MafA and MafB regulate genes critical to beta-cells in a unique temporal manner

Abstract

Objective: Several transcription factors are essential to pancreatic islet β-cell development, proliferation, and activity, including MafA and MafB. However, MafA and MafB are distinct from others in regard to temporal and islet cell expression pattern, with β-cells affected by MafB only during development and exclusively by MafA in the adult. Our aim was to define the functional relationship between these closely related activators to the β-cell.

Research design and methods: The distribution of MafA and MafB in the β-cell population was determined immunohistochemically at various developmental and perinatal stages in mice. To identify genes regulated by MafB, microarray profiling was performed on wild-type and MafB(-/-) pancreata at embryonic day 18.5, with candidates evaluated by quantitative RT-PCR and in situ hybridization. The potential role of MafA in the expression of verified targets was next analyzed in adult islets of a pancreas-wide MafA mutant (termed MafA(ΔPanc)).

Results: MafB was produced in a larger fraction of β-cells than MafA during development and found to regulate potential effectors of glucose sensing, hormone processing, vesicle formation, and insulin secretion. Notably, expression from many of these genes was compromised in MafA(ΔPanc) islets, suggesting that MafA is required to sustain expression in adults.

Conclusions: Our results provide insight into the sequential manner by which MafA and MafB regulate islet β-cell formation and maturation.

FIG. 1.

MafA and MafB expression is dynamically regulated in developing and adult β-cells. A: The percentage of β-cells coexpressing MafA or MafB was assessed at E14.5, E18.5, P14, and P28 by quantification of the number of insulin⁺ cells costaining with either MafA or MafB. Sections spanning the entire pancreas of wild-type mice were used; the results are presented as the mean ± SEM. B and C: MafB is sustained in a fraction of the insulin⁺ cells in MafA^Δpanc islets. Wild-type (B) and mutant (C) pancreatic sections were stained for MafB (green) and insulin (red). Notably, MafB⁺ is not found in wild-type insulin⁺ cells, but is found in MafA^ΔPanc. Yellow arrowhead denotes representative MafB⁺ insulin⁺ cells, and white arrowhead denotes MafB⁺ insulin⁻ cells. Nuclei were stained with YO-PRO-1 in blue. MafB was found in 33.4 ± 5.6% of MafA^Δpanc insulin⁺ cells (n = 3). (A high-quality digital representation of this figure is available in the online issue.)

FIG. 2.

Islet zinc transporter Slc30a8 expression is activated by MafB during embryogenesis and by MafA in islet β-cells. Slc30a8 transcripts (green) were detected by in situ analysis in wild-type insulin⁺ (red, A and C) and glucagon⁺ (red, B and D) cells at E15.5 and E18.5. Expression is still detected in the remaining insulin⁺ cells (E), but lost in glucagon⁺ (F) cells in MafB^−/− pancreata. No Slc30a8 expression was detected in the MafA and MafB compound mutant (MafA^ΔPanc; MafB^−/−; G and H). I: RT-PCR data illustrate the change in Slc30a8 mRNA expression between 3-month-old MafA^Δpanc and wild-type islets. *P < 0.05 (n = 3). Slc30a8 was detected in islet β-cells (J) and α-cells (K) of 12-week-old wild-type islets by immunofluorescence. Expression was profoundly reduced in MafA^ΔPanc β-cells (L) and unaffected in α-cells (M). The (L) Slc30a8⁺ insulin⁻ cells labeled with white arrows correspond to (M) Slc30a8⁺ glucagon⁺ cells denoted by yellow arrows. Scale bar = 20 μm. Nuclei were stained in blue in panels J–M. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 3.

G6pc2 expression is compromised in adult islets lacking MafA. A: Quantitative RT-PCR analysis revealed that G6pc2 mRNA levels were significantly downregulated in adult MafA^ΔPanc islets. *P < 0.05 (n = 3). G6pc2 expression (green) in islet insulin⁺ cells (B–D) (red) was shown by immunostaining to be profoundly reduced in MafA^Δpanc islets (E–G). Scale bar = 20 μm. Nuclei were stained with YO-PRO-1 (blue). (A high-quality digital representation of this figure is available in the online issue.)

FIG. 4.

Nnat expression in β-cells is regulated only by MafB. Appreciable levels of Nnat (green) were detected in insulin⁺ (red, A, yellow arrows), insulin⁻ (A, white arrows), but not glucagon⁺ (red, B) cells by in situ analysis. Nnat was essentially restricted to β-cells by E18.5 in the wild-type (G and H) and the remaining insulin⁺ cells in the MafB^−/− and MafA^ΔPanc;MafB^−/− mutants (I and L). Scale bar = 20 μm. Nnat expression was unaffected in MafA^Δpanc islets (M). Yellow arrows denote Nnat⁺hormone⁺ cells and white arrows denote Nnat⁺hormone⁻ cells. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 5.

Rbp4 is induced in the embryonic MafB^−/− and islet MafA^ΔPanc mutants. Rbp4 transcripts (green) are present in some (A and G) insulin⁺ (red, yellow arrows) and (B and H) glucagon⁺ (red) cells (A and B, yellow arrows) at both E15.5 and E18.5, with expression also found in (I) somatostatin⁺ δ-cells (red, yellow arrows) by E18.5. The number of hormone⁺ Rbp4⁺ cells increased in MafB^−/− and MafA^ΔPanc;MafB^−/− mutant pancreata (C–F; J–O). P: Rbp4 mRNA expression was elevated in 12-week MafA^ΔPanc islets. S–X: The Rbp4 protein is found in somatostatin (SST)⁺ δ-cells in (S) wild-type and (W) MafA^ΔPanc islets, and not (Q and U) insulin⁺, (R and V) glucagon⁺, or (T and X) pancreatic peptide⁺ cells. Scale bar = 20 μm. Nuclei were stained in blue in panels Q–W. White arrows denote Rbp4⁺ hormone⁻ cells. (A high-quality digital representation of this figure is available in the online issue.)