Self-Organization in Limb Pattern Formation--Test of a Hypothesis

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0090499NewmanThe objective of this project is to test a hypothesis for how the cells of the vertebrate limb bud generate the quasi-periodic array of cartilage rods and nodules that serve as the primordia of the limb skeleton in all such species. The hypothesis is that the "self-organizing" properties of limb mesenchymal cells that lead them to generate spatiotemporally arranged precartilage condensations and cartilage nodules in vitro are also utilized in vivo in the generation of the limb skeletal pattern. To test this idea key gene regulatory processes will be identified that are intrinsic to limb bud mesenchyme which provide the basis for its ability to generate precartilage condensations in a spatiotemporally-controlled fashion. Because the extracellular matrix protein fibronectin is a major mediator of precartilage cell condensation both in vitro and in vivo, DNA constructs will be designed to contain the chicken fibronectin gene promoter linked to a readily visualized reporter molecule, Green Fluorescent Protein. The length of normal promoter sequence that directs authentic condensation-associated gene expression in vitro and then mutate the minimal promoter to determine which cis-acting sites are essential for this activity will be identified. This will permit the delineation of candidate sites for action of developmentally relevant transcription factors such as Fast-1. The response of these constructs to added growth and differentiation factors such as TGF-B2, and FGF-2, -4 and -8 will be used to help identify endogenous factors that regulate fibronectin expression. Once the relevant determinants have been characterized in vitro, they will be introduced into embryonic limbs by electroporation or detergent-mediated transfection to ascertain whether or not the establishment of fibronectin "prepatterns" for the skeletal primordia in vivo employ the same fibronectin regulatory pathways utilized in the self-organizing processes responsible for the generation of these patterns in vitro.