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. 2007 Mar 6;104(10):3853-8.
doi: 10.1073/pnas.0700013104. Epub 2007 Feb 22.

MafB is required for islet beta cell maturation

Isabella Artner  1 , Bruno Blanchi, Jeffrey C Raum, Min Guo, Tomomi Kaneko, Sabine Cordes, Michael Sieweke, Roland Stein
Affiliations

MafB is required for islet beta cell maturation

Isabella Artner et al. Proc Natl Acad Sci U S A. .

Abstract

Pancreatic endocrine cell differentiation depends on transcription factors that also contribute in adult insulin and glucagon gene expression. Islet cell development was examined in mice lacking MafB, a transcription factor expressed in immature alpha (glucagon(+)) and beta (insulin(+)) cells and capable of activating insulin and glucagon expression in vitro. We observed that MafB(-/-) embryos had reduced numbers of insulin(+) and glucagon(+) cells throughout development, whereas the total number of endocrine cells was unchanged. Moreover, production of insulin(+) cells was delayed until embryonic day (E) 13.5 in mutant mice and coincided with the onset of MafA expression, a MafB-related activator of insulin transcription. MafA expression was only detected in the insulin(+) cell population in MafB mutants, whereas many important regulatory proteins continued to be expressed in insulin(-) beta cells. However, Pdx1, Nkx6.1, and GLUT2 were selectively lost in these insulin-deficient cells between E15.5 and E18.5. MafB appears to directly regulate transcription of these genes, because binding was observed within endogenous control region sequences. These results demonstrate that MafB plays a previously uncharacterized role by regulating transcription of key factors during development that are required for the production of mature alpha and beta cells.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
MafB−/− pancreata have fewer insulin+ and glucagon+ cells. (A) Immunofluorescence staining for insulin (green) and glucagon (red) was performed in wild-type and MafB−/− pancreas at E10.5 (glucagon only), E12.5, E13.5, and E15.5. Insulin+ cells are indicated by an arrow at E12.5. Notably, insulin expression was delayed until E13.5 in the MafB mutant, whereas glucagon was observed at all stages. (B) Quantification of insulin+ and glucagon+ cells in wild-type (black bars) and MafB−/− (gray bars) embryos. Because the wild-type and heterozygous MafB embryos showed no difference in hormone cell numbers (data not shown), both were incorporated into the “wild-type” data. The average number of cells per section is shown ± SD, with the asterisk denoting the significance between wild-type and mutant embryos (P < 0.02). (Scale bars: 20 μM.)
Fig. 2.
Fig. 2.
Total endocrine cell numbers are unchanged in MafB−/− pancreata. (A) Immunofluorescence staining of E15.5 wild-type and MafB mutant pancreas sections with α-Isl1 (green), α-insulin (red), α-Pax6 (green), and/or α-glucagon (red). Nuclei were stained with YoPro1 (blue). In the mutant, sections containing a larger number of glucagon and insulin producing cells were selected to illustrate Isl1 and Pax6 coexpression, and not the quantitative decrease in insulin+ or glucagon+ cells found between wild-type and MafB−/− mice. (B) Quantification of Isl1+ cells in wild-type (black bars) and MafB−/− (gray bars) embryos. No significant difference was found in Isl1+ cell numbers at E12.5, E13.5, E15.5, and E18.5. The average number of cells per section is shown ± SD. (C) The real-time PCR mRNA levels of isl1, pax6, and neuroD1 are shown as the percentage of wild type (100%) at E15.5 and E18.5. (Scale bar: 20 μM.)
Fig. 3.
Fig. 3.
α and β cell identity markers are maintained in hormone− β cells at E15.5. (A) Double immunofluorescence detection of MafA (green) and insulin (red), at E15.5 in wild-type and MafB−/− embryos. Nuclei were stained with YoPro1 (blue). The percentage of total cells expressing MafA and insulin (MafA+/INS+) is shown. (Scale bar: 20 μM.) (B) Relative mRNA level of various α and β cell markers at E15.5 and E18.5 in MafB-deficient pancreata. MafB−/− mRNA levels are shown as the percent of wild type. The asterisk denotes the significance between wild-type and mutant mRNA levels (P < 0.05). (C) ChIP analysis was performed on E18.5 pancreata treated with α-MafB antibody. The precipitated chromatin was analyzed by PCR for insulin (−378/−46), glucagon (−353/+7) and PEPCK (−434/−96) control region sequences. As controls, PCRs were run with input chromatin (1:200 dilution) and DNA obtained after treatment with rabbit IgG or no antibody. (D) MafA mRNA levels in βTC3 cells infected with Ad:GFP (AdGFP:βTC3) or Ad:MafB (AdMafB:βTC3). The real-time PCR data represent the normalized fold difference relative to GFP alone. (E) Chromatin from AdGFP or AdMafB-infected β TC3 cells was precipitated with α-MafB or IgG and analyzed by PCR with mafA (−8120 to −7750), GLUT2 (−1012 to −667), and nkx6.1 (−480 to −141) control region primers. rVISTA and TRANSFAC analysis of upstream GLUT2 and nkx6.1 sequences identified within the primer spanned regions many potential large Maf binding sites very near [GLUT2 (40)] or within [Nkx6.1 (41)] transcriptional regulatory domains.
Fig. 4.
Fig. 4.
Pdx1 and GLUT2 are not detected in hormone− β cells at E18.5. (A) Double immunofluorescence staining of wild-type and MafB−/− E15.5 and E18.5 pancreata. Pdx1 (green) and Nkx6.1 (green) are detected in both insulin+ (red) and insulin− endocrine cells in E15.5 MafB−/− embryos. At E18.5, Pdx1 expression is only retained in insulin+ cells, whereas Nkx6.1 expression was lost in some insulin− β cells. The percentage of Pdx1+/insulin+ or Nkx6.1+/insulin+ cells in wild-type (black bars) and MafB mutant embryos (gray bars) within the total Pdx1+ or Nkx6.1+ cell population is shown in the histogram. Only cells producing high levels of Pdx1+ were counted in the analysis, because these represent β precursor cells (42). Nuclei were stained with YoPro1 (blue). (B) Analysis of Nkx2.2 (green), Pdx1 (blue), and insulin (red) coexpression in E18.5 wild-type and MafB−/− pancreata. Nkx2.2 expression is unchanged from wild type in E15.5 and E18.5 MafB−/− embryos (Fig. 3B and SI Fig. 6). The arrows depict the location of Nkx2.2/Pdx1/insulin-expressing cells, with their percentage in wild type (black) and mutant (gray) shown in the graph. The number of Nkx2.2+ only cells increased in MafB mutant pancreata to 37% (wild type, 22%) at the expense of Nkx2.2+/Pdx1+/insulin+ cells, whereas the level of Pdx1+/Nkx2.2+ cells remained constant (E18.5: wild type, 3% of all Nkx2.2+ cells; MafB−/−, 6%). (C) GLUT2 (green) is expressed in all wild-type insulin+ (red) cells at E18.5. Nuclei were stained with YoPro1 (blue). In contrast, MafB mutant pancreata have decreased numbers of GLUT2+/INS+ cells. The histogram depicts the percentage of wild-type (black bars) and MafB−/− (gray bars) GLUT2+/INS+ cells. The arrows indicate the region magnified in Inset. The sections were selected to illustrate insulin and marker gene coexpression. (Scale bars: 20 μM.)
Fig. 5.
Fig. 5.
Potential role of MafB in β cell development. The model proposes that MafB is required for β cell differentiation and maturation, as supported by the loss of first wave insulin+ cells and the delay in appearance of hormone-producing cells until MafA is made in the MafB mutant. The ≈75% decline in insulin mRNA in MafA+/insulin+ cells (dashed line illustrates the minor role of MafA in insulin production) also indicates that MafB is the principal insulin activator during β cell development. Our results also suggest that MafB is directly required for inducing mafA transcription in β precursor cells and is a primary regulator of pdx1, nkx6.1, and GLUT2 expression in maturing β cells.
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