Abstract

The exact mechanisms that regulate limb outgrowth the mouse embryo are unknown. Although there are several models, we favor a hypothesis where cells become polarized by signals secreted from the AER which orient their cell migration and/or divisions causing limb outgrowth. Clonal analysis has provided a mechanism to study cell behavior. We have generated a targeting construct containing the Fgf8 inhibitor, Sprouty2, in order to generate mutant clones for behavioral analyses in the limb. In order to more effectively study clonal behavior we report the modification of a novel clonal analysis approach, exo-utero surgery. We have modified, enhanced and proven that this technique can be used successfully in mouse embryos in which we directly apply 4-OHtamoxifen to the limb to induce YFP or β-gal reporter genes in limb mesenchyme. Using this method, we can closely control the timing and location of the induced clones and observe cell behavior during embryonic limb development. Phenotypes of Wnt5a-/- and Ror2-/- exhibit shortened limbs suggesting they function in a similar pathway. Wnt5a and Ror have been found to "colocalize" in the growing limb bud and have also been shown to bind in vitro. Here we show preliminary results about Wnt5a and Ror2 in vivo association by immunoprecipitation of limb bud extracts.

Degree

College and Department

Life Sciences; Physiology and Developmental Biology

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