STUDIES ON THE MECHANISMS OF LIMB POSITIONING

1. 肢芽被认为是在背腹交界区形成的。我们首先通过在侧翼区域形成异位边界证明了肢体芽实际上是在背腹边界形成的。此外，我们还证实了肢芽是在en -1阳性区和en -1阴性区交界处形成的。已知成纤维细胞生长因子（FGF）可诱导额外肢体。然而，在之前的研究中，只形成了一个额外的肢体。我们发现，当使用fgf -2浸泡的头部时，形成了两个额外的肢体。前翼区形成的肢体在近远端模式下是完整的，而后躯干区域形成的肢体是截断的。FGF-4和FGF-8只诱导了一个额外的肢体。尽管在FGF-4和FGF-8小珠附近未观察到这种异位表达，但FGF-2诱导音hedgehog基因（shh）在小珠附近异位表达。两个异位肢似乎是由shh和fgf-10.3两个异位表达域的形成形成的。为了分析背腹边界顶端外胚层脊（AER）形成的分子机制，我们建立了肢体芽外胚层的离体培养体系。肢体芽外胚层培养4 d，观察AER细胞与非AER细胞的分选。在单层培养中形成了许多fgf-8正脊状结构。该系统将适用于背-腹交界区AER形成的分析。

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.