Sox9-Haploinsufficiency Causes Glucose Intolerance Figure 7. Sox9+/Dpan mice exhibit compensatory postnatal beta-cell proliferation. (A) Immunofluorescence staining of control and Sox9+/Dpan pancreata for insulin and glucagon reveals normal islet architecture in Sox9+/Dpan mice. (B) Normal glucose stimulated insulin secretion of isolated islets from Sox9+/Dpan mice (B; n = 7). (C) In Sox9+/Dpan mice, beta-cell mass is reduced by ,30% compared to control littermates at 6 and 16 weeks of age (n = 3), consistent with pancreatic insulin content in 6-week-old Sox9+/Dpan mice being 70% that of control siblings (D; n = 11). (E) Serum insulin levels are comparable in Sox9+/Dpan mice and control littermates (n = 11). (F, G) Beta-cell proliferation, measured by BrdU incorporation into insulin+ (Ins+) cells, is increased in Sox9+/Dpan mice at 2 weeks, but not at 6 weeks of age (n = 3). Scale bars: 50 mm. Values are shown as mean 6 S.E.M.; * = P,0.05. doi:10.1371/journal.pone.0023131.g007 to Ngn3, Sox9 regulates a network of developmental transcription factors upstream of Ngn3, including Hnf6, Foxa2, and Tcf2. These findings, reviewed most recently by Pan and Wright [31], while intriguing, demanded further validation in an in vivo model. Our finding that neither Hnf6, Foxa2 nor Tcf2 are dysregulated in the embryonic pancreas following Sox9 ablation suggests that this same transcriptional network is not conserved in pancreatic progenitor cells in vivo. Our in vivo findings suggest a revised model of the regulatory interactions between key developmental transcription factors in pancreatic progenitor cells (Fig. 8). Importantly, our study uncovers a previously unknown role for Sox9 in regulating Pdx1 expression during the major time window of endocrine cell differentiated in mice. Whether or not Sox9 regulates Pdx1 expression directly is currently unclear. In the early pancreatic bud, Pdx1 expression is initially maintained upon Sox9 inactivation in vivo [13] and only lost at e12.5, which argues against a role for Sox9 in directly regulating Pdx1 at early pancreatic stages. However, it is possible that Sox9 controls Pdx1 expression at early and later developmental stages by distinct mechanisms and that Sox9 deletion after e13 has a more immediate effect on Pdx1 expression. Pdx1 gene transcription is controlled by different PLoS ONE | www.plosone.org 9 August 2011 | Volume 6 | Issue 8 | e23131