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Mafa and Mafb Regulate Genes Critical To ORIGINAL ARTICLE MafA and MafB Regulate Genes Critical to ␤-Cells in a Unique Temporal Manner Isabella Artner,1,2 Yan Hang,1 Magdalena Mazur,2 Tsunehiko Yamamoto,1 Min Guo,1 Jill Lindner,1,3 Mark A. Magnuson,1,3 and Roland Stein1 OBJECTIVE—Several transcription factors are essential to pan- type 2 diabetic patients as discussed by others (1–3). The creatic islet ␤-cell development, proliferation, and activity, in- significance of these proteins was recently reinforced cluding MafA and MafB. However, MafA and MafB are distinct upon observing their expression during the stepwise dif- from others in regard to temporal and islet cell expression ferentiation of human embryonic stem cells to ␤-like cells pattern, with ␤-cells affected by MafB only during development (4,5) and the reprogramming of adult acinar cells to ␤-like and exclusively by MafA in the adult. Our aim was to define the cells upon misexpression of a unique subset of transcrip- functional relationship between these closely related activators to the ␤-cell. tion factors, specifically MafA, Pdx1, and Ngn3 (6). Among the transcription factors vital to the pancreas, RESEARCH DESIGN AND METHODS—The distribution of there are instances when members of the same gene MafA and MafB in the ␤-cell population was determined immu- family contribute to ␤-cell formation, including winged- nohistochemically at various developmental and perinatal stages helix/forkhead (e.g., FoxA1/2) (7–9), NK6 homeodomain in mice. To identify genes regulated by MafB, microarray profil- ing was performed on wild-type and MafBϪ/Ϫ pancreata at (Nkx6.1 and Nkx6.2) (10–12), paired box homeodomain embryonic day 18.5, with candidates evaluated by quantitative (Pax4/6) (13–17), and basic leucine-zipper (MafA and RT-PCR and in situ hybridization. The potential role of MafA in MafB) (18,19) proteins. FoxA1/2, Nkx6.1/6.2, and Pax4/6 are expressed broadly in pancreatic epithelial cells in both the expression of verified targets was next analyzed in adult ϩ Ϫ islets of a pancreas-wide MafA mutant (termed MafA⌬Panc). islet hormone and hormone cells before or near the ␤ onset of pancreatic morphogenesis (3) and then become RESULTS—MafB was produced in a larger fraction of -cells confined to more specific cellular domains (e.g., Nkx6.1 than MafA during development and found to regulate potential ␤ effectors of glucose sensing, hormone processing, vesicle forma- [ ] and Pax6 [all islet cells]) or disappear entirely late in tion, and insulin secretion. Notably, expression from many of development (Pax4, Nkx6.2). The large MafA/B factors are ⌬Panc distinct in being produced relatively later in development these genes was compromised in MafA islets, suggesting ϩ that MafA is required to sustain expression in adults. and essentially only in hormone cells. Thus, MafB is present in developing ␣-(glucagonϩ) cells, ␤-cells, and a CONCLUSIONS—Our results provide insight into the sequen- ϩ Ϫ ␤ very small number of Ngn3 islet hormone progenitors tial manner by which MafA and MafB regulate islet -cell ␣ formation and maturation. Diabetes 59:2530–2539, 2010 and then becomes restricted to -cells soon after birth (20,21). MafA is found exclusively in developing and adult insulinϩ cells, with expression first detected at embryonic day (E) 13.5 during the secondary and principal wave of ϩ ␤ insulin cell production (22). nsulin-secreting islet -cells play a pivotal role in the A comparison of the properties of islet-enriched tran- regulation of fuel metabolism. Loss or dysfunction ␤ scription factor mutant mice reveals a novel role for MafA of -cells causes an imbalance in glucose homeosta- and MafB in ␤-cell maturation and function. Thus, islet ␣, Isis that leads to the development of diabetes. Efforts ␤, ␦, ε, or pancreatic polypeptide producing cells are either aimed at understanding the molecular processes underly- ␤ lost or respecified in most transcription factor knockout ing -cell development and function have provided per- mice (1–3), whereas the principal defect in MafBϪ/Ϫ spectives into new diabetes treatment strategies. For embryos is reduced insulin and glucagon expression (18). example, a hierarchy of transcription factors has been Furthermore, this change does not affect total endocrine found to regulate ␤-cell differentiation during development cell numbers, supporting a distinct importance in one or and adult islet cell function, a few of which are mutated in more late steps in ␣- and ␤-cell differentiation (18). In contrast, MafA is solely required for glucose-regulated 1 insulin secretion in adult islet ␤-cells and is not involved in From the Department of Molecular Physiology and Biophysics, Vanderbilt ϩ ϩ University School of Medicine, Nashville, Tennessee; the 2Lund Center for islet cell development (19). Notably, MafB insulin cells Stem Cell Biology and Cell Therapy, Lund University, Lund, Sweden; and generated during human embryonic stem cell differentia- the 3Center for Stem Cell Biology, Vanderbilt University School of Medicine, ϩ ϩ Nashville, Tennessee. tion were dysfunctional until becoming MafA insulin Corresponding authors: Roland Stein, [email protected], and Isa- (5), suggesting that the transition from MafB to MafA is bella Artner, [email protected]. critical to ␤-cell function. Received 8 February 2010 and accepted 30 June 2010. Published ahead of print at http://diabetes.diabetesjournals.org on 13 July 2010. DOI: 10.2337/ To obtain a mechanistic understanding of the associa- db10-0190. tion between MafB and MafA in ␤-cell formation and I.A. and Y.H. contributed equally to this study. function, gene-profiling studies were performed with E18.5 © 2010 by the American Diabetes Association. Readers may use this article as pancreata from wild-type, MafBϪ/Ϫ, MafA⌬Panc, and long as the work is properly cited, the use is educational and not for profit, ⌬Panc Ϫ/Ϫ ⌬Panc and the work is not altered. See http://creativecommons.org/licenses/by MafA ;MafB embryos. (MafA mice were gen- -nc-nd/3.0/ for details. erated by crossing MafAfl/fl mice with transgenic mice The costs of publication of this article were defrayed in part by the payment of page 5.5 charges. This article must therefore be hereby marked “advertisement” in accordance producing Cre recombinase from the Pdx1 promoter with 18 U.S.C. Section 1734 solely to indicate this fact. fragment early in development and in a pancreas-wide 2530 DIABETES, VOL. 59, OCTOBER 2010 diabetes.diabetesjournals.org I. ARTNER AND ASSOCIATES TABLE 1 PCRs were performed using the SYBR Green (with dissociation curve) The mRNA expression profile of E18.5 wild-type and mutant program on an Applied Biosystems 7900 machine. All reactions were per- pancreata formed in duplicate with reference dye normalization, and median cycling threshold values were used for analysis. Primer sequences are available on % of wild-type request. MafA⌬panc MafBϪ/Ϫ MafA⌬panc;MafBϪ/Ϫ Immunohistochemistry and in situ hybridization. Tissue fixation, embed- ding, and immunofluorescence labeling were performed as previously de- MafA 1 Ϯ 1* 16 Ϯ 1* 2 Ϯ 1* scribed (25). The primary antibodies used were guinea pig ␣-insulin (1:2,000; MafB 168 Ϯ 1* 10 Ϯ 6* 9 Ϯ 4* Linco Research), mouse ␣-insulin (1:2,000; Invitrogen), guinea pig ␣-glucagon Insulin 99 Ϯ 16 34 Ϯ 18* 11 Ϯ 3* (1:2,000; Linco Research), sheep ␣-somatostatin (1:2,000; American Research), ␣ ␣ Glucagon 132 Ϯ 23 55 Ϯ 28* 31 Ϯ 10* guinea pig -pancreatic polypeptide (1:2,000; Linco Research), rabbit -MafB (1:10,000; Bethyl Laboratories), rabbit ␣-MafA (1:1,000, Bethyl Laboratories), Isl1 112 Ϯ 12 118 Ϯ 50 117 Ϯ 42 rabbit ␣-Slc30a8 (1:1,000, Mellitech), mouse ␣-Rbp4 (1:1,000, Abnova), rabbit Slc30a8 90 Ϯ 23 4 Ϯ 3* 0 Ϯ 0* ␣ Ϯ Ϯ Ϯ -G6PC2 (1:500, a gift from Dr. John Hutton at the University of Colorado), G6pc2 59 20* 18 9* 4 0* rabbit ␣-Pdx1 (1:5,000, a gift from Dr. Chris Wright at Vanderbilt University), Rbp4 84 Ϯ 21 196 Ϯ 54* 188 Ϯ 21* mouse ␣-Isl1 (1:300, Developmental Studies Hybridoma Bank), rabbit Nnat 50 Ϯ 11* 31 Ϯ 7* 26 Ϯ 4* ␣-Nkx6.1 (1:1,000, Beta Cell Biology Consortium), and rabbit ␣-Pax6 (1:200, Covance Research Products). The secondary Cy2-, Cy3-, or Cy5-conjugated Data are means Ϯ SEM. *Change from wild-type littermates is ␣ ␣ ␣ Ͻ donkey -rabbit, -guinea pig, and -sheep IgGs were obtained from Jackson significant, P 0.05. ImmunoResearch Laboratories. Nuclear counterstaining was performed using YoPro1 or DAPI (Invitrogen). Immunofluorescence images were acquired pattern.) Multiple genes were differentially expressed in using confocal microscopy (LSM510, Carl Zeiss). Ϫ/Ϫ ⌬Panc the MafB mutant, but not in MafA mutant, consis- In situ hybridization assays were performed using Neuronatin (base pair tent with the much more critical role of MafB in ␣- and 119 to 1,163 relative to coding ATG) and Slc30a8 (base pair 166 to 1,269) gene ␤-cell development (18,21). Significantly, MafB-dependent probes according to Henrique et al. (26). The Rbp4 cDNA plasmid (clone ID genes were associated with adult ␤-cell function, including IMAGp998P0711141Q1) was obtained from the resource center of the human glucose sensing and insulin secretion. Interestingly, many genome project (www.rzpd.de) and used to generate the probe (base pair of these target genes were affected in a similar manner in 295–927). ⌬Panc Quantification and statistical analysis. Immunolabeling for insulin and adult MafA islets, even though MafB was retained in ␮ ϩ MafA (or MafB) was performed on serial 6- m pancreatic sections that were a fraction of the mutant insulin cell population. These collected at 60-␮m (i.e., at E14.5), 84-␮m (E18.5), or 150-␮m (postnatal) findings provide insight into why the ␤-cell is dysfunc- intervals. All insulinϩ cells were imaged by confocal microscopy and pre- tional in MafA mutant mice, and they illustrate the unusual sented as the percentage of MafAϩinsulinϩ or MafBϩinsulinϩ to total insulinϩ cells.
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