EPITHELIAL/MESENCHYMAL INTERACTIONS IN LIMB PATTERNING

这项拟议研究的长期目标是了解来自发育中肢芽的三个信号中心的信息如何在空间和发育时间上整合，以产生有图案的羊膜肢体。为了做到这一点，(I)必须发现目前未知的信令中心路径的组件，以及(Ii)确定整合单调信令中心效应器的手段。在过去的资助期间，我们定义了无肢突变体的分子生物学。我们提出的假设是，鸡的无肢突变体是利用差异克隆技术研究这两个在胚胎模式形成中的重要问题的重要工具。减去无肢型和野生型肢芽常见的cDNA，可以得到一个丰富的信号通路成分的cdna文库。初步结果有力地支持了该假说的正确性。根据初步数据，我们提出了以下三个具体目标。1)继续筛选信号中心途径丰富的cDNA文库，分离在空间和时间受限模式中表达的新的cDNA，并通过对已知肢体模式基因的上位分析和病毒技术的错误表达来分析它们在肢体模式形成中的作用。2)确定线虫异时性基因LIN-41的鸡(C)同源基因在肢体发育中的作用；在最初的筛选中分离到了clin-41。3)确定在最初筛选中分离的Wnt7a-成纤维细胞生长因子反应基因在背侧-腹侧信号通路中已知成分下游的功能中的作用；或者，在平行通路中的作用，其功能被认为是维持肢体芽中的Sonic Hedgehog信号。这项拟议的研究将产生关于胚胎发育中细胞信号的新信息。通过确定一个假定的鸡异时基因在肢体发育中的作用，我们将能够更好地理解沿肢体近端-远端轴的发育时机是如何被控制的。这可能会在分子水平上导致对青光眼等畸形的洞察。同样，通过定义独特的WNT7a-成纤维细胞生长因子反应基因的作用，我们将显著促进对这一复杂途径如何在肢体发育中发挥作用的理解。

The long-term objective of the proposed research is to understand how information from the three signaling centers of the developing limb bud is integrated in space and developmental time to produce the patterned amniote limb. In order to do this (i) components of the signaling center pathways that are presently unknown must be discovered, and (ii) a means of integrating the monotonic signaling center effectors determined. In the past grant period, we defined the molecular biology of a limbless mutant. We propose the hypothesis that the chicken limbless mutant is an important tool for using differential cloning techniques to study these two significant problems in embryonic pattern formation. Subtraction of cDNAs common to limbless and wild-type limb buds yielded a cDNA library enriched for signaling pathway components. Preliminary results give strong support to the validity of the hypothesis. Following up on the preliminary data we propose the following three specific aims. 1) Continue to screen the signaling center pathway-enriched cDNA library to isolate novel cDNAs that are expressed in spatially and temporally restricted patterns and to analyze them for their role in limb pattern formation by epistatic analysis to known limb pattern genes and misexpression using viral techniques. 2) Determine the role in limb development of a chicken (c) homolog of the C. elegans heterochronic gene Lin-41 which we propose as a timing gene along the proximal-distal axis of the developing limb; cLin-41 was isolated in the initial screen. 3) Determine the role of a Wnt7a-FGF responsive gene isolated in the initial screen for its function in the dorsal-ventral signaling pathway downstream of known components in this pathway; or, alternatively, in a parallel pathway whose function is proposed to maintain Sonic hedgehog signaling in the limb bud. The proposed research will produce new information on cell signaling in embryonic development. By defining the role of a putative chicken heterochronic gene in limb development we will be able to better understand how developmental timing along the limb proximal-distal axis is controlled. This could lead to insights at the molecular level of malformations such as phocomelia. Similarly, by defining the role of the unique Wnt7a-FGF responsive gene we will significantly advance understanding of how this complex pathway functions in limb development.