Figure 8. Contrasting the previously suggested Sox9 transcrip- tional regulatory network during pancreas development to the network based on genetic evidence. (A) Proposed Sox9 regulated genes based on in vitro evidence [15]. (B) Revised model based on this study as well as previous genetic in vivo studies [12,19,33,34,35,47]. Note that a solid line and arrow does not necessarily indicate direct gene regulation. doi:10.1371/journal.pone.0023131.g008 enhancers [32] and occupancy of these enhancers by different transcription factors might dynamically change during develop- ment. Genome wide DNA occupancy studies for Sox9 in progenitor cells will determine which of the genes that are regulated by Sox9 in vivo are direct transcriptional target genes. Genetic studies have previously shown that similar to Sox9, Hnf6 also functions upstream of Pdx1 [33] and is important for the expression of Ngn3 [34]. Furthermore, Hnf6 has been shown to regulate the expression of Tcf2 (MODY5) [19], a transcription factor that is also required for Ngn3 expression [35]. We show that even though all three factors are co-expressed in the epithelial progenitor cords during the major wave of endocrine cell formation, loss of Sox9 does not affect the expression of Tcf2 or Hnf6. This suggests that Sox9 regulates Pdx1 and Ngn3 expression independent of Tcf2 and Hnf6. While the requirement for Ngn3 in endocrine development is well established, the network of transcription factors required for its activation has not been comprehensively identified, nor their interactions clearly defined. Here, we reveal that Sox9 is required for proper Ngn3 induction through several possible mechanisms. Previous observations led us to suggest a mechanism through which Sox9 induces Ngn3 expression. Wang et al. have recently shown that Ngn3 expression in pancreatic progenitors must attain a critical high ‘‘Ngn3Hi’’ threshold level to initiate an endocrine developmental program in those cells [36]. It is conceivable that Sox9 activates Ngn3 in a dosage-sensitive manner such that a Sox9Hi expression level is crucial for activation of a Ngn3Hi expression level sufficient to induce endocrine differentiation. In support of this hypothesis, we observed a significant reduction in the number of Ngn3Hi cells in Sox9-haploinsufficient mice [14]. Additionally, Pdx1, which we show is expressed in a Sox9- dependent manner, has also been reported to activate Ngn3 in coordination with Hnf6 [12]. Thus, Sox9 contributes to acquisition of the Ngn3Hi state by two mechanisms: (1) by directly activating Ngn3 and (2) by reinforcing Ngn3 expression through positive regulation of Pdx1. Since no functional role has thus far been demonstrated for Sox9 in endocrine cell maturation [14], those progenitors that attain sufficiently high Ngn3 expression to initiate an endocrine program should differentiate normally, so that, albeit reduced in numbers, the adult beta-cells of Sox9- haploinsufficient pancreata are mature and fully functional. Consistent with this notion, we found that islets from Sox9- haploinsufficient mice exhibit normal GSIS in vitro. Sox9-Haploinsufficiency Causes Glucose Intolerance The role of Sox9 in maintaining adult glucose homeostasis In humans, Sox9-haploinsufficiency is associated with the syndrome campomelic dysplasia (CD). Although pancreatic islet abnormalities have been reported in neonatal CD cases [37], the early lethality of the condition precludes the manifestation of metabolic defects. However, as pancreas-specific expression of Sox9 is governed by enhancer region E1, located approximately 28 kb 59 of the transcription initiation site in humans [38], it is conceivable that enhancer-specific Sox9 mutations could cause a diabetic phenotype in humans. The viability of mice displaying pancreas-specific heterozygous loss of Sox9 afforded us the unique opportunity of studying the effects of reduced Sox9 dosage on pancreatic endocrine function in adult mice, to test whether Sox9 is a potential MODY or diabetes susceptibility gene. While the majority of MODY genes play important functional roles in adult beta-cells [16,17,27,39,40], Tcf2/MODY5 is the exception. Like Sox9, Tcf2 is excluded from beta-cells and instead, is expressed in the pancreatic progenitor epithelium during embryonic development, becoming restricted to duct and centroacinar cells in adulthood [3,4]. Pancreatic hypoplasia, pancreatic atrophy, defective expression of Glut2, and impaired insulin secretion have been reported in human cases of Tcf2 mutation [35,41]. This suggests that dysregulation of factors not expressed in beta-cells but expressed in their precursors, can cause diabetes independently of beta-cell dysfunction, due presumably to pancreatic endocrine dysgenesis. While we found no evidence for pancreatic Tcf2 expression being Sox9-dependent, our data suggest that Sox9-haploinsufficiency might itself manifest in a MODY phenotype. Furthermore, the fact that Sox9 regulates expression of the MODY4 gene Pdx1 during development raises the possibility that the diabetic phenotype caused by heterozygous Pdx1 mutations [7] could in part be attributable to impaired beta- cell development. While we found a 50% reduction in Sox9 gene dosage to cause glucose intolerance in mice, Sox9+/Dpan mice did not develop overt diabetes. This raises the question of how severely reduced pancreatic Sox9 levels might affect glucose homeostasis in humans. Interestingly, attempts to generate mouse models for human MODY have revealed that mutations in mouse homologues of human MODY genes do not always phenocopy the human condition. While Tcf2-haploinsufficient humans display diabetes and severe pancreatic endocrine defects [42,43], partially mirroring our findings in Sox9+/Dpan mice, Tcf2+/2 mice do not display diabetic symptoms [44]. Additionally, the MODY3 phenotype is not recapitulated in Hnf1a+/2 mice, although Hnf1a2/2 mice do become diabetic [17]. One possible explanation for the discrepancy between the metabolic effects of MODY gene mutations in mouse and humans is that the beta-cells of mice are able to better compensate for the reduction in gene dosage than those of humans. Consistent with this idea, we observed compensatory beta-cell proliferation in Sox9+/Dpan mice, which led to a significant, albeit not complete, recovery of beta-cell mass during the early postnatal period. Because human beta-cells have a much lower proliferative capacity than murine beta-cells [45], it is predicted that reduced Sox9 levels in humans will have more profound effects on glucose homeostasis than observed in Sox9-haploinsufficient mice. Although to this point Sox9 has not emerged as a type 2 diabetes susceptibility gene from genome-wide association studies (GWAS) [46], other genes with known roles in human diabetes, such as Pdx1, also failed to show an association with type 2 diabetes in GWAS. Additional studies are therefore required to determine whether mutations in Sox9 are associated with diabetes in humans. PLoS ONE | www.plosone.org 10 August 2011 | Volume 6 | Issue 8 | e23131