beta-catenin regulated genes during skeletogenesis

The hallmark of vertebrates is that they posses an endoskeleton. This skeleton is formed during development from mesenchymal derived cells, giving rise to chondrocytes, osteoblasts and cell contributing to the joints. Over the past years, enormous progress has been made in elucidating the transcription factors required for the differentiation of chondrocytes and osteoblasts from mesenchymal precursors. Far less is known, about the signaling pathways controlling these lineage decisions. Recently we have shown that the Wnt/ß-catenin pathway is involved in these processes. Activation of the pathway leads to stabilization of ß-catenin and its accumulation in the nucleus, where it interacts with members of the TCF/LEF transcriptionfactor family to activate the transcription of target genes. When it is active this pathway negatively regulates the differentiation of mesenchymal cells into a "skeletal precursor". In essence, ß-catenin levels need to be low in mesenchymal cells in order for them to undergo differentiation into skeletal precursor cells, which express key regulators of skeletal development such as Sox9, Runx2, Sox5, Sox6 and extracellular matrix molecules such as Col2a1. Furthermore, we have shown that ß-catenin levels need to be elevated in uncommitted osteoblast precursor cells as well as in joint interzone cells in order to actively suppress the chondrogenic potential of these cells and to allow them to mature into osteoblasts and joint cells, respectively. The goal of the work proposed here is to identify the target genes of ß-catenin during the different steps of lineage differentiation. We will do this by expression profiling (microarray and Affimetrix) and by ChIP analyses of material originating from primary cells (mesenchymal precusrsors, osteoblast precursors and chondrocytes) of mice homozygous for conditional ß-catenin loss- and gain-of function alleles, which enable us to alter the ß-catenin levels using Cre-recombination. Our specific aims are to identify the direct targets of ß-catenin in different populations of skeletal cells and to analyze their function in those cells. We are motivated by the expectation that this will help us to further elucidate the key processes in skeletal development and that it will eventually lead to new reagents / targets that might be useful for directed differentiation of stem cells into distinct skeletal lineages.

The vertebrate skeleton consists of over 200 skeletal elements (bones), which differ in shape and size. Most of which are formed by the process of endochondral ossification; whereby first a cartilaginous template prefiguring the future bone is formed, which is than later during embryonic development being remodelled into bone. This process needs to be coordinated on multiple levels. Firstly, it needs to be ensured that the primary cartilaginous skeletal elements form only at specific locations within the body. Secondly, since the cell types, which contribute to the cartilaginous skeleton (chondrocytes) and those differentiating into bone forming cells (osteoblasts) share a common precursor, mechanisms must exist which determine whether the precursor cell will develop into a chondrocyte or osteoblast. And lastly, the further differentiation of chondrocytes within the cartilage template needs to be coupled to the differentiation of bone forming cells in the surrounding of the cartilage template where the future bone collar will form, but also at the transition zone where chondrocytes are removed and trabecular bone is being formed. In the past, work from our group has shown that the Wnt/beta-catenin pathway plays an important role in at least the first two processes by exerting an anti-chondrogenic activity. The present project aimed to elucidate the molecular processes underlying this anti-chondrogenic activity of active Wnt/beta-catenin signaling. We undertook a transcriptome-analysis to identify potential beta-catenin regulated genes using mesenchymal cells of the early embryonic limb anlage and chondrocytes of the limb. Within the pool of de- regulated genes we primarily focused on transcription factors, which could serve as potential mediators of the anti- chondrogenic effect of increased beta-catenin levels or could act in concert with a beta-catenin/Tcf transcriptional complex inhibiting the differentiation of cells into chondro-cytes or leading to their de-differentiation. We independently verify the candidates from the transcriptome-analysis using in vitro and in vivo approaches. The promoter regions of some selected potential target genes were further analyzed using a bioinformatics approach and in vitro functional assays. The transcription factor Sox9, which is a master regulator of chondrogenesis, was one of the genes that was down-regulated in both assays. Among the transcription factors, which were up-regulated in both assays was Tcf1, which acts in a complex with beta-catenin. This was particularly interesting, as our promoter studies revealed that Sox9 can be repressed by a beta-catenin/Tcf1 complex specifically. Two other transcription factors, which were positively regulated and direct targets of a beta-catenin/Tcf or /Lef-complex, were also capable to down-regulate Sox9-promoter activity in vitro. Thus, making them likely candidates to act in concert with beta- catenin mediating the anti-chondrogenic effect of Wnt/beta-catenin signalling.