Form and pattern arise during vertebrate embryogenesis because groups of cells differentiate under specific spatial and temporal constraints imposed by reciprocal tissue interactions. Central to this research is the hypothesis that such interactions are mediated in part by unique sets of cell surface and extracellular proteins. We plan to test this hypothesis using the developing avian limb. Specifically, the aim of this research is to identify and characterize extracellular and cell surface proteins that are (i) restricted to specific regions of the limb bud, (ii) correlated in the time of their appearance with key morphogenetic events and (iii) affected by mutations that disrupt limb morphogenesis. Proteins that meet these criteria are very likely to be controlling factors in morphogenesis. Cell surface and matrix proteins will be identified and compared in (1) uncommitted, proliferating limb mesoderm vs. differentiation proximal mesoderm in normal embryos and in talpid2 mutants which form polysyndactylous limbs, (2) inductively active apical extodermal ridge vs. non-ridge extoderm from normal embryos and ectoderm from the transient limb buds of limbless mutants which fails to form an apical ridge. Proteins will be labeled in vivo with 35S-methionine, 3H-fucose or 32p-orthophosphate and cell surface and matrix proteins and glycoproteins will be selectively labeled in vitro with 125I/lodogen or 3H-borohydride/periodate. Extracellular, cell surface and plasma membrane proteins will be further distinguished by selective enzymatic digestion of intact tissues, by selective solubilization and by plasma membrane isolation. We believe that the limbless and talpid mutants provide excellent model systems for the analysis of inherited limb defects in higher organisms and that the characterization of cell surface and extracellular proteins in both normal and mutant limb development will significantly enhance our understanding of the molecular bases of tissue interactions. We hope that these studies will conribute to a better understanding of human congenital limb defects.
