Neural crest development is regulated by the transcription factor Sox9

The neural crest is a transient migratory population of stem cells derived from the dorsal neural folds at the border between neural and non-neural ectoderm. Following induction, prospective neural crest cells are segregated within the neuroepithelium and then delaminate from the neural tube and migrate into the periphery, where they generate multiple differentiated cell types. The intrinsic determinants that direct this process are not well defined. Group E Sox genes (Sox8, Sox9 and Sox10) are expressed in the prospective neural crest and Sox9 expression precedes expression of premigratory neural crest markers. Here, we show that group E Sox genes act at two distinct steps in neural crest differentiation. Forced expression of Sox9 promotes neural-crest-like properties in neural tube progenitors at the expense of central nervous system neuronal differentiation. Subsequently, in migratory neural crest cells, SoxE gene expression biases cells towards glial cell and melanocyte fate, and away from neuronal lineages. Although SoxE genes are sufficient to initiate neural crest development they do not efficiently induce the delamination of ectopic neural crest cells from the neural tube consistent with the idea that this event is independently controlled. Together, these data identify a role for group E Sox genes in the initiation of neural crest development and later SoxE genes influence the differentiation pathway adopted by migrating neural crest cells.     

Analysis of the molecular cascade responsible for mesodermal limb chondrogenesis: Sox genes and BMP signaling

Here, we have studied how Sox genes and BMP signaling are functionally coupled during limb chondrogenesis. Using the experimental model of TGFbeta1-induced interdigital digits, we dissect the sequence of morphological and molecular events during in vivo chondrogenesis. Our results show that Sox8 and Sox9 are the most precocious markers of limb cartilage, and their induction is independent and precedes the activation of BMP signaling. Sox10 appears also to cooperate with Sox9 and Sox8 in the establishment of the digit cartilages. In addition, we show that experimental induction of Sox gene expression in the interdigital mesoderm is accompanied by loss of the apoptotic response to exogenous BMPs. L-Sox5 and Sox6 are respectively induced coincident and after the expression of Bmpr1b in the prechondrogenic aggregate, and their activation correlates with the induction of Type II Collagen and Aggrecan genes in the differentiating cartilages. The expression of Bmpr1b precedes the appearance of morphological changes in the prechondrogenic aggregate and establishes a landmark from which the maintenance of the expression of all Sox genes and the progress of cartilage differentiation becomes dependent on BMPs. Moreover, we show that Ventroptin precedes Noggin in the modulation of BMP activity in the developing cartilages. In summary, our findings suggest that Sox8, Sox9, and Sox10 have a cooperative function conferring chondrogenic competence to limb mesoderm in response to BMP signals. In turn, BMPs in concert with Sox9, Sox6, and L-Sox5 would be responsible for the execution and maintenance of the cartilage differentiation program.     

L-Sox5 and Sox6 proteins enhance chondrogenic miR-140 microRNA expression by strengthening dimeric Sox9 activity

Sox9 plays a critical role in early chondrocyte initiation and promotion as well as repression of later maturation. Fellow Sox family members L-Sox5 and Sox6 also function as regulators of cartilage development by boosting Sox9 activation of chondrocyte-specific genes such as Col2a1 and Agc1; however, the regulatory mechanism and other target genes are largely unknown. MicroRNAs are a class of short, non-coding RNAs that act as negative regulators of gene expression by promoting target mRNA degradation and/or repressing translation. Analysis of genetically modified mice identified miR-140 as a cartilage-specific microRNA that could be a critical regulator of cartilage development and homeostasis. Recent findings suggest Sox9 promotes miR-140 expression, although the detailed mechanisms are not fully understood. In this study we demonstrate that the proximal upstream region of pri-miR-140 has chondrogenic promoter activity in vivo. We found an L-Sox5/Sox6/Sox9 (Sox trio) response element and detailed binding site in the promoter region. Furthermore, detailed analysis suggests the DNA binding and/or transactivation ability of Sox9 as a homodimer is boosted by L-Sox5 and Sox6. These findings provide new insight into cartilage-specific gene regulation by the Sox trio.     

Identification of Sox8 as a modifier gene in a mouse model of Hirschsprung disease reveals underlying molecular defect

Mice carrying heterozygous mutations in the Sox10 gene display aganglionosis of the colon and represent a model for human Hirschsprung disease. Here, we show that the closely related Sox8 functions as a modifier gene for Sox10-dependent enteric nervous system defects as it increases both penetrance and severity of the defect in Sox10 heterozygous mice despite having no detectable influence on enteric nervous system development on its own. Sox8 exhibits an expression pattern very similar to Sox10 with occurrence in vagal and enteric neural crest cells and later confinement to enteric glia. Loss of Sox8 alleles in Sox10 heterozygous mice impaired colonization of the gut by enteric neural crest cells already at early times. Whereas proliferation, apoptosis, and neuronal differentiation were normal for enteric neural crest cells in the gut of mutant mice, apoptosis was dramatically increased in vagal neural crest cells outside the gut. The defects in enteric nervous system development of mice with Sox10 and Sox8 mutations are therefore likely caused by a reduction of the pool of undifferentiated vagal neural crest cells. Our study suggests that Sox8 and Sox10 are jointly required for the maintenance of these vagal neural crest stem cells.     

Microbiota-derived lipopolysaccharide retards chondrocyte hypertrophy in the growth plate through elevating Sox9 expression

Accumulating data show that the cytotoxicity of bacterial lipopolysaccharides (LPS) from microbiota or infection is associated with many disorders observed in the clinics. However, it is still obscure whether or not embryonic osteogenesis is affected by the LPS exposure during gestation. Using the early chicken embryo model, we could demonstrate that LPS exposure inhibits chondrogenesis of the 8-day chicken embryos by Alcian Blue-staining and osteogenesis of 17-day by Alcian Blue and Alizarin Red staining. Further analysis of the growth plates showed that the length of the proliferating zone (PZ) increases whereas that of the hypertrophic zone (HZ) decreased following LPS exposure. However there is no significant change on cell proliferation in the growth plates. Immunofluorescent staining, western blot analysis, and quantitive polymerase chain reaction revealed that Sox9 and Col2a1 are highly expressed at the messenger RNA level and their protein products are also abundant. LPS exposure causes a downregulation of Runx2 and Col10a1 expression in 8-day hindlimbs, and a suppression of Runx2, Col10a1, and Vegfa expression in 17-day phalanges. Knocking down Sox9 in ATDC5 cells by small interfering RNA transfection lead to the expression reduction of Col2a1, Runx2, and Col10a1, implying the vital role of Sox9 in the process of LPS-induced delay in the transition from proliferating chondrocytes to hypertrophic chondrocytes in the growth plate. In the presence of LPS, the antioxidant defense regulator nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is highly expressed, and the activities of superoxide dismutase 1 (SOD1), SOD2, and glutaredoxin rise in 17-day phalanges and ADTC5 cells. Simultaneously, an increase of intracellular ROS is observed. When Nrf2 expression was knocked down in ATDC5 cells, the expressions of Sox9, Col2a1, Runx2, Col10a1, and Vegfa were also going down as well. Taken together, our current data suggest that LPS exposure during gestation could restrict the chondrocytes conversion from proliferating to hypertrophic in the growth plate, in which LPS-induced Sox9 plays a crucial role to trigger the cascade of downstream genes by excessive ROS production and Nrf2 elevation.