Sox9 coordinates a transcriptional network in pancreatic progenitor cells - PubMed (original) (raw)

Sox9 coordinates a transcriptional network in pancreatic progenitor cells

F C Lynn et al. Proc Natl Acad Sci U S A. 2007.

Abstract

During pancreas development, both the exocrine and endocrine lineages differentiate from a common pool of progenitor cells with similarities to mature pancreatic duct cells. A small set of transcription factors, including Tcf2, Onecut1, and Foxa2, has been identified in these pancreatic progenitor cells. The Sry/HMG box transcription factor Sox9 is also expressed in the early pancreatic epithelium and is required for normal pancreatic exocrine and endocrine development in humans. In this study, we found Sox9 in mice specifically expressed with the other progenitor transcription factors in both pancreatic progenitor cells and duct cells in the adult pancreas. Sox9 directly bound to all three genes in vitro and in intact cells, and regulated their expression. In turn, both Foxa2 and Tcf2 regulated Sox9 expression, demonstrating feedback circuits between these genes. Furthermore, Sox9 activated the expression of the proendocrine factor Neurogenin3, which also depends on the other members of the progenitor transcription network. These studies indicate that Sox9 plays a dual role in pancreatic progenitor cells: both maintaining a stable transcriptional network and supporting the programs by which these cells differentiate into distinct lineages.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Sox9 expression in the mouse pancreas by immunofluorescence. Staining for Sox9 (red) is shown with Pdx1 (green; B, D, and E), glucagon (green; A and C), mucin (green; F), and Neurogenin3 (green, G and H) at e10.5 (A and B), e12.5 (C and D), e15.5 (G and H), and adult (F). In E, arrowheads indicate examples of cells with high expression of Pdx1. In F, arrowheads indicate examples of duct cells coexpressing Sox9 and mucin; arrow indicates an example of intraacinar cells expressing mucin but not Sox9. In G and H, arrowheads indicate examples of cells coexpressing Sox9 and Neurogenin3. (H Inset) Red and green channels are shown separately. Du, duodenum; DP, dorsal pancreas. (Scale bars, 50 μM.)

Fig. 2.

Fig. 2.

Sox9 coexpression with Neurogenin3 in the mouse embryonic pancreas. Immunofluorescence staining for Sox9 (red) is shown with Neurogenin3 (green) at e14.5 imaged by confocal microscopy. Arrowheads indicate examples of cells coexpressing Sox9 and Neurogenin3. (Scale bars, 50 μM.)

Fig. 3.

Fig. 3.

Sox9 regulates Neurog3 expression. (A) Fragments of the human NEUROG3 promoter with the indicated 5′ endpoints driving luciferase expression were cotransfected into αTC1.6 cells together with plasmids expressing either the Sox9 cDNA or no cDNA (Control). Luciferase activities were determined 48 h after transfection and expressed relative to the activity in cells transfected with the promoterless reporter construct and the empty expression plasmid. Results are expressed as the mean ± SEM of data from experiments performed in triplicate on at least three separate occasions. (B) Chromatin IP studies were performed by immunoprecipitating cross-linked chromatin with antiserum against Sox9 or with control IgG. Four fragments of the mouse Neurog3 promoter outlined in the line drawing were amplified by PCR from the precipitates or the input DNA. (C) DNA binding of _in vitro_-translated Sox9 or luciferase (Control) protein was tested by EMSA. Double-stranded, radiolabeled oligonucleotide probes contained the binding sites shown in the line figure in B and a consensus Sox9-binding site (S9C). (D) Stable mPAC L20 cell lines expressing an shRNA directed against Sox9 or a control shRNA were infected with a Mash1 adenovirus to induce Neurogenin3 expression. Neurogenin3 protein levels were assessed by Western blot 48 h after infection.

Fig. 4.

Fig. 4.

Sox9 regulates the other progenitor cell transcription factors. (A) DNA binding of _in vitro_-translated Sox9 or luciferase (Control) protein was tested by EMSA. Double-stranded, radiolabeled oligonucleotide probes contained binding sites from the indicated promoters and a consensus Sox9-binding site (S9C). (B) chromatin IP studies were performed by immunoprecipitating cross-linked chromatin with antiserum against Sox9 or with control IgG. 4 fragments of the indicated promoters were amplified by PCR from the precipitates or the input DNA. (C) mPAC L20 cells were transfected with synthetic double-stranded siRNA directed against Sox9 or with a control siRNA. Protein levels were assessed by Western blot 48 h after transfection. (D) Quantification is shown for Western blots. Data represent the mean ± SEM of three independent experiments; statistical analyses were carried out by using one-way ANOVA, followed by the Newman–Keuls post hoc test. Asterisks indicate significant difference (P < 0.001) from control conditions.

Fig. 5.

Fig. 5.

Foxa2 and Tcf2 regulate Sox9 expression. mPAC L20 cells were transfected with a control siRNA or siRNAs against Foxa2 and Tcf2 at a concentration of 10 nM. Protein levels were assessed by Western blot 48 h after transfection. (B) Quantification for Western blots shown in A. Data represent the mean ± SEM of four independent experiments.

Fig. 6.

Fig. 6.

Model for the role of Sox9 in pancreatic progenitor cells. The model in A shows the transcription factors expressed at different steps of differentiation from gut endoderm to mature pancreatic duct cells (modified from refs. and 31). The model in B shows the regulatory relationships among Sox9 and the other transcription factors in pancreatic progenitor cells. Dashed lines indicate relationships supported only by in vitro DNA-binding studies.

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