Sox9 -Haploinsufficiency Causes Glucose Intolerance in Mice Claire L. Dubois., Hung Ping Shih., Philip A. Seymour, Nisha A. Patel, James M. Behrmann, Victoria Ngo, Maike Sander* Department of Pediatrics and Cellular & Molecular Medicine, University of California San Diego, La Jolla, California, United States of America Abstract The HMG box transcription factor Sox9 plays a critical role in progenitor cell expansion during pancreas organogenesis and is required for proper endocrine cell development in the embryo. Based on in vitro studies it has been suggested that Sox9 controls expression of a network of important developmental regulators, including Tcf2/MODY5, Hnf6, and Foxa2, in pancreatic progenitor cells. Here, we sought to: 1) determine whether Sox9 regulates this transcriptional network in vivo and 2) investigate whether reduced Sox9 gene dosage leads to impaired glucose homeostasis in adult mice. Employing two genetic models of temporally-controlled Sox9 inactivation in pancreatic progenitor cells, we demonstrate that contrary to in vitro findings, Sox9 is not required for Tcf2, Hnf6, or Foxa2 expression in vivo. Moreover, our analysis revealed a novel role for Sox9 in maintaining the expression of Pdx1/MODY4, which is an important transcriptional regulator of beta-cell development. We further show that reduced beta-cell mass in Sox9-haploinsufficient mice leads to glucose intolerance during adulthood. Sox9-haploinsufficient mice displayed 50% reduced beta-cell mass at birth, which recovered partially via a compensatory increase in beta-cell proliferation early postnatally. Endocrine islets from mice with reduced Sox9 gene dosage exhibited normal glucose stimulated insulin secretion. Our findings show Sox9 plays an important role in endocrine development by maintaining Ngn3 and Pdx1 expression. Glucose intolerance in Sox9-haploinsufficient mice suggests that mutations in Sox9 could play a role in diabetes in humans. Citation: Dubois CL, Shih HP, Seymour PA, Patel NA, Behrmann JM, et al. (2011) Sox9-Haploinsufficiency Causes Glucose Intolerance in Mice. PLoS ONE 6(8): e23131. doi:10.1371/journal.pone.0023131 Editor: Kathrin Maedler, University of Bremen, Germany Received March 16, 2011; Accepted July 7, 2011; Published August 2, 2011 Copyright: 2011 Dubois et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from the NIH/NIDDK (RO1 DK078803 and RO1 DK68471) to M.S., by postdoctoral fellowships from the JDRF to P.A.S. and H.P.S., and a graduate student fellowship from the CIRM to C.L.D and N.A.P. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: masander@ucsd.edu . These authors contributed equally to this work. Introduction Both proper neogenesis of mature endocrine cells during embryonic pancreas development and the maintenance of an adequate number of functional islets during adulthood are necessary for normal glucose homeostasis. During development, endocrine and exocrine (ductal and acinar) cells of the pancreas derive from multipotent progenitor cells (MPCs) expressing the transcription factors Pdx1 [1], Ptf1a [2], Sox9 [3] and Tcf2 (Hnf1b) [4]. Furthermore, all five endocrine cell types, including the insulin+ beta-cells, arise from a common transient endocrine progenitor marked by expression of the transcription factor Neurogenin 3 (Ngn3) [1]. Proper allocation of MPCs to the endocrine lineage is achieved by induction of Ngn3 [5], which is tightly governed by a complex transcriptional network involving Notch signaling as well as transcription factors, such as Tcf2, Foxa2 (Hnf3b), and Hnf6. While it is well established that Pdx1 (also known as maturity onset diabetes of the young [MODY] 4 [6,7,8]) is required for MPC specification as well as for proper adult islet function [9,10,11], Pdx1 has also recently been shown to regulate Ngn3 directly in cooperation with Hnf6 [12]. We have shown previously that the MPC marker and maintenance factor Sox9 [3,13,14] governs endocrine development in a dosage-dependent manner [14]. Pancreas-specific Sox9- haploinsufficiency in Pdx1-Cre; Sox9+/flox (Sox9+/Dpan) mice results in a 50% decrease in both Ngn3+ endocrine progenitors and consequently, endocrine cells at birth [14]. While reduced in numbers, immunohistochemical analysis of beta-cell markers suggested that beta-cells are properly differentiated in Sox9- haploinsufficient mice. Though Sox9 has been shown to bind the Ngn3 promoter in embryonic pancreas in vivo [14], in vitro studies have implied that it also regulates Hnf6, Tcf2, and Foxa2 and is therefore critical for activating and/or maintaining an entire network of endocrine differentiation genes [15]. However, because early deletion of Sox9 in pancreatic progenitors results in developmental arrest prior to the onset of endocrine cell differentiation [13], the role of Sox9 in regulating key endocrine differentiation genes in vivo has not been studied. By employing temporally controlled Sox9 inactivation strategies in mice, we thus sought to determine which components of the pancreatic transcriptional network are regulated by Sox9 in vivo. MODY is a disease caused by mutations in autosomal dominant genes: MODY1–5 result from mutations in Hnf4a, glucokinase, Hnf1a, Pdx1, and Tcf2, respectively. Most MODY genes are expressed in the mature islet; their downregulation is associated with loss of islet function, manifesting in diabetes [9,16,17,18]. PLoS ONE | www.plosone.org 1 August 2011 | Volume 6 | Issue 8 | e23131