Sox9-Haploinsufficiency Causes Glucose Intolerance Results Sox9 expression parallels that of a network of factors upstream of Ngn3 Previously, we showed that Sox9+/Dpan embryos exhibit a 50% reduction in the number of Ngn3+ endocrine progenitors and consequently, all mature endocrine cell types [14]. However, it remains unclear why endocrine cells are reduced in Sox9+/Dpan embryos and how Sox9 orchestrates endocrine cell development in conjunction with other transcriptional regulators that control endocrine cell differentiation. It has been reported that Sox9 regulates the transcription factors Hnf6, Tcf2, and Foxa2, in the mPAC pancreatic tumor cell line [15]. However, it has not been studied whether Sox9 controls the expression of these endocrine differentiation genes during development. Therefore, to test whether this regulation occurs in vivo, we first examined whether the expression domain of Sox9 coincides with those of these transcription factors in MPCs. At e12.5, when the pancreatic epithelium predominantly comprises undifferentiated progenitors, Sox9 is widely co- expressed with Pdx1 (Fig. 1A-A0). At this stage, Sox9+ cells are also intimately associated with cells expressing osteopontin (Spp-1), which, like mucin-1, marks the apical aspect of cells lining the forming luminal network within a still-stratified epithelium [25] (Fig. 1B). Paralleling the expression domain of Sox9, Hnf6 similarly marks cells lining the Spp-1-delineated epithelial lumen Figure 1. Sox9 is co-expressed with Hnf6, Foxa2, Tcf2, and Pdx1 in progenitor cords of the developing pancreas. Immunofluorescence staining of e12.5 (A–D) and e15.5 (E–H) wild-type pancreata reveals extensive overlap of the Sox9+ domain with Hnf6 (B, F), Foxa2 (C, G), and Tcf2 (D, H). (B, F) Osteopontin (Spp1), which marks the apical surface of cells in the lumen of the epithelial cords, is used as a reference for the Sox9 and Hnf6 expression domains. (A) Sox9 is widely coexpressed with Pdx1 throughout the pancreatic epithelium at e12.5. Pdx1+/Sox92 cells at this stage are insulin/glucagon+ (ins+glu) (A9-A0, white arrowheads). (E) At e15.5, Sox9+ cells are found restricted to the inner epithelial layer and are weakly Pdx1+ (E9-E0, yellow arrowheads). At this time, differentiated cells expressing high levels of Pdx1 are insulin/glucagon+, but Sox92 (E9-E0, white arrowheads). Similarly, Foxa2+/Sox92 cells producing insulin/glucagon are present at e12.5 (C9-C0, white arrowheads), as well as at e15.5 (G9-G0, white arrowheads). (D) At e12.5, Sox9 and Tcf2 widely colocalize throughout the pancreatic epithelium. (H) By e15.5, some cells that are Sox9+/Tcf22 are visible at the distal tips of the epithelial cords (H9-H0, white arrowheads). Scale bars: 50 mm. doi:10.1371/journal.pone.0023131.g001 PLoS ONE | www.plosone.org 3 August 2011 | Volume 6 | Issue 8 | e23131