A far-upstream (-70 kb) enhancer mediates Sox9 auto-regulation in somatic tissues during development and adult regeneration.

SOX9 encodes a transcription factor that presides over the specification and differentiation of numerous progenitor and differentiated cell types, and although SOX9 haploinsufficiency and overexpression cause severe diseases in humans, including campomelic dysplasia, sex reversal and cancer, the mechanisms underlying SOX9 transcription remain largely unsolved. We identify here an evolutionarily conserved enhancer located 70-kb upstream of mouse Sox9 and call it SOM because it specifically activates a Sox9 promoter reporter in most Sox9-expressing somatic tissues in transgenic mice. Moreover, SOM-null fetuses and pups reduce Sox9 expression by 18-37% in the pancreas, lung, kidney, salivary gland, gut and liver. Weanlings exhibit half-size pancreatic islets and underproduce insulin and glucagon, and adults slowly recover from acute pancreatitis due to a 2-fold impairment in Sox9 upregulation. Molecular and genetic experiments reveal that Sox9 protein dimers bind to multiple recognition sites in the SOM sequence and are thereby both necessary and sufficient for enhancer activity. These findings thus uncover that Sox9 directly enhances its functions in somatic tissue development and adult regeneration through SOM-mediated positive auto-regulation. They provide thereby novel insights on molecular mechanisms controlling developmental and disease processes and suggest new strategies to improve disease treatments.

Sox9 directs hypertrophic maturation and blocks osteoblast differentiation of growth plate chondrocytes.

The transcription factor Sox9 is necessary for early chondrogenesis, but its subsequent roles in the cartilage growth plate, a highly specialized structure that drives skeletal growth and endochondral ossification, remain unclear. Using a doxycycline-inducible Cre transgene and Sox9 conditional null alleles in the mouse, we show that Sox9 is required to maintain chondrocyte columnar proliferation and generate cell hypertrophy, two key features of functional growth plates. Sox9 keeps Runx2 expression and ß-catenin signaling in check and thereby inhibits not only progression from proliferation to prehypertrophy, but also subsequent acquisition of an osteoblastic phenotype. Sox9 protein outlives Sox9 RNA in upper hypertrophic chondrocytes, where it contributes with Mef2c to directly activate the major marker of these cells, Col10a1. These findings thus reveal that Sox9 remains a central determinant of the lineage fate and multistep differentiation program of growth plate chondrocytes and thereby illuminate our understanding of key molecular mechanisms underlying skeletogenesis.

Sox6 is necessary for efficient erythropoiesis in adult mice under physiological and anemia-induced stress conditions.

BACKGROUND: Definitive erythropoiesis is a vital process throughout life. Both its basal activity under physiological conditions and its increased activity under anemia-induced stress conditions are highly stimulated by the hormone erythropoietin. The transcription factor Sox6 was previously shown to enhance fetal erythropoiesis together and beyond erythropoietin signaling, but its importance in adulthood and mechanisms of action remain unknown. We used here Sox6 conditional null mice and molecular assays to address these questions. METHODOLOGY/PRINCIPAL FINDINGS: Sox6fl/flErGFPCre adult mice, which lacked Sox6 in erythroid cells, exhibited compensated anemia, erythroid cell developmental defects, and anisocytotic, short-lived red cells under physiological conditions, proving that Sox6 promotes basal erythropoiesis. Tamoxifen treatment of Sox6fl/flCaggCreER mice induced widespread inactivation of Sox6 in a timely controlled manner and resulted in erythroblast defects before reticulocytosis, demonstrating that impaired erythropoiesis is a primary cause rather than consequence of anemia in the absence of Sox6. Twenty five percent of Sox6fl/flErGFPCre mice died 4 or 5 days after induction of acute anemia with phenylhydrazine. The others recovered slowly. They promptly increased their erythropoietin level and amplified their erythroid progenitor pool, but then exhibited severe erythroblast and reticulocyte defects. Sox6 is thus essential in the maturation phase of stress erythropoiesis that follows the erythropoietin-dependent amplification phase. Sox6 inactivation resulted in upregulation of embryonic globin genes, but embryonic globin chains remained scarce and apparently inconsequential. Sox6 inactivation also resulted in downregulation of erythroid terminal markers, including the Bcl2l1 gene for the anti-apoptotic factor Bcl-xL, and in vitro assays indicated that Sox6 directly upregulates Bcl2l1 downstream of and beyond erythropoietin signaling. CONCLUSIONS/SIGNIFICANCE: This study demonstrates that Sox6 is necessary for efficient erythropoiesis in adult mice under both basal and stress conditions. It is primarily involved in enhancing the survival rate and maturation process of erythroid cells and acts at least in part by upregulating Bcl2l1.

Synovial joint morphogenesis requires the chondrogenic action of Sox5 and Sox6 in growth plate and articular cartilage.

The mechanisms underlying synovial joint development remain poorly understood. Here we use complete and cell-specific gene inactivation to identify the roles of the redundant chondrogenic transcription factors Sox5 and Sox6 in this process. We show that joint development aborts early in complete mutants (Sox5(-/-)6(-/-)). Gdf5 and Wnt9a expression is punctual in articular progenitor cells, but Sox9 downregulation and cell condensation in joint interzones are late. Joint cell differentiation is unsuccessful, regardless of lineage, and cavitation fails. Sox5 and Sox6 restricted expression to chondrocytes in wild-type embryos and continued Erg expression and weak Ihh expression in Sox5(-/-)6(-/-) growth plates suggest that growth plate failure contribute to this Sox5(-/-)6(-/-) joint morphogenesis block. Sox5/6 inactivation in specified joint cells and chondrocytes (Sox5(fl/fl)6(fl/fl)Col2Cre) also results in a joint morphogenesis block, whereas Sox5/6 inactivation in specified joint cells only (Sox5(fl/fl)6(fl/fl)Gdf5Cre) results in milder joint defects and normal growth plates. Sox5(fl/fl)6(fl/fl)Gdf5Cre articular chondrocytes remain undifferentiated, as shown by continued Gdf5 expression and pancartilaginous gene downregulation. Along with Prg4 downregulation, these defects likely account for joint tissue overgrowth and incomplete cavitation in adult mice. Together, these data suggest that synovial joint morphogenesis relies on essential roles for Sox5/6 in promoting both growth plate and articular chondrocyte differentiation.

Generation of mice harboring a Sox5 conditional null allele.

Sox5 belongs to the Sry-related HMG box gene family, which encodes transcription factors controlling cell fate and differentiation in many lineages. Sox5 produces a long L-Sox5 protein in neuronal, glial, neural crest, cartilage, and other cells, and a short Sox5 protein in spermatids. Sox5(-/-) mice have revealed essential roles for L-Sox5 in development but their neonatal death has prevented postnatal studies. We show here that we have generated mice harboring a conditional null allele for L-Sox5 (Sox5(fl+)) by flanking the fifth coding exon with loxP sites. Cre recombinase-mediated conversion of Sox5(fl+) into Sox5(fl-) abolishes L-Sox5 expression. Expectedly, Sox5(fl+/fl+) mice are indistinguishable from wildtype mice, and Sox5(fl-/fl-) mice from Sox5(-/-) mice. Moreover, the chondrodysplasia of Sox5(fl+/fl+)Sox6(fl+/fl+)Prx1Cre mice demonstrates that the two redundant chondrogenic Sox genes can be efficiently inactivated in a cell type-specific manner. This Sox5 conditional null allele will be valuable in further uncovering the in vivo roles of L-Sox5.

The three SoxC proteins--Sox4, Sox11 and Sox12--exhibit overlapping expression patterns and molecular properties.

The group C of Sry-related high-mobility group (HMG) box (Sox) transcription factors has three members in most vertebrates: Sox4, Sox11 and Sox12. Sox4 and Sox11 have key roles in cardiac, neuronal and other major developmental processes, but their molecular roles in many lineages and the roles of Sox12 remain largely unknown. We show here that the three genes are co-expressed at high levels in neuronal and mesenchymal tissues in the developing mouse, and at variable relative levels in many other tissues. The three proteins have conserved remarkable identity through evolution in the HMG box DNA-binding domain and in the C-terminal 33 residues, and we demonstrate that the latter residues constitute their transactivation domain (TAD). Sox11 activates transcription several times more efficiently than Sox4 and up to one order of magnitude more efficiently than Sox12, owing to a more stable alpha-helical structure of its TAD. This domain and acidic domains interfere with DNA binding, Sox11 being most affected and Sox4 least affected. The proteins are nevertheless capable of competing with one another in reporter gene transactivation. We conclude that the three SoxC proteins have conserved overlapping expression patterns and molecular properties, and might therefore act in concert to fulfill essential roles in vivo.

Generation of mice harboring a Sox4 conditional null allele.

Sox4 belongs to the family of Sry-related HMG box transcription factors, which specify cell fate and differentiation in many lineages. Sox4 is widely expressed in the embryo and controls such processes as neuronal tissue, lymphocyte, heart, and bone development. Sox4-null mice die at embryonic day 14 from heart malformation. This early lethality has therefore limited studies on Sox4 functions. We show here that we have generated mice harboring a Sox4 conditional null allele (Sox4fl+) by flanking the entire coding region with loxP sites. Sox4fl+/fl+ mice are indistinguishable from wildtype mice and produce the wildtype Sox4 protein at a normal level. Sox4fl+ is efficiently converted into a null allele (Sox4fl-) by Cre recombinase in somatic and germ-line cells, and Sox4fl-/fl- embryos die from the same heart defects as Sox4-/- mice. This Sox4 conditional null allele will thus be a valuable tool to further uncovering Sox4 functions in various processes in vivo.

Generation of mice harboring a Sox6 conditional null allele.

Sox6 belongs to the family of Sry-related HMG box transcription factors, which determine cell fate and differentiation in various lineages. Sox6 is expressed in several tissues, including cartilage, testis, neuronal, and erythropoietic tissues. Mice lacking Sox6 have revealed critical roles for Sox6 in several of these tissues, but their multiple defects and early lethality has limited studies in specific cell types and in postnatal mice. We show here that we have generated mice harboring a Sox6 conditional null allele (Sox6(fl+)) by flanking the second coding exon with loxP sites. This allele encodes wildtype Sox6 protein, is expressed normally, and is efficiently converted into a null allele (Sox6(fl-)) by Cre-mediated recombination in somatic and germ cells. Sox6(fl+/fl+) mice are indistinguishable from wildtype mice, and Sox6(fl-/fl-) mice from Sox6(-/-) mice. These Sox6 conditional null mice will thus be valuable for further uncovering the roles of Sox6 in various processes in vivo.

Sox5 and Sox6 are needed to develop and maintain source, columnar, and hypertrophic chondrocytes in the cartilage growth plate.

Sox5 and Sox6 encode Sry-related transcription factors that redundantly promote early chondroblast differentiation. Using mouse embryos with three or four null alleles of Sox5 and Sox6, we show that they are also essential and redundant in major steps of growth plate chondrocyte differentiation. Sox5 and Sox6 promote the development of a highly proliferating pool of chondroblasts between the epiphyses and metaphyses of future long bones. This pool is the likely cellular source of growth plates. Sox5 and Sox6 permit formation of growth plate columnar zones by keeping chondroblasts proliferating and by delaying chondrocyte prehypertrophy. They allow induction of chondrocyte hypertrophy and permit formation of prehypertrophic and hypertrophic zones by delaying chondrocyte terminal differentiation induced by ossification fronts. They act, at least in part, by down-regulating Ihh signaling, Fgfr3, and Runx2 and by up-regulating Bmp6. In conclusion, Sox5 and Sox6 are needed for the establishment of multilayered growth plates, and thereby for proper and timely development of endochondral bones.