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BIOLOGICAL SCIENCES / DEVELOPMENTAL BIOLOGY
MafB is required for islet 
cell maturation
*Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, 723 Light Hall, Nashville, TN 37232; Centre d'Immunologie de Marseille-Luminy, Centre National de la Recherche Scientifique–Institut National de la Santé et de la Recherche Médicale–Universite Mediterrane, Campus de Luminy, Case 906, 13288 Marseille Cedex 09, France; and Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, ON, Canada M5G 1X5
Communicated by Donald F. Steiner, The University of Chicago, Chicago, IL, January 2, 2007 (received for review December 6, 2006)
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(glucagon+) and 
(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– 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 and cells.
insulin | MafA | pancreas development
, , 
, 
, and pancreatic polypeptide (PP) cells, which produce the hormones glucagon, insulin, somatostatin, ghrelin, and PP, respectively. Collectively, these hormones regulate both fuel and energy metabolism, with insulin and glucagon essential to controlling glucose homeostasis (1). Thus, glucagon secreted from cells stimulates the mobilization of glucose through gluconeogenesis and glycogenolysis, whereas cell secreted insulin promotes glucose storage. Defects in and cell function play a significant role in the ability of individuals with diabetes to maintain glycemic control.
Characterization of the and cell enriched transcription factors involved in regulating insulin and glucagon expression has revealed not only their significance to islet function, but also to pancreas organogenesis. For example, Pdx1 is necessary for the growth of the endocrine and exocrine compartments, with pancreatic agenesis observed in pdx1 mutant mice (2, 3). Pdx1 has also been proposed to be a master regulator of cell activity, in part based on the severe diabetic phenotype observed upon deletion of this factor from cells in animals, a phenotype at least caused by reduced insulin and glucose transporter type 2 (GLUT2) expression (4).
In contrast to the profound deficiencies in pancreatic endocrine and exocrine cell development observed in the total Pdx1 knockout, the loss of Pax6 (5, 6) or BETA2 [NeuroD1 (7)] only affects endocrine cell production. In fact, changes in endocrine cell development are commonly found in transcription factor knockout models. Thus, loss of Ngn3 leads to a severe reduction in endocrine progenitor cells (8), whereas Nkx6.1-deficient animals display a selective loss of cells (9). In contrast, there is a switch in cell lineage commitment in Arx (10), Pax4 (11), Pax6 (12), and Nkx2.2 (11) null mutant mice. The notable exceptions are MafA and Brn4 (13), with a deficiency only observed in adult MafA–/– mice, which were glucose intolerant and developed diabetes (14).
The absence of a developmental phenotype in MafA–/– animals was particularly surprising, because this factor is expressed exclusively within the insulin+ cell population produced after embryonic day (E) 13.5 that mature to become islet cells (15). The Maf basic leucine-zipper-containing transcription factor family consists of two distinct subfamilies, which differ by the presence of an N-terminal transactivation domain in a large Maf (i.e., MafB, MafA, c-Maf, and NRL) that is not found in a small Maf (i.e., MafF, MafG, and MafK). In contrast to MafA, all other closely related members of the large Maf family are involved in processes associated with cell specification, including in the brain [i.e., MafB (16)], kidney [MafB (17, 18)], and immune system [cMaf (19) and MafB (18, 20)].
MafB is expressed in all developing insulin- and glucagon-producing cells, and then in a restricted fashion in adult cells (21, 22). MafB is also capable of activating insulin- and glucagon-driven transcription in non-hormone-expressing cell types in vitro (21, 23, 24), indicating a possible redundancy with MafA during cell differentiation. Here we show that MafB is critical for and cell differentiation, because the number of insulin+ and glucagon+ cells were substantially reduced in MafB mutant animals throughout embryogenesis. Strikingly, no difference was found between the wild type and MafB mutant in total endocrine cell numbers, which were assessed by pan-endocrine Islet1 (Isl1), NeuroD1, and Pax6 expression. In addition, the level of many (e.g., Arx, Nkx2.2, Brn4) and (Nkx2.2, Nkx6.1) cell markers was unchanged in and cells (both hormone-expressing and -deficient) at E15.5. However, insulin production was delayed until the onset of MafA expression, and several other gene products required for cell maturation and function were selectively reduced in hormone– cells by E18.5 (Pdx1, MafA, Nkx6.1, GLUT2). These results revealed that MafB is required for the generation of the physiologically functional and cell population by directly activating hormone gene transcription and key regulators of cell differentiation and function.
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cells in the adult islet (21, 22). The importance of MafB in endocrine cell formation was examined in homozygous MafB mutant animals, which die at birth because of central apnea [MafB–/– (16)] or renal failure [krENU/krENU (17)]. Glucagon+ cells first appear in the mouse at E9.5, followed by ghrelin+ cells at E10.5 (11), insulin+ cells at E11.5 (25), and somatostatin+ and PP+ at E15.5 and E18.5, respectively (26). MafB is expressed in the distinct first- and second-phase insulin+ and glucagon+ cells made during development, with only second-phase cells producing the and cells that mature and populate the islet (27).
The size and gross appearance of the MafB–/– embryonic pancreata was normal, and insulin- and glucagon-producing cells were detected (Fig. 1A). However, the number of glucagon+ cells was drastically lower at all embryonic stages when compared with the wild type (Fig. 1B). Moreover, insulin+ cells were not detected until E13.5, and their number was reduced to <50% of wild type (Fig. 1B). Glucagon+ cell numbers were also significantly lower at E15.5 in krENU/krENU embryos, in which krENU is a hypomorphic allele of MafB (wild type, 39.1 ± 6.7 glucagon+ cells per section; krENU/krENU, 19.8 ± 6.0), and trended toward fewer insulin+ cells (wild type, 63.3 ± 23.5; krENU/krENU, 40.7 ± 26.8). In contrast, the number of somatostatin- and PP-producing cells was not changed from wild type in either MafB–/– or krENU/krENU pancreata (data not shown). There was also no replacement of and cells with the normally minor ghrelin+ cell population (data not shown), an abnormality observed in Pax6 (12), Nkx2.2, and Pax4 null mutants (11).
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and Cell Differentiation Markers Are Present in Hormone– Endocrine Cells.
Many of the transcription factors required in differentiation and hormone expression are predominantly expressed within one adult islet cell type. For example, MafA, Nkx6.1, and Pdx1 are enriched in cells, whereas Arx, Brn4, and MafB are principally in cells, and Nkx2.2 is found in both and cells (reviewed in ref. 30). The expression pattern of these cell identity markers was next used to evaluate the differentiation state of the endocrine cells produced in MafB–/– mice.
The endocrine cell population was analyzed immunohistochemically for cells expressing MafA, Nkx2.2, Brn4, or both a transcription factor and hormone (MafA/insulin, Nkx2.2/insulin, Brn4/glucagon). The number of cells producing a given transcription factor was set to 100% in this analysis. Nkx2.2 and Brn4 were detected in both hormone– and hormone+ endocrine cells of mutant pancreata at E15.5 [ supporting information (SI) Fig. 6 ]. The percentage of cells expressing both hormone and transcription factor was decreased in MafB mutant pancreatic tissue, whereas cells producing only Nkx2.2+ or Brn4+ increased (SI Fig. 6). The total number of Nkx2.2 and Brn4 producing cells appears to be unchanged despite the presence of fewer insulin+ and glucagon+ cells in MafB mutant embryos. In addition, the mRNA levels for other key and cell gene products were unchanged in E15.5 MafB mutant samples (e.g., Isl1, NeuroD1, and Pax6 in Fig. 2C; Pdx1, GLUT2, ARX, glucokinase, and Pax4 in Fig. 3B). These results demonstrated that hormone– endocrine cells express many and cell differentiation markers in the MafB mutant.
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and cells (22). Our inability to detect a change in islet development in cMaf –/– animals is also consistent with a much more limited role than MafB in hormone gene transcription or endocrine pancreatic cell formation (data not shown). These results strongly suggest that large Maf factors, principally, if not exclusively MafA and MafB, are critical to insulin transcription during pancreas development.
MafB Binds Within the insulin and glucagon Transcriptional Control Region of Endocrine Cells.
Not only was insulin and glucagon protein expression lost in 
50% of and cells, hormone mRNA levels were reduced even further to 27% of wild type (Fig. 3B). Chromatin immunoprecipitation (ChIP) studies were next performed to determine whether MafB binds within both the insulin and glucagon control regions of wild-type E18.5 pancreata. -MafB precipitated the large Maf element spanning sequences of both genes, whereas normal IgG or no antibody treatment did not (Fig. 3C). Additionally, -MafB did not precipitate control region sequences for PEPCK, a gene not expressed in the pancreas. This analysis further supports a direct role for MafB in insulin and glucagon activation in and precursor cells.
MafB Stimulates mafA Transcription.
To examine whether the loss of MafA in insulin– endocrine cells results from the direct actions of MafB on mafA transcription, MafB was over-expressed in insulinoma TC3 cells. Adenoviral-mediated MafB (Ad:MafB) does not affect endogenous insulin, pdx1, or 
-actin expression under these conditions (21), whereas mafA mRNA levels were induced 15-fold (Fig. 3D). Presumably, transcription of insulin and pdx1 is already maximal in cell lines. ChIP analysis demonstrated that MafB was capable of binding to the cell-specific control domain of mafA found between base pairs –8118 and –7750 (Fig. 3E) (31), with binding at the base pair –7810/–7782 element detected in gel-shift assays (SI Fig. 7). The combined data support a role for MafB in mafA transcription during cell development.
Pdx1 and GLUT2 Are only Produced in Insulin+ Cells in E18.5 MafB–/– Embryos.
The maturation of cells in regards to glucose responsiveness and the promotion of high insulin expression occurs during late gestation in mice (32). Thus, cells do not become organized into islet structures, obtain high insulin production capacity, or produce significant amounts of membrane-associated GLUT2 until around E18.5 (33). During this period, Pdx1 and Nkx6.1 transcription factor expression becomes principally restricted to developing cells, with much higher Pdx1 produced in this cell population than surrounding acinar cells (42).
Pdx1 and Nkx6.1 mRNA and protein expression is maintained at wild-type levels in the insulin– endocrine cell population at E15.5 (Figs. 3B and 4A), despite the 50% decrease in insulin+ cell number in MafB mutant embryos. However, pdx1 mRNA levels decreased significantly by E18.5 (Fig. 3B), whereas the percentage of mutant Pdx1+/insulin+ cells was the same as wild type (Fig. 4A). In addition, pan endocrine marker expression was unaffected within this developmental time frame (Fig. 2). These results strongly indicated that pdx1 mRNA expression was restricted to insulin+ cells in E18.5 MafB–/– endocrine cells, and that sustaining high Pdx1 expression involves MafB actions in precursor cells.
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cell precursor population, immunohistochemical staining for Nkx2.2, Pdx1, and insulin was performed at E18.5. Nkx2.2 is expressed in and cell precursors (34) and serves as a positive control in this analysis, because its mRNA and protein levels were unchanged at E15.5 and E18.5 in MafB–/– pancreata (Figs. 3B and 4B, and SI Fig. 6). As predicted Pdx1 was selectively lost in insulin– cells, the percentage of cells expressing Nkx2.2+/Pdx1+/insulin+ decreased by E18.5 in MafB mutant embryos from 75% to 57% (Fig. 4B), whereas the percentage of cells expressing Nkx2.2 alone increased from 22% to 37% (Fig. 4B). These results demonstrate that Pdx1 is selectively lost from the insulin– endocrine cell population by E18.5, whereas Nkx2.2 expression is maintained in the MafB mutant. We conclude that the loss of Pdx1 reflects the importance of MafB in maintaining expression through activation at the large Maf response element present in the pdx1 regulatory region (35), whereas retention in insulin+ cells represents the actions of MafA.
In contrast to Pdx1, Nkx6.1 expression was still detected in both insulin+ and insulin– cells in MafB–/– embryos at E18.5 (Fig. 4A). However, Nkx6.1 mRNA levels were reduced in the mutant (Fig. 3B). ChIP analysis performed over the nkx6.1 regulatory region in Ad:MafB-infected TC3 cells supports that MafB directly regulates transcription (Fig. 3E). Interestingly, the E18.5 MafB–/– embryo also had a higher proportion of Nkx6.1+ only cells, implying that immature cells accumulate in the absence of MafB.
A hallmark of cell maturation is the establishment of membrane bound GLUT2. At E18.5, GLUT2 expression was undetected in hormone– endocrine cells and was present in only 56% of the remaining insulin+ cells (Fig. 4C). Consistent with a loss of GLUT2 in insulin+ cells, the E18.5 mutant pancreata contained but 32% of the wild-type GLUT2 mRNA level (Fig. 3B). This effect is unlikely to simply be due to the reduction in Pdx1, a known transcriptional regulator (4), because GLUT2 expression was lost in many insulin and Pdx1 producing cells. The ability to detect MafB binding to upstream GLUT2 regulatory sequences supports a direct role for this factor in activation (Fig. 3E). GLUT2 expression is also decreased in adult MafA-deficient animals (14), further indicating a crucial role for large Maf factors in GLUT2 transcription in cells. These results demonstrate that MafB activates genes like GLUT2 and pdx1 that are critical to cell maturation.
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and cell differentiation with previously uncharacterized control properties. A notable phenotype of MafB-deficient animals was the loss of 50% of insulin+ and glucagon+ cells while retaining the endocrine cell population and expression of and cell identity markers in both hormone– and hormone+ endocrine cells during early islet cell development. Significantly, MafA expression was only detected in insulin+ cells and pdx1 and nkx6.1 levels were specifically reduced in hormone– endocrine cells between E15.5 and E18.5 cells. MafB also profoundly impacted GLUT2 expression by E18.5, resulting in reduced levels in insulin+ cells and complete loss in hormone– cells.
Importantly, the defects in and cell development in the MafB null animals are very different from other mouse models with impaired endocrine cell development, which often impact islet cell production by affecting cell lineage commitment, differentiation, and/or the generation of endocrine progenitors. In contrast, MafB appears to be a key regulator of and cell maturation, by directly affecting transcription of genes enriched in these cells and crucial to function (Fig. 5). Thus, MafB did not affect the expression of genes broadly expressed in islet cell types (e.g., Pax6, Isl1, and NeuroD1) but only those selectively present in or cells (e.g., : glucagon; : insulin, MafA, and Pdx1).
Our data also suggests the presence of at least two distinct cell populations within the developing pancreas. In one, transcription of many key cell genes is principally dependent on MafB (e.g., insulin, pdx1, mafA, and GLUT2), whereas it is only partially dependent in the other cell population because of the presence of MafA (Fig. 5). The presence of a Maf transcription factor appears to be key to insulin transcription, because the production of insulin+ cells was delayed until the onset of MafA expression in the MafB–/– embryos. MafB may play a similar important role in cell differentiation, although this was not apparent in our analysis of the comparatively few cell-enriched products available (i.e., Arx and Brn4). Our findings also strongly indicate that MafB is the principal regulator of insulin and glucagon transcription during development.
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Immunohistochemistry.
Immunofluorescence and confocal image analyses were performed on paraffin sections as described (23). The primary antibodies used were rabbit -MafA (1:2,000; Bethyl Laboratories, Montgomery, TX), rabbit -Pdx1 (1:10,000; Chris Wright, Vanderbilt University Medical School), guinea pig -insulin (1:2,000; Linco Research, St. Charles, MO), guinea pig -glucagon (1:2,000; Linco Research), rabbit -glucagon (1:2,000, Linco Research), rabbit -Brn4 (1:1,000; Palle Serup, Hagedorn Research Institute, Gentofte, Denmark), rabbit -Nkx6.1 (1:1,000; Palle Serup), mouse -Nkx2.2 (1:500; Developmental Studies Hybridoma Bank, Iowa City, IA), mouse -Isl1 (1:300 Developmental Studies Hybridoma Bank), rabbit -GLUT2 (1:1,000; Chemicon, Billerica, MA), and rabbit -Pax6 (1:300; Covance, Princeton, NJ). Secondary antibodies were Cy3- or Cy5-conjugated donkey -guinea pig, -mouse, and -rabbit IgG (1:500; Jackson ImmunoResearch, West Grove, PA). Nuclear counterstaining was performed by using YoPro1 (Molecular Probes, Eugene, OR). Fluorescent images were captured with a Zeiss LSM 510 confocal microscope, using an optical depth of 1 µm.
Adenovirus Infection and RNA Analysis.
Purified recombinant mouse MafB (Ad:MafB) and the GFP control (Ad:GFP) adenoviruses (21) were used to infect TC3 (2 x 106) cells for 5 h at a multiplicity of infection of 100. Total RNA was prepared from TC3-infected cells and E15.5 embryonic pancreas anlagen from wild-type and MafB–/– mice by using the RNeasy kit (Qiagen, Valencia, CA). Isolated RNAs were subjected to DNaseI treatment (Versagene RNA DNase kit; Gentra Systems, Minneapolis, MN). Reverse-transcript PCR reagents (TaqMan; Applied Biosystems, Foster City, CA) were used to generate cDNAs from 0.5 µg of DNaseI-treated RNA. PCR mixes were assembled by using SYBR Green Master Mix reagents (Applied Biosystems), and reactions were performed and analyzed by using the Applied Biosystems Prism 7000 sequence detection system and software. The real-time PCR cDNA primer sets for ARX, Brn4, cMaf, -actin, glucagon, -glucokinase, GLUT2, insulin II, MafA, MafB, BETA2, Pax4, Pax6, Nkx2.2, Nkx6.1, and 18s rRNA are available upon request. Relative changes were calculated by the comparative 
Ct method in which -actin was used for normalization (38).
ChIP Assay.
TC3 cells (2 x 107) infected with Ad:GFP or Ad:MafB at a multiplicity of infection of 100 and E18.5 pancreata were formaldehyde cross-linked, and the sonicated protein–DNA complexes were isolated under conditions described previously (23, 39). Sonicated chromatin was incubated 12–14 h at 4°C with -MafB, and the complexes were isolated with A/G-agarose (Santa Cruz Biotechnology, Santa Cruz, CA). PCR was performed on 1/10 of the purified immunoprecipitated DNA by using Ready-to-Go PCR beads (Amersham Pharmacia Biotech, Rockville, MD) and 15 pmol of the mafA Region 3 (31), insulin (23), PEPCK, glucagon (21), GLUT2 (40), and Nkx6.1 (41) transcriptional control region primers. PCR primer sequence information is available upon request. Amplified products were resolved on a 1.4% agarose gel in Tris-acetate EDTA buffer containing ethidium bromide.
Statistical Analysis.
The entire pancreas anlagen from several animals (n 
3 for each genotype) were used to obtain a representative average of the number of endocrine or hormone+ cells. Immunofluorescence staining was performed on 6-µm sections; positive cells were counted every eighth section throughout the pancreas at E15.5 and E18.5, every fifth section at E12.5 and E13.5, and on all sections at E10.5. The average cell number per section was determined for each individual pancreas (all sections containing pancreatic tissue were examined). The percentage of single-positive (Pdx1+, Nkx2.2+, MafA+, Nkx6.1+, Brn4+) cells to double-positive (Pdx1+/INS+, Nkx2.2+/INS+, Nkx6.1+/INS+, MafA+/INS+, Brn4+/GLU+) or triple-positive (Nkx2.2+/Pdx1+/INS+) cells was measured by counting expressing cells in at least 20 random microscopy fields. Mean differences were tested for statistical significance by using the Student's two tail t test.
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Abbreviations: En, embryonic day n.
To whom correspondence should be addressed. E-mail: roland.stein{at}vanderbilt.edu
Freely available online through the PNAS open access option.
Author contributions: I.A. and R.S. designed research; I.A., B.B., J.C.R., M.G., and T.K. performed research; S.C. and M.S. contributed new reagents/analytic tools; I.A. and J.C.R. analyzed data; and I.A. and R.S. wrote the paper.
The authors declare no conflict of interest.
This article contains supporting information online at www.pnas.org/cgi/content/full/0700013104/DC1.
© 2007 by The National Academy of Sciences of the USA
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