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. 2016 Jun 21;15(12):2637-2650.
doi: 10.1016/j.celrep.2016.05.040. Epub 2016 Jun 9.

Threshold-Dependent Cooperativity of Pdx1 and Oc1 in Pancreatic Progenitors Establishes Competency for Endocrine Differentiation and β-Cell Function

Kathryn D Henley #  1   2 , Diana E Stanescu #  3   4 , Peter A Kropp #  2   5 , Christopher V E Wright  1   2 , Kyoung-Jae Won  3 , Doris A Stoffers #  3 , Maureen Gannon #  1   2   5   6   7
Affiliations

Threshold-Dependent Cooperativity of Pdx1 and Oc1 in Pancreatic Progenitors Establishes Competency for Endocrine Differentiation and β-Cell Function

Kathryn D Henley et al. Cell Rep. .

Abstract

Pdx1 and Oc1 are co-expressed in multipotent pancreatic progenitors and regulate the pro-endocrine gene Neurog3. Their expression diverges in later organogenesis, with Oc1 absent from hormone+ cells and Pdx1 maintained in mature β cells. In a classical genetic test for cooperative functional interactions, we derived mice with combined Pdx1 and Oc1 heterozygosity. Endocrine development in double-heterozygous pancreata was normal at embryonic day (E)13.5, but defects in specification and differentiation were apparent at E15.5, the height of the second wave of differentiation. Pancreata from double heterozygotes showed alterations in the expression of genes crucial for β-cell development and function, decreased numbers and altered allocation of Neurog3-expressing endocrine progenitors, and defective endocrine differentiation. Defects in islet gene expression and β-cell function persisted in double heterozygous neonates. These results suggest that Oc1 and Pdx1 cooperate prior to their divergence, in pancreatic progenitors, to allow for proper differentiation and functional maturation of β cells.

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Figures

Figure 1
Figure 1. Combined heterozygous reduction in Pdx1 and Oc1 gene dosage has a broad impact on the transcriptional network of endocrine pancreas progenitors
(A) Hierarchical clustering of 2331 differentially expressed genes in individual pancreata at e15.5 from Pdx1LacZ/+ (n=4), Oc1+/− (n=3), and Pdx1LacZ/+;OC1+/− mice (n=5) compared to WT (n=4); (B) Venn diagram depicting the number of altered genes in the Pdx1LacZ/+, Oc1+/− and Pdx1LacZ/+;Oc1+/−; (C) Endocrine system development and function gene ontology category in each genotype, according to negative log of p-value from Fisher exact test. The numbers above each column represent the number of genes enriched in each category; (D) Heat map of endocrine development and function genes. Up- or down-regulated genes with false discovery rate less than 0.1 and fold change higher than 0.5 versus WT are highlighted with bold black borders. (See also Tables S1 and S2)
Figure 2
Figure 2. Reduced number and altered location of Neurog3-expressing endocrine progenitors in DH mice at e15.5
WT, Pdx1LacZ/+, Oc1+/− and DH pancreata were immunolabeled for (A,E,F) Neurog3 or (B) Pax6 (in red), Pdx1 (green), and E-cadherin (blue). (C,D) Quantification for Neurog3 and Pax6. White arrows: delaminated Neurog3+ progenitors; yellow arrowheads: Neurog3+ progenitors within developing trunk. A and B are at 20X magnification. Scale bar represents 100 μm. E and F are at 40X magnification. Scale bar represents 100 μm. p-value for all marked comparisons was <0.05 by One-Way Anova with Tukey correction. *p=0.0019. (See also Figure S1)
Figure 3
Figure 3. Defective differentiation of DH hormone-expressing cells at e15.5
WT, Pdx1LacZ/+, Oc1+/− and DH pancreata were immunolabeled for insulin (A), glucagon (B), MafA (C), or MafB (D) in red, E-Cadherin (green), and DAPI (blue). Images are at 20X magnification. Scale bar represents 100 μm. p-value for all marked comparisons was <0.05 by One-Way Anova with Tukey correction. *p<0.05. (See also Figure S3)
Figure 4
Figure 4. Defects in glucose homeostasis and islet gene expression in DH mice at P1
Body weight (A) and ad lib feeding blood glucose measurements (B). (C-F) Insulin (green) and glucagon (red) immunolabeling of pancreatic sections from WT (C), SH (D,E) and double heterozygotes (F). (G) β cell mass, (H) α cell mass, and (I) α/β cell ratio. (J) Total pancreatic insulin content. †p>0.10, ‡p=0.069, *p <0.05, **p<0.01 by One-Way Anova with Tukey correction.
Figure 5
Figure 5. Combined Pdx1 and Oc1 heterozygosity leads to dramatic alterations in neonatal islet gene expression
WT, Pdx1LacZ/+, Oc1+/− and Pdx1LacZ/+;Oc1+/− islets were analyzed for gene expression of hormones (A), endocrine-associated transcription factors (B), and secretory functional genes (C). p-value for marked comparisons was <0.05 by Kruskal-Wallace Test followed by two-tailed Student's T-Test. *: WT v. Pdx1LacZ/+;Oc1+/−; #: WT v. Pdx1LacZ/+; ^: WT v. Oc1+/−; @: Pdx1LacZ/+ v. Pdx1LacZ/+;Oc1+/−; $: Oc1+/− v. Pdx1LacZ/+;Oc1+/−; ?: Pdx1LacZ/+ v. Oc1+/−. ND = not detected. (See also Figure S5)
Figure 6
Figure 6. Increased α- and β-cell proliferation at e18.5 in DH pancreata
Representative images of proliferating β cells (A; red: Glut2, green: Ki67) and α-cells (B; red: glucagon, green: Ki67) at e18.5. α-cell Proliferation at e18.5. Arrows point to proliferating hormone+ cells. (C) quantification of β-cell proliferation, (D) quantification of α-cell proliferation. Scale bar = 100μm; *: p<0.05; ***: p<0.001.
Figure 7
Figure 7. Early reductions in Pdx1 and Oc1 lead to persistent alterations in islet gene expression at weaning
WT, Pdx1LacZ/+, Oc1+/− and DH islets were analyzed for gene expression of hormones (A), endocrine-associated transcription factors (B), and secretory functional genes (C) at P28. p-value for marked comparisons were <0.05 by Kruskal-Wallace Test followed by two-tailed Student's T-Test. #: WT v. Pdx1LacZ/+; $: Oc1+/− v. Pdx1LacZ/+;Oc1+/−. (See also Figure S6)
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