MafB is required for islet beta cell maturation

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.

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.

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.

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.

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.

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.