Tcf2 however, is not expressed in beta-cells. Mirroring the expression pattern of Sox9, it is expressed in the MPC population, then confined to ductal and centroacinar cells in adult pancreas [3,19]. Thus, MODY can occur not only as a result of mutations in genes expressed in adult beta-cells, but also, due to mutations in genes expressed in the progenitors from which they arise. Tcf2 exemplifies that defects in the embryonic development of beta-cells can manifest in diabetes later in life. On the basis of the findings that: 1) Sox9-haploinsufficient mice are born with half the normal complement of pancreatic endocrine cells [14]; 2) Sox9 colocalizes with Tcf2 in embryonic and adult pancreas [3], and 3) Sox9 regulates Tcf2 in vitro [15], we sought to explore whether reduced Sox9 activity could result in a diabetic phenotype during adulthood. Here, we identify a novel role for Sox9 in the regulation of the transcriptional network upstream of Ngn3, specifically in the maintenance of Pdx1 (MODY4) expression. Contrary to previous in vitro studies we find that Sox9 is dispensable for expression of Tcf2, Hnf6, and Foxa2 in the developing pancreas. Additionally, by characterizing the adult phenotype of Sox9-haploinsufficient mice, we show that reduction of Sox9 gene dosage causes glucose intolerance. Sox9-haploinsufficient mice did not progress to overt diabetes, likely owing to a compensatory postnatal increase in beta-cell proliferation and mass observed in Sox9+/Dpan mice. Methods Ethics Statement All animal experiments described herein were approved by the University of California, Irvine and San Diego Institutional Animal Care and Use Committees (protocol numbers 2001– 2420 and S08215, respectively). Mouse Strains and Husbandry Pdx1-Cre; Sox9flox/flox (Sox9Dpan/Dpan) and Sox9+/Dpan mice were generated and maintained as previously described [14]. Gt(RO- SA)26Sortm1(cre/Esr1)Nat/J (hereon abbreviated to R26CreTM) mice [20] were obtained from Jackson Laboratory (JAX) and main- tained on a C57BL/6J background before breeding to Sox9flox/flox mice. In all experiments, Cre2 littermates served as controls. Embryos were harvested from timed matings in which noon on the day of vaginal plug appearance was considered as e0.5. Sox9- ablation in R26CreTM; Sox9flox/flox (Sox9De13/De13) and Sox9+/De13 mice was induced by intraperitoneal (i.p.) injection of 3 mg/40 g body weight tamoxifen (Sigma) dissolved in corn oil (Sigma) into e12.5 or e14.5 pregnant dams. Since recombination in this system occurs within 6 hours of tamoxifen administration [21,22], R26CreTM; Sox9flox/flox mice will be denoted as Sox9De13/De13 or Sox9De15/De15, respectively. For BrdU labeling of 2-week-old mice, 50 mM BrdU (Sigma) was injected i.p. 6 hours before sacrifice. In 6-week-old mice, BrdU labeling was achieved by administration of 1 mg/mL BrdU in the drinking water for one week before sacrifice. High-fat diet-fed mice were fed a 60% high-fat diet from Bio-Serv (F3282). Histological Analysis and Beta-Cell Mass Measurements Tissue preparation, immunochemistry, imaging, and morpho- metric analysis were performed as previously described [14]. Beta- cell mass was calculated as follows: (insulin+ area/total pancreatic area) multiplied by pancreatic weight. For beta-cell proliferation measurements, BrdU and insulin co-positive cells were counted and expressed relative to total insulin+ cells. A minimum of 50 islets were analyzed per animal. At least 3 mice were analyzed for each experimental group. Sox9-Haploinsufficiency Causes Glucose Intolerance The following primary antibodies were used at the given dilutions: rabbit anti-Sox9 (Chemicon), 1:1000; goat anti-Sox9 (Santa Cruz), 1:100; guinea pig anti-Pdx1 (kindly provided by C. V. E. Wright, Vanderbilt University, Nashville TN), 1:10,000; rabbit anti-Hnf6 (Santa Cruz), 1:200; goat anti-Spp1 (R&D Systems), 1:1000; goat anti-Foxa2 (Santa Cruz), 1:200; goat anti-Tcf2 (Santa Cruz), 1:100; rat anti-E-cadherin (Sigma), 1:1000; guinea pig anti- insulin (DAKO), 1:1000; mouse anti-glucagon (DAKO), 1:10,000 and mouse anti-BrdU (Chemicon), 1:200. Secondary antibodies were diluted 1:2000 (Jackson ImmunoResearch or Invitrogen). Quantitative RT-PCR and Western Blot At e15.5, RNA was extracted from pancreata of nine embryos per experimental group and three pancreata per genotype were pooled. Each PCR was run in triplicate. RNA isolation, cDNA synthesis and quantitative (q)RT-PCR were performed and analyzed as previously described [14]. Primers (59-39) used were: Sox9 forward: AGACTCA- CATCTCTCCTAATGCT and reverse: ACGTCGGTTTTGG- GAGTGG; Foxa2 forward: AGGCACTGCGCTTCACTCC and reverse: CTCATTCCAGCGCCCACATAG; Ngn3 forward: AATGATCGGGAGCGCAATCG and reverse: CGCAGGGT- CTCGACCTTTG; Pdx1 forward: GATGAAATCCACCAAA- GCTCA and reverse: AGAATTCCTTCTCCAGCTCCA; Hnf6 forward: GGCAACGTGAGCGGTAGTTT and reverse: TTG- CTGGGAGTTGTGAATGC; Tcf2 forward: GCCTGAAC- CAATCCCACCTC and reverse: TGACTGCTTTTGTCTGT- CATGT. Protein for Western blots was obtained, processed, transferred to nitrocellulose, and incubated with primary antibody as previously described [14]. Pancreata from 3 mice were pooled per sample. Signal was detected and quantified using the Odyssey infrared imaging system and reagents (LI-COR Technologies). Values were calculated relative to GAPDH expression. Metabolic Assays Intraperitoneal glucose tolerance tests (IPGTTs) were per- formed on mice after 16 hours of fasting. Mice were injected i.p. with 1.5 mg/g body weight dextrose solution and their blood glucose was measured at the outset as well as 20, 40, 60, 90, and 120 minutes post-challenge. For in vitro glucose stimulated insulin secretion (GSIS) assays, islets were isolated as previously described [14] and cultured overnight at 37uC in RPMI 1640 (Mediatech) supplemented with 4.8 mM D-glucose (Fisher), 10 mM HEPES (Sigma), 2 mM glutamine (Gibco), 1 mM sodium pyruvate (Sigma), 10% FBS (Sigma) and 1% penicillin/streptomycin (Mediatech). Sets of 30 islets were incubated in 500 ml KRBH buffer [23] containing 2.8 mM D-glucose for 1 hour at 37uC. Buffer was replaced with KRBH containing 16.7 mM D-glucose for an additional 2-hour incubation. Buffer was collected and islets were sonicated in acid ethanol. Insulin content was separately determined in the supernatant and the islet cell fraction and the percentage of secreted insulin calculated as: 100*(secreted insulin/ secreted insulin+islet cell insulin), as previously described [24]. Whole pancreas extracts were obtained by homogenizing whole pancreata in acid ethanol, incubating overnight at 4uC, and harvesting the supernatant. Serum was collected from the blood of 16-hour fasted mice. In all cases, insulin content was determined by mouse insulin ELISA (ALPCO Diagnostics). Statistical Analysis All values are shown as mean 6 standard error of the mean (S.E.M.); p values were calculated using unpaired two-tailed Student’s t test; p,0.05 was considered significant. PLoS ONE | www.plosone.org 2 August 2011 | Volume 6 | Issue 8 | e23131