EPITHELIAL/MESENCHYMAL INTERACTIONS IN LIMB PATTERNING

拟议研究的长期目标是了解来自发育中的肢芽的三个信号中心的信息如何在空间和发育时间中整合以产生图案化的肢芽。 为了做到这一点，（i）目前未知的信号传导中心通路的组件必须被发现，和（ii）整合确定的单调信号传导中心效应器的手段。 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，鸡无肢突变体是一个重要的工具，利用差异克隆技术来研究这两个重要的问题，在胚胎模式的形成。 减去无肢和野生型肢芽共同的cDNA产生了富含信号通路组分的cDNA文库。 初步结果有力地支持了假设的有效性。 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，以分离以空间和时间限制模式表达的新cDNA，并使用病毒技术通过对已知肢体模式基因和错误表达的上位性分析来分析它们在肢体模式形成中的作用。 2)确定C的鸡（c）同源物在肢体发育中的作用。线虫异时基因Lin-41，我们提出它是沿着发育肢体的近端-远端轴沿着的定时基因; cLin-41在初始筛选中被分离。 3)确定在初始筛选中分离的Wnt 7a-FGF反应基因在该途径中已知组分的背腹信号传导途径下游的功能中的作用;或者，在其功能被提议为维持肢芽中的Sonic hedgehog信号传导的平行途径中。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时基因在肢体发育中的作用，我们将能够更好地了解如何沿沿着肢体近端-远端轴的发育时序控制。 这可能会导致在分子水平上的见解畸形，如短肢畸形。 同样，通过定义独特的Wnt 7a-FGF反应基因的作用，我们将大大推进对这一复杂途径在肢体发育中如何发挥作用的理解。

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.