P.L.H., P.M., and W.R. from progenitor cells expressing the transcription factor Ngn3 (1). Ngn3 regulates specification of the four endocrine cell lineages as a function of specific developmental time windows (2). A complex network of transcription factors directs the differentiation of Ngn3+ progenitors into mature endocrine cells (3). Important factors implicated in -cell development include NeuroD1, Nkx2.2, Pax4, Nkx6.1, MafA, and Pdx1 (3). NeuroD1, encoded by an Ngn3-regulated gene, is required for the formation of -cells (4). Nkx2.2 functions downstream of NeuroD1 and promotes commitment of cells to the , , and PP lineages at the expense of the -cell lineage (5,6). A balance between Arx and Pax4 expression controls specification of / versus / precursors (7). Nkx6.1 is expressed in cells committed to the -lineage and participates in the developmental program leading to the generation of mature -cells (8). Mature -cells acquire the capacity to synthesize and secrete insulin in response to variations in blood glucose levels. Important components of the glucose-sensing and insulin secretion machinery include the Glut-2 glucose transporter and the glucose sensor glucokinase. Several transcription factors have been implicated in the acquisition of mature -cell functions, including Pdx1, MafA, and NeuroD1 (4,9,10). There is growing evidence that Rfx transcription factors are implicated in islet development. You will find seven Rfx factors (Rfx1CRfx7) in mammals (11C13). With the exception of Rfx5, which is a well-known regulator in the immune system (14), the functions of mammalian Rfx factors have only started to emerge recently (15C19). Rfx6 was recently demonstrated to be crucial for islet development in zebra fish, mice, and humans (18,19). We had reported earlier that pancreatic Rfx3 expression is restricted to islets and detected in Ngn3+ progenitors and , , , and PP cells (20). Islets of perinatal expression. Finally, we recognized the glucokinase gene as a direct target of Rfx3. These results show that Rfx3 is required for the differentiation and function of mature -cells, and that it is a key regulator of glucokinase expression. RESEARCH DESIGN AND METHODS Mice. Data for allele in which exon 3 is usually flanked by sequences (deletion (with mice (21). mice. E0.5 was defined as the morning when a vaginal plug was detected. Genotyping was carried out as explained (16). Mice were on a TCS 401 C57BL/6 background. Experiments were approved by the Federal and Cantonal veterinary government bodies. Staining of sections and morphometry. For E13.5 and E15.5, pancreases were cut, respectively, into three or five consecutive series of 10 sections. For E17.5 and E19.5, pancreases were cut into seven consecutive series of 10 sections. Measurements were performed using one section from each series. Immunostaining of frozen sections was performed by standard procedures. Antibodies and secondary reagents are indicated in supplementary Table 1, available in the online appendix at http://diabetes.diabetesjournals.org/cgi/content/full/db09-0986/DC1. Apoptotic cells were revealed by Tdt-mediated dUTP nick end labeling (TUNEL) staining (Roche). Stained sections were visualized by confocal microscopy. Cell TCS 401 counting and morphometry were performed using Mertamorph v6.2 (Universal Imaging Corporation). Labeled cells were quantified within Pdx1+ cells (E13.5 and E15.5) or 4,6-diamidino-2-phenylindole dihydrochloride (DAPI)-stained cells (E17.5 and E19.5). Islet purification. Mouse islets were isolated as explained (22). Human islets (purity 90%, viability 95%) were provided by the Islet Cell Rabbit polyclonal to LRRC15 Resource Center of Geneva (Juvenile Diabetes Research Foundation, European islet distribution program). Quantitative RT-PCR. Total RNA was extracted from pancreas with RNeasy packages (QIAGEN, Switzerland) and from Min6 cells and purified islets with TRIzol (Invitrogen). Quantitative RT-PCR (qRT-PCR) was performed as explained (20). Results were normalized TCS 401 using TATA-binding protein (The best results were obtained with the following: 5-CGCGTCCCCAACACTGGAGGAAATTACTTTCAAGAGAAGTAATTTCCTCCAGTGTTTTTTTGGAAAT-3 (sense) and 5-CGATTTCCAAAAAAACACTGGAGGAAATTACTTCTCTTGAAAGTAATTTCCTCCAGTGTTGGGGA-3 (antisense). Min6 cells were transduced as explained (24). GFP expression was utilized for assessing transduction efficiencies and purifying.