EPITHELIAL/MESENCHYMAL INTERACTIONS IN LIMB PATTERNING

本研究的长期目标是了解来自发育中的肢体芽的三个信号中心的信息如何在空间和发育时间上整合，从而产生羊膜肢体的图案。为了做到这一点，(i)必须发现目前未知的信号中心通路的组成部分，以及（ii）确定一种整合单调信号中心效应器的方法。在过去的拨款期间，我们定义了一个无肢突变体的分子生物学。我们提出假设，鸡无肢突变体是利用差异克隆技术研究胚胎模式形成中这两个重要问题的重要工具。减去无肢和野生型肢芽共有的cDNA，得到一个富含信号通路成分的cDNA文库。初步结果有力地支持了这一假设的有效性。根据初步数据，我们提出以下三个具体目标。1)继续筛选富含信号中心通路的cDNA文库，分离出以空间和时间受限模式表达的新cDNA，并通过对已知肢体模式基因的上位性分析和利用病毒技术的错表达来分析它们在肢体模式形成中的作用。2)确定鸡肢体发育中的作用(c)线虫异慢性基因Lin-41的同源物，我们认为这是一个沿着发育肢体的近端-远端轴的定时基因；在初始筛选中分离出clin41。3)确定初始筛选中分离的Wnt7a-FGF应答基因在背腹侧信号通路下游已知组分中的作用；或者，在一个平行的通路中，其功能被认为是维持肢体芽中的Sonic hedgehog信号。这项研究将为胚胎发育中的细胞信号传递提供新的信息。通过定义一个假定的鸡异慢性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-远端轴的发育时间是如何被控制的。这可能会导致在分子水平上的畸形，如光面畸形的见解。同样，通过确定独特的Wnt7a-FGF反应基因的作用，我们将显著推进对这一复杂途径在肢体发育中的作用的理解。

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.