The diabetes gene Pdx1 regulates the transcriptional network of pancreatic endocrine progenitor cells in mice
Jennifer M. Oliver-Krasinski, Margaret T. Kasner, Juxiang Yang, Michael F. Crutchlow, Anil K. Rustgi, Klaus H. Kaestner, Doris A. Stoffers
Jennifer M. Oliver-Krasinski, Margaret T. Kasner, Juxiang Yang, Michael F. Crutchlow, Anil K. Rustgi, Klaus H. Kaestner, Doris A. Stoffers
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Research Article Metabolism

The diabetes gene Pdx1 regulates the transcriptional network of pancreatic endocrine progenitor cells in mice

Abstract

Heterozygous mutations in the gene encoding the pancreatic homeodomain transcription factor pancreatic duodenal homeobox 1 (PDX1) are associated with maturity onset diabetes of the young, type 4 (MODY4) and type 2 diabetes. Pdx1 governs the early embryonic development of the pancreas and the later differentiation of the insulin-producing islet β cells of the endocrine compartment. We derived a Pdx1 hypomorphic allele that reveals a role for Pdx1 in the specification of endocrine progenitors. Mice homozygous for this allele displayed a selective reduction in endocrine lineages associated with decreased numbers of endocrine progenitors and a marked reduction in levels of mRNA encoding the proendocrine transcription factor neurogenin 3 (Ngn3). During development, Pdx1 occupies an evolutionarily conserved enhancer region of Ngn3 and interacts with the transcription factor one cut homeobox 1 (Hnf6) to activate this enhancer. Furthermore, mRNA levels of all 4 members of the transcription factor network that regulates Ngn3 expression, SRY-box containing gene 9 (Sox9), Hnf6, Hnf1b, and forkhead box A2 (Foxa2), were decreased in homozygous mice. Pdx1 also occupied regulatory sequences in Foxa2 and Hnf1b. Thus, Pdx1 contributes to specification of endocrine progenitors both by regulating expression of Ngn3 directly and by participating in a cross-regulatory transcription factor network during early pancreas development. These results provide insights that may be applicable to β cell replacement strategies involving the guided differentiation of ES cells or other progenitor cell types into the β cell lineage, and they suggest a molecular mechanism whereby human PDX1 mutations cause diabetes.

Authors

Jennifer M. Oliver-Krasinski, Margaret T. Kasner, Juxiang Yang, Michael F. Crutchlow, Anil K. Rustgi, Klaus H. Kaestner, Doris A. Stoffers

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Figure 5

Pdx1 regulates Ngn3, at least in part, via its direct occupancy of a conserved upstream enhancer of Ngn3.

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Pdx1 regulates Ngn3, at least in part, via its direct occupancy of a con...
(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).