and arrowheads mark single-positive Ngn3 cells. Scale bar: 10 μm. (D) Diagram depicting conserved TT/AAT sites in the mouse Ngn3 promoter (black and red boxes). The region homologous to the previously described human Ngn3 Cluster 1 enhancer that contains binding sites for Hnf1, Foxa2, and Hnf6 is indicated in red. (E) ChIP with Pdx1 antisera performed on at least 65 pooled wild-type E13.5 pancreata per experiment (n = 3; *P < 0.05). (F) Promoter reporter assays were performed in HepG2 cells. Cells were transfected with expression vectors for Pdx1, Pdx1ΔC or empty vector, and the promoter reporter Ngn3(–3379 to –3227)-tkluc (n = 3; ††P < 0.05 versus empty vector, P < 0.05 versus full-length Pdx1).

Jennifer M. Oliver-Krasinski, et al. J Clin Invest. 2009 July 1;119(7):1888-1898.
6.
Figure 1

Figure 1. Derivation of Pdx1ΔC/ΔC mice. . From: The diabetes gene Pdx1 regulates the transcriptional network of pancreatic endocrine progenitor cells in mice .

(A) Targeting strategy to replace the codon encoding Ser210 in the mouse Pdx1 locus with a termination codon (asterisk), thereby preventing translation of the C terminus. Schematic shows both exons (black rectangles), the 5′ diphtheria toxin A (DTA) gene, and the loxP-flanked 3′ neomycin resistance–thymidine kinase (NeoR-tk) cassettes (white rectangles). Black arrows indicate the location of genotyping primers. White arrows denote the location of loxP sites. X indicates sites of homologous recombination. (B) Predicted domain structure of the truncated Pdx1(1–210) protein, termed Pdx1ΔC, is represented below the wild-type full-length form of Pdx1(1–284). TAD, transactivation domain; HD, homeodomain. (CF) Pdx1+/+ and Pdx1ΔC/ΔC E13.5 pancreas stained with antisera raised against either N-terminal (N-term; red) or C-terminal (C-term; green) Pdx1 epitopes. Scale bar: 10 μm. (G) Pdx1 mRNA levels in total pancreas from Pdx1+/+, Pdx1+/ΔC, and Pdx1ΔC/ΔC pancreata measured at E13.5 (n = 7–8 per genotype; *P = 0.0001). (H) Representative Western blot of E13.5 total pancreas protein from Pdx1ΔC/ΔC, Pdx1+/ΔC, and Pdx1+/+ littermate controls using N-terminal–specific Pdx1 (top panel) and cyclophilin B (cyclo; bottom panel) antisera. Both full-length Pdx1 and truncated Pdx1ΔC are indicated. Quantitation from 3 separate Western blots (n = 9 animals per group) is shown in I. P < 0.00001.

Jennifer M. Oliver-Krasinski, et al. J Clin Invest. 2009 July 1;119(7):1888-1898.
7.
Figure 4

Figure 4. Specific defect in endocrine progenitor specification in Pdx1ΔC/ΔC mice. . From: The diabetes gene Pdx1 regulates the transcriptional network of pancreatic endocrine progenitor cells in mice .

(AC) Global reduction in endocrine lineages at E17.5 in Pdx1ΔC/ΔC mice. (A) Immunofluorescence for insulin (green). Scale bar: 20 μm. (B) Pancreatic area occupied by insulin-positive β cells. Values are expressed relative to wild-type mice (n = 5–9 per genotype; *P < 0.05, **P < 0.0005, P < 0.05). (C) Percentage pancreatic area at E17.5 occupied by the 5 endocrine cell types (n = 6–8 animals per genotype, 3 sections per animal; P < 0.05 compared with both other groups). PP, pancreatic polypeptide. (D and E) Normal epithelial area in Pdx1ΔC/ΔC mice. (D) Pancreatic epithelial area measured from 3 (E13.5) or 5 (E11.5) H&E-stained sections. (E) Relative mRNA levels of Hnf1a, Hnf4a, and Ptf1a in E13.5 pancreas (n = 7–9 per genotype). (F and G) Reduction in Ngn3+ pancreatic endocrine progenitor cells in Pdx1ΔC/ΔC mice. (F) Immunostaining for Ngn3 (brown) with hematoxylin counterstain (blue). Scale bar: 50 μm. (G) Number of Ngn3+ cells per unit of epithelial area (E11.5) or pancreatic area (E13.5–P1) quantified from 3 (E13.5–P1) or 5 (E11.5) tissue sections per animal (n = 4–8 per genotype; *P < 0.05 compared with wild-type mice; P < 0.05 compared with Pdx1+/ΔC mice). (H and I) Ngn3+ endocrine progenitors replicate normally in Pdx1ΔC/ΔC mice. (H) Representative example of Ki67+Ngn3+ double-positive cell. Arrows point to the same cell in all panels. Scale bar: 10 μm. (I) Percentage of total Ngn3+ cells that are Ki67+ or BrdU+; more than 100 Ngn3+ cells per animal were counted (n = 4–5 per genotype).

Jennifer M. Oliver-Krasinski, et al. J Clin Invest. 2009 July 1;119(7):1888-1898.

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