(Fig. 1B-B9). At e12.5, Sox9 also widely co-localizes with Foxa2 (Fig. 1C-C0) and Tcf2 (Fig. 1D-D0). Some cells already expressing glucagon and/or insulin with elevated Foxa2 levels but little or no Sox9 were also present (Fig. 1C-C0). By e15.5, during the major window of pancreatic differentiation (termed the secondary transition), Sox9 becomes exclusively restricted to the monolayered, polarized epithelial ‘‘cords’’ (Fig. 1E–H0), from which Ngn3+ endocrine progenitors and their descendents as well as mature ductal cells arise [3,4]. In the progenitor epithelium, Sox9 co-localized with Pdx1 (Fig. 1E9,E0, yellow arrowheads). Notably, Sox9+ progenitors displayed lower levels of Pdx1 expression than the Sox92 hormone+-cells that have delaminated from the progenitor cell epithelium (Fig. 1E9,E0, yellow versus white arrowheads) [26]. Sox9+ progenitors also expressed Foxa2 at e15.5 (Fig. 1G-G0). As previously reported [4], progenitor cells of the e15.5 epithelial cords were additionally marked by Tcf2 (Fig. 1H). However, at the distal tips of the lumens, we consistently observed cells that expressed high levels of Sox9, but little or no Tcf2 (Fig. 1H9,H0, arrowheads). These distal Sox9+ cells presumably constitute acinar progenitors [3]. Together, this expression analysis shows that Sox9+ progenitors express Pdx1, Hnf6, Foxa2, and Tcf2 during the major period of endocrine cell differentiation. Sox9 is required for the maintenance of Pdx1 Next, we examined whether Sox9 regulates the expression of the transcription factors Pdx1, Hnf6, Foxa2, and Tcf2 in the pancreatic progenitor epithelium. We have shown that Pdx1-Cre-mediated inactivation of both Sox9flox alleles leads to pancreatic hypoplasia (Fig. 2B,D,F,H,J; [13]). Although we saw no obvious decrease in Pdx1 expression at e10.5 in Sox9Dpan/Dpan pancreata [13], by e12.5, there was a striking decrease in Pdx1 immunofluorescence intensity in Sox9-deleted cells (Fig. 2C,D). In contrast to our earlier analysis [13], some mosaicism was apparent in the deletion of Sox9 by Pdx1- Cre. As such, robust Pdx1 expression was maintained only in those progenitor cells that retained Sox9 (Fig. 2D, arrowheads). Together, the retention of Pdx1 in Sox9+ cells and the gradual loss of Pdx1 in Sox9-deleted cells suggest that Sox9 is required for the maintenance of Pdx1 expression in pancreatic progenitors. Con- trastingly, at e12.5, the expression of Hnf6, Foxa2, and Tcf2 appeared unaffected by Sox9 deletion (Fig. 2E–J). It has been proposed that Sox9 interacts with Hnf6, Foxa2, and Tcf2 in a transcriptional network that stabilizes pancreatic progenitor gene expression, but also cooperates in activating Ngn3, which disrupts the network in order to drive endocrine differentiation [15]. To investigate whether Sox9 differentially regulates these transcription factors during the major period of endocrine cell neogenesis in vivo, we examined the expression of Hnf6, Foxa2, and Tcf2, as well as Pdx1 in Sox9-deficient pancreas at e15.5. However, the acute pancreatic hypoplasia resulting from Pdx1-Cre-mediated Sox9 ablation precluded us from dissecting later roles of Sox9 in pancreatic differentiation. To overcome this obstacle, we utilized a ubiquitously expressed tamoxifen-inducible Cre line, R26CreTM, to ablate Sox9 after e12.5 and then examine the effects at e15.5 in Sox9De13/De13 mice. R26CreTM-mediated deletion of Sox9 by intraperitoneal admin- istration of tamoxifen at e12.5 in pregnant dams resulted in efficient ablation of Sox9 protein in ,90% of the luminal epithelial cells comprising the Sox9+ domain at e15.5 (Fig. 3A,B). Since Sox9 was inactivated after the period of rapid progenitor cell expansion, overall pancreatic organ size and morphogenesis were not significantly affected in Sox9De13/De13 mice at e15.5 (Fig. 3A– J; data not shown). Mirroring our earlier observation of Pdx1 loss following Pdx1-Cre-mediated Sox9 deletion (Fig. 2C–D), Sox9-Haploinsufficiency Causes Glucose Intolerance immunofluorescence analysis revealed a dramatic decrease in the number of Pdx1+ cells at e15.5 in Sox9De13/De13 pancreata (Fig. 3C,D). This decrease was predominantly observed in the luminal epithelial cells and was accompanied by a marked reduction in the number of newly-formed Pdx1+/insulin+ cells (Fig. 3D). Since Pdx1 is critical for endocrine cell differentiation and regulates expression of the endocrine differentiation factor Ngn3 [12], this finding suggests that Sox9-dependent regulation of Pdx1 expression in the progenitor cell epithelium after e13.5 is critical for the major wave of beta-cell differentiation that occurs between e14 and birth. While Sox9 deletion had a profound effect on Pdx1 expression, the expression of Hnf6, Foxa2, and Tcf2 appeared unaffected by the loss of Sox9 (Fig. 3E–J). To determine whether Sox9 might control the expression of Hnf6, Foxa2, Tcf2, and Pdx1 at later time points, when Sox9 becomes restricted to the pancreatic ducts (Fig. 3K), we induced Sox9 deletion by injecting pregnant dams with tamoxifen at e14.5 and analyzed the embryos at e18.5 in Sox9De15/De15 mice. Similar to the inactivation performed at e12.5, R26CreTM-mediated deletion of Sox9 by tamoxifen injection at e14.5 resulted in efficient ablation of Sox9 protein (Fig. 3K,L). At e18.5, Pdx1 expression is largely restricted to the endocrine cell compartment (Fig. 3M; [26]). Sox9 deletion Figure 2. Pancreas-specific Sox9-deletion results in loss of Pdx1 by e12.5. Immunofluorescence staining of control (A, C, E, G, I) and Sox9Dpan/Dpan pancreata (B, D, F, H, J) at e12.5. Pdx1-Cre-mediated Sox9- flox recombination efficiently ablates Sox9 (B), resulting in pancreatic hypoplasia (B, D, F, H, J). (A, B, E–J) E-cadherin (Ecad) was used to visualize the Sox92 pancreatic epithelium. While loss of Sox9 does not affect expression of Hnf6 (F), Foxa2 (H), or Tcf2 (J), Pdx1 expression is reduced (D). (D) Mosaic recombination results in an unrecombined population of Sox9+ cells, which retains high levels of Pdx1 (white arrowheads). Scale bars: 50 mm. doi:10.1371/journal.pone.0023131.g002 PLoS ONE | www.plosone.org 4 August 2011 | Volume 6 | Issue 8 | e23131