The factors necessary to maintain organ-specific progenitor cells are poorly understood and yet of extreme clinical importance. Here, we identify the transcription factor SOX9 as the first specific marker and maintenance factor of multipotential progenitors during pancreas organogenesis. In the developing pancreas, SOX9 expression is restricted to a mitotically active, Notch-responsive subset of PDX1 ؉ pluripotent progenitors and is absent from committed endocrine precursors or differentiated cells. Similar to Notch mutations, organ-specific Sox9 inactivation in mice causes severe pancreatic hypoplasia resulting from depletion of the progenitor cell pool. We show that Sox9 maintains pancreatic progenitors by stimulating their proliferation, survival, and persistence in an undifferentiated state. Our finding that SOX9 regulates the Notcheffector HES1 suggests a Notch-dependent mechanism and establishes a possible genetic link between SOX factors and Notch. These findings will be of major significance for the development of in vitro protocols for cell replacement therapies.

The HMG-box transcription factor Sox9 is expressed in the intestinal epithelium, specifically, in stem/progenitor cells and in Paneth cells. Sox9 expression requires an active β-catenin–Tcf complex, the transcriptional effector of the Wnt pathway. This pathway is critical for numerous aspects of the intestinal epithelium physiopathology, but processes that specify the cell response to such multipotential signals still remain to be identified. We inactivated the Sox9 gene in the intestinal epithelium to analyze its physiological function. Sox9 inactivation affected differentiation throughout the intestinal epithelium, with a disappearance of Paneth cells and a decrease of the goblet cell lineage. Additionally, the morphology of the colon epithelium was severely altered. We detected general hyperplasia and local crypt dysplasia in the intestine, and Wnt pathway target genes were up-regulated. These results highlight the central position of Sox9 as both a transcriptional target and a regulator of the Wnt pathway in the regulation of intestinal epithelium homeostasis.

SRY-box containing gene 9 (Sox9) and scleraxis (Scx) regulate cartilage and tendon formation, respectively. Here we report that murine Scx+/Sox9+ progenitors differentiate into chondrocytes and tenocytes/ligamentocytes to form the junction between cartilage and tendon/ligament. Sox9 lineage tracing in the Scx+ domain revealed that Scx+ progenitors can be subdivided into two distinct populations with regard to their Sox9 expression history: Scx+/Sox9+ and Scx+/Sox9− progenitors. Tenocytes are derived from Scx+/Sox9+ and Scx+/Sox9− progenitors. The closer the tendon is to the cartilaginous primordium, the more tenocytes arise from Scx+/Sox9+ progenitors. Ligamentocytes as well as the annulus fibrosus cells of the intervertebral discs are descendants of Scx+/Sox9+ progenitors. Conditional inactivation of Sox9 in Scx+/Sox9+ cells causes defective formation in the attachment sites of tendons/ligaments into the cartilage, and in the annulus fibrosus of the intervertebral discs. Thus, the Scx+/Sox9+ progenitor pool is a unique multipotent cell population that gives rise to tenocytes, ligamentocytes and chondrocytes for the establishment of the chondro-tendinous/ligamentous junction.

In the presence of the Y-chromosomal gene Sry, the bipotential mouse gonads develop as testes rather than as ovaries. The autosomal gene Sox9, a likely and possibly direct Sry target, can induce testis development in the absence of Sry. Sox9 is thus sufficient but not necessarily essential for testis induction. Mutational inactivation of one allele of SOX9/Sox9 causes sex reversal in humans but not in mice. Because Sox9(-/-) embryos die around Embryonic Day 11.5 (E11.5) at the onset of testicular morphogenesis, differentiation of the mutant XY gonad can be analyzed only ex vivo in organ culture. We have therefore conditionally inactivated both Sox9 alleles in the gonadal anlagen using the CRE/loxP recombination system, whereby CRE recombinase is under control of the cytokeratin 19 promoter. Analysis of resulting Sox9(-/-) XY gonads up to E15.5 reveals immediate, complete sex reversal, as shown by expression of the early ovary-specific markers Wnt4 and Foxl2 and by lack of testis cord and Leydig cell formation. Sry expression in mutant XY gonads indicates that downregulation of Wnt4 and Foxl2 is dependent on Sox9 rather than on Sry. Our results provide in vivo proof that, in contrast to the situation in humans, complete XY sex reversal in mice requires inactivation of both Sox9 alleles and that Sox9 is essential for testogenesis in mice.