resulted in a noticeable reduction of Pdx1+ cells, suggesting that Sox9 continues to be required for endocrine cell formation at later stages of development (Fig. 3N). This finding is consistent with the observation that Sox9+ cells give rise to new endocrine cells until birth [3]. Mirroring our results obtained in embryos after Sox9 deletion at earlier time points, ablation of Sox9 during late embryogenesis did not affect the expression of Hnf6, Foxa2, and Tcf2 (Fig. 3O–T). Quantitative RT-PCR analysis confirmed the requirement of Sox9 for Pdx1 expression, revealing a significant, dose-dependent decrease in Pdx1 transcript levels with progressive loss of Sox9 in pancreata of Sox9+/+RSox9+/De13RSox9De13/De13 mice at e15.5 (Fig. 4A). Likewise, and consistent with our findings in Sox9+/Dpan mice [14] (Fig. 4B), Ngn3 mRNA levels were dependent upon Sox9 gene dosage (Fig. 4A,B). Reflecting the reduction in mRNA levels, whole pancreata of e15.5 Sox9De13/De13 mice also exhibited a 60% reduction in Pdx1 protein levels compared with those of control littermates (Fig. 4C,D). Concordant with the results of our immunofluorescence analysis (Fig. 3E–J), Hnf6, Foxa2, and Tcf2 Sox9-Haploinsufficiency Causes Glucose Intolerance transcript and protein levels at e15.5 were unaffected by deletion of Sox9 at ,e13 (Fig. 4A,C,D). Unlike Sox9+/De13 mice (Fig. 4A), Sox9+/Dpan mice displayed a slight, but significant reduction in Hnf6 and FoxA2 mRNA levels at e15.5 (Fig. 4B). Our findings reveal that Sox9 is crucially required for maintaining Pdx1 and Ngn3 expression in the pancreatic progenitor cell epithelium during the major wave of endocrine cell differentiation. Since Pdx1 has been shown to regulate the expression of Ngn3 and, in turn, beta-cell formation [12], Sox9- dependent regulation of Pdx1 is a critical component of the transcriptional network governing endocrine and beta-cell devel- opment. However, as Pdx1 levels are not affected by Sox9- halpoinsufficiency, direct regulation of Ngn3 by Sox9 appears to be the critical factor that accounts for reduced endocrine cell mass in pancreata of Sox9-haploinsufficient mice at birth. Importantly, the fact that Hnf6, Foxa2, and Tcf2 expression were not affected by deletion of Sox9 argues strongly that, contrary to the in vitro findings of Lynn et al. [15], Sox9 does not regulate these upstream regulators of Ngn3 expression during pancreas organogenesis. Figure 3. Sox9 deletion during endocrine cell neogenesis results in reduced numbers of Pdx1+ cells. Immunofluorescence staining of control (A, C, E, G, I, K, M, O, Q, S) and R26-CreER; Sox9flox/fox embryos (B, D, F, H, J, L, N, P, E, T) injected with tamoxifen at e12.5 and analyzed at e15.5 (Sox9De13/De13) or injected with tamoxifen at e14.5 and analyzed at e18.5 (Sox9De15/De15). Sox9 expression is lost in the majority of E-cadherin+ (Ecad+) epithelial cells (B, L). Deletion of Sox9 results in decreased expression of Pdx1 (D, N) as well as a reduction in the number of insulin+ (Ins+) cells, while Hnf6 (F, P), FoxA2 (H, R), and Tcf2 (J, T) expression is unaffected. Scale bar: 50 mm. doi:10.1371/journal.pone.0023131.g003 PLoS ONE | www.plosone.org 5 August 2011 | Volume 6 | Issue 8 | e23131