UBC Theses and Dissertations
Delineating the molecular mechanisms regulating chondrogenesis Karamboulas, Konstantina
Sox9, (SRY-type HMG box), has been shown to play a critical role throughout chondrogenesis. Haploinsufficiency of Sox9 in humans leads to a skeletal malformation syndrome known as campomelic dysplasia. To understand the regulation of Sox9 during chondrogenesis, the developing mouse limb was used to identify and characterize regulatory regions within the Sox9 promoter. Luciferase-based reporter assays in mouse revealed a proximal promoter spanning – 2 kb from the transcriptional start site, while mobility shift assays demonstrated that a CCAAT motif is involved in the transactivation of Sox9. Moreover, luciferase-based reporter assays revealed a proximal promoter spanning – 4 kb in Fugu rubripes, and potential regulatory regions spanning the remainder of the promoter. Comparison of mammalian Sox9 upstream intergenic sequences to that of Fugu has identified 5 conserved regions that are contained within 18 kb of upstream Fugu sequence. Analysis of the transcriptional activity of these sequences has led to the identification of regulatory elements within the Sox9 promoter. Several studies also provide evidence of a role for wingless (WNT) and bone morphogenetic (BMP) signaling molecules in the regulation of chondrogenesis. TCF/LEF-LacZ reporter mice show activated canonical WNT signaling distributed throughout the embryonic age (E) 9.5 forelimb. At later stages, LacZ expression becomes confined to distal regions of the limb bud. Previous studies have demonstrated that canonical WNTs inhibit chondrogenesis. Our studies demonstrate that treatment of cultures derived from E11.5 proximal limb buds with the canonical WNT, WNT3a, inhibits chondrogenesis. However, treatment of cultures derived from E9.5 and distal E11.5 limb buds with WNT3a stimulates chondrogenesis. Quantitative PCR (qPCR) also demonstrates that WNT3a modulates a number of genes expressed throughout chondrogenesis. To gain insights into BMP function in the early limb, we have characterized BMP action in sub-populations of cells from the E10.5 limb. Surprisingly, BMPs were found to inhibit cartilage formation in immature cells, while promoting cartilage formation in more mature cells. Transcriptional profiling coupled with qPCR and time course analyses revealed that the extent of induction of Gatas by BMPs was associated with its stimulatory versus inhibitory activity. Further, SOX9 activity was inhibited following over-expression of Gatas.
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