STUDIES ON THE MECHANISMS OF LIMB POSITIONING

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1. Limb bud is thought to be formed in the dorso-ventral boundary region. We first demonstrated that the limb bud is actually formed in the dorso-ventral boundary by forming ectopic bounadry in the flank region. Furthermore, we demonstrated that the limb bud is formed in the boundary between En-l-positive and En-1-negative regions.2. Fibroblast Growth Factor (FGF) is known to induce additional limbs. However, in the previous study only one additional limb was formed. We found that two additional limbs were formed when FGF-2-soaked bead was applied. The limb formed in the anterior flank region was complete in proximodistal pattern but the limb formed in the posterior trunk region was truncated.FGF-4 and FGF-8 induced only one additional limb. FGF-2 induced ectopic expression of sonic hedgehog(shh) near the bead, although no such ectopic expression was observed near the FGF-4 and FGF-8 beads. Two ectopic limbs seem to be formed by the formation of two ectopic expression domains of shh and fgf-10.3. To analyze the molecular mechanisms of apical ectodermal ridge (AER) formation in the dorsal-ventral boundary, we established an in vitro culture system of limb bud ectoderm. The limb bud ectoderm can be cultured for 4 days, and the sorting-out of AER cells from non-AER cells was observed. Many fgf-8-positive ridge-like structures were formed in the monolayer culture. This system will be suitable for the analysis of AER formation in the dorsal-ventral boundary.