STUDIES ON THE MECHANISMS OF LIMB POSITIONING

1.肢芽被认为是在背腹交界区形成的。我们首先证明了肢芽实际上是通过在侧腹区域形成异位边界而在背腹边界形成的。此外，我们还证实了肢芽形成于En-1阳性和En-1阴性区域的交界处.成纤维细胞生长因子（FGF）是已知的诱导额外的肢体。然而，在之前的研究中，只形成了一个额外的肢体。我们发现，当应用FGF-2浸泡的珠粒时，形成了两个额外的肢。在前胁区形成的肢是完整的近远侧模式，但在后躯干区形成的肢被截断。FGF-4和FGF-8只诱导了一个额外的肢。FGF-2诱导珠附近的音刺猬（shh）的异位表达，虽然没有这样的异位表达附近的FGF-4和FGF-8珠观察。两个异位肢似乎是由shh和fgf-10两个异位表达域的形成而形成的。为探讨背腹交界处顶端外胚层嵴形成的分子机制，建立了肢芽外胚层体外培养体系。肢芽外胚层可培养4天，观察到AER细胞从非AER细胞中分选出来。在单层培养中形成许多fgf-8阳性的脊状结构。该系统将适用于分析背腹边界AER的形成。

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.