Two lines of evidence suggest that the Sry-related gene Sox9 is important for chondrogenesis in mammalian embryos. Sox9 mRNA is expressed in chondrogenic condensations in mice, and mutations in human SOX9 are known to cause skeletal dysplasia. We show here that mouse SOX9 protein is able to bind to a SOX/SRY consensus motif in DNA and contains a modular transcriptional activation domain, consistent with a role for SOX9 as a transcription factor acting on genes involved in cartilage development. One such gene is Col2a1, which encodes type II collagen, the major structural component of cartilage. We have compared, in detail, the expression of Sox9 and Col2a1 during mouse development. In chondrogenic tissues the expression profiles of the two genes were remarkably similar. Coexpression was detected in some nonchondrogenic tissues such as the notochord, otic vesicle, and neural tube, but others such as heart and lung differed in their expression of the two genes. Immunohistochemistry using an antibody specific for SOX9 revealed that expression of SOX9 protein mirrored the distribution of Sox9 mRNA. Our results suggest that SOX9 protein is involved in the regulation of Col2a1 during chondrogenesis, but that this regulation is likely to depend on additional cofactors.

We report the complete genomic organization of the human WNT-5A gene, which encodes a cysteine-rich growth factor involved in cell-cell signaling during growth and differentiation. The gene comprises five exons with the terminal exon coding for a large 3-untranslated region of Ϸ6.5 kilobase pairs and utilizes multiple polyadenylation signals to generate at least four discrete transcripts. We discovered a new leader exon interrupted by a 411-base pair intron that was retained in our original cDNA cloning. The promoter region was located in a GpC-rich island and harbored numerous cis-acting elements including several GC boxes and Sp1, AP1, and AP2 binding motifs. It lacked TATA or CAAT boxes typical of housekeeping and growth factor genes. In support of this, primer extension revealed two transcription start sites. Transient cell transfection assays showed functional promoter activity for the 3.9-kilobase pair 5-flanking region. Interestingly, internal and 5 deletions revealed that the distal promoter was not required for full transcriptional activity and that the first 631 base pairs of WNT-5A harbored the strongest promoter activity. Using a panel of rodent-human hybrid DNAs carrying portions of chromosome 3p, we mapped the gene to 3p14.2-p21.1, between a constitutional and a familial renal cell carcinoma-associated translocation. In situ hybridization analyses of early human embryos at 28 -42 days of gestation revealed that WNT-5A transcripts were not restricted to the developing brain and limbs but were also observed in the mesenchyme bordering the pharyngeal clefts and pouches and in the developing gonads and kidneys. The relatively high expression in the celomic epithelium and in the precursors of follicles and seminiferous tubules suggest a novel role for WNT-5A in germ-cell differentiation. This study provides the molecular basis for discerning the regulation of the WNT-5A gene and offers the opportunity to investigate genetic disorders linked to this important gene.