New molecular concepts on pattern formation in limb morphogenesis.

在发育中的肢芽前后轴形成方面，发现了一个在肢芽中具有独特表达模式的基因mario，该基因在肢芽的前后轴形成过程中起着至关重要的作用，并参与了包括mario和bmp在内的分子级联反应。通过HoxD基因表达分析，发现爪蟾前肢趾数不是1234，而是2345。在肢芽近端-远端轴形成方面，详细的命运图显示，在早期肢芽中，未来的自足区和未来的舟足区大部分重叠，但随着发育的进行，重叠越来越小。Hoxa13/Hoxall表达谱证实了这些结果。同时，表达分析显示远端肢体芽的分子状态是渐进式变化的。一系列的移植实验表明，轴向结构的某些因素决定了肢体类型的身份。不同水平的体块移植诱导了Tbx4/5表达的转变，抑制了后肢芽的生长。这些结果表明，肢体场的定位及其身份不是在侧板中胚层中以自主的方式指定的，而是组织-组织相互作用是重要的。研究了shh表达的表观遗传调控，结果表明shh表达与shh增强子序列的DNA甲基化之间存在良好的相关性（shh表达组织在增强子区域的DNA甲基化状况较低），提示shh增强子区域的表观遗传控制在肢体发育和再生过程中对shh表达起作用。

In terms of the anterior-posterior axis formation in a developing limb bud, a gene, mario, which has unique expression pattern in the limb bud, has a crucial role in the antero-posterior patterning, and a part of the molecular cascade for the patterning, including mario and bmp, was identified. Also, digit number in Xenopus forelimb is identified as not 1234 but 2345 by expression analysis of HoxD genes.In terms of the proximal-distal axis formation in the limb bud, a detailed fate map showed that future autopod region and future zeugopod region is largely overlapped in an early limb bud, but that the overlap is getting smaller as the development proceeds. Hoxa13/Hoxall expression profile confirmed these results from the fate map. Also, the expression analysis revealed that the molecular condition in the distal limb bud is changing progressively.A series of transplantation experiments suggested that a factor(s) from the axial structure, somites determines limb-type identity. Transplantation of different level of somites induced transition of Tbx4/5 expression and inhibited outgrowth of the hindlimb bud. These results suggest that positioning of limb field and its identity are specified not in an autonomous manner in the lateral plate mesoderm but that the tissue-tissue interactions are important.Epigenetic regulation of shh expression was examined, and the results indicated a well correlation between shh expression and DNA methylation of shh enhancer sequence (shh-expressing tissues had low condition of DNA methylation in the enhancer region), suggesting that epigenetic control of shh enhancer region plays a role in shh expression during limb development and regeneration.