The prevention and treatment of birth defects are two of the most important aims of medical research. Fundamental to achieving these goals is an understanding of the genetic basis of normal embryogenesis. The developing chick limb is a powerful model for studying this process due to the wealth of embryological studies and the ease of molecular manipulations. Recently, key regulatory genes have been identified which are important for the generation of distinct polarity in all three cardinal axes of the limb. The secreted factor Wnt7a and the transcription factor Lmx-1 are necessary and sufficient to generate the pattern of tendons and muscles found on the dorsal side of the limb. With this information as a foundation, more detailed questions arise: 1) What role do these factors play in early limb bud formation? 2) To what degree are Lmx-1 and Wnt7a functions coordinates with anterioposterior (A/P) and proximodistal (P/D) pattern formation? 3) How does Lmx-1 dorsalize muscles? With these questions in mind, we propose to study three important aspects of Wnt7a and Lmx-1 function. 1) Lmx-1 and Wnt7a may function early in limb formation by forming developmental compartments which restrict cell movement across the D/V border. We will monitor cell movements by performing a clonal analysis in ovo. We will investigate differences in adhesion by performing cell sorting experiments in vitro. If these assays suggest that compartments do exist, then we will misexpress Lmx-1 or Wnt7a both in ovo and in vitro to determine if they regulate compartment formation. 2) A number of gain and loss of function experiments suggest that Wnt7a and Lmx-1 may regulate and be regulated by factors controlling A/P and P/D pattern formation. We will ectopically express Wnt7a and Lmx-1 in ovo and assay their ability to induce or maintain factors governing A/P of D/P polarity. Likewise, we will determine whether these same A/P and P/D factors can maintain Lmx-1 expression. 3) Lmx-l can generate dorsal muscle pattern. Whether it must be expressed in the muscle precursors or their adjacent mesenchymal cells is unknown. We will investigate this by targeting Lmx-1 expression to myogenic precursors or he surrounding mesenchyme, and then assessing the ability of these cells to generate dorsal muscle pattern.
