The cellular repair response to damaged articular cartilage that has been characterized by traditional light microscopic technology suggests reactivation of the same chondrocyte lineage that initially formed the growth plate cartilage. However identifying the cellular components of the lineage within adult cartilage is not possible with available technology, nor is it possible to test the hypothesis that cells within the articular cartilage are derived from a distinctly different limb of the chondrocyte lineage. This proposal is designed to introduce a new tool set of genetic markers for lineage studies in the chondrocytes that form the articular and growth plate cartilage. Based on our prior experience of building and validating promoter-GFP reporter transgenic mice for studying the osteoprogenitor lineage, a similar strategy is proposed for the chondrocyte lineage. The plan integrates input from four different investigators and combines the molecular skills of recombineering and BAC transgenesis, early limb developmental biology and GFP fluorescence imaging in living cell and fixed histological section. The genetic units for driving GFP have been chosen from early limb development studies as likely candidates to identify cells at defined stages of differentiation. A proven protocol for the rapid production of BAC-GFP constructs has been selected. The methods for the rapid screening of BAC transgenic mice, obtained from existing colonies or produced in house, will identify genetic units which can either alone or in combination be used to identify a definable level of chondrocyte differentiation. The hypothesis that this technology driven grant will test is that the chondrocytes of the articular cartilage have a distinctly different pathway of differentiation than the chondrocytes that form the growth plate. Specifically, articular chondrocytes develop from the interzone cells of the developing joint which ultimately give rise to many of the structures of the synovial joint. Following this reasoning, the genetic unit that will be tested for the growth plate chondrocytes will include: mesenchymal chondrogenic progenitor cells in the developing limb, FGF10; prechondrocytes (precartilage condensation), Sox9 and Col1a1; proliferating chondrocytes, Sox9 and Col2a1; prehypertrophic chondrocytes, IHH; hypertrophic chondrocytes, Col10a1. The genetic units to identify cell of the articular lineage will include: pre-interzone cells, Dlx5 and Col2a1; interzone cells, Gdf5; early articular chondrocytes, Pg4 (lubricin) and mature articular cells, tnc (tenascin c) and Col2a1. The validated genetic units for lineage identification produced from this proposal will be made available to the chondrocyte community and will form the basis for more advanced expression constructs for fate mapping, directed and inducible expression, multiplexing and vector delivery. ? ?
Visual markers that report the level of differentiation within the chondrocyte lineage will be a significant advance for studying diseases of articular cartilage. Knowing the relative distribution of cells from progenitor to fully differentiated, particularly if articular and growth plate cartilage arise from a different branch, will give fundamental insight into cellular pathogenesis and point to agents which might alter lineage progression in a positive direction. However the greatest application of the GFP reporters may be in regenerative medicine in which progenitor cells from a donor (marked with one reporter color) are implanted into the articular cartilage of an injured host (marked with another color). This approach will indicate the success of the donor to differentiate to the appropriate cell type and integrate with the host articular chondrocytes. Current markers cannot make this distinction that has restrained progress in developing chondrocyte progenitors that can be directed to form articular cartilage. The GFP tool set that this grant will produce will be the first step in a more knowledge based approach to regenerative medicine for diseases of the joint. ? ? ?
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