Epithelial/Mesenchymal Interactions in Limb Patterning

描述(申请人提供)：这项研究的长期目标是了解极化活动区(ZPA)如何在空间和发育时间内发挥作用，以控制前后肢(A/P)的模式。小鸡和小鼠发育中的肢体提供了一个系统，在这个系统中，可以将实验操作与分子和遗传方法相结合，以了解模式结构是如何指定、确定和实现的。在过去的十年中，肢芽信号中心的效应分子被定义。然而，信号中心是如何建立的，以及效应器分子的确切靶点是什么，人们对此知之甚少。尤其是ZPA，通过分泌蛋白Sonic Hedgehog(SHH)起作用。从ZPA的启动，到对靶细胞的信号传递，再到SHH下游的间指作为信号中心的功能，有三个目的。利用实验和分子技术，我们将：确定ZPA中Shh表达所必需的鸡和小鼠保守的Lmbr1内含子5区域的最小序列(S)；以缺失缺失的肢体为模型系统，分析SHH信号对靶基因表达和肢体表型发育的影响；并测试交叉点作为信号中心通过Tbx基因和骨形态发生蛋白信号发挥作用。为了实现这些目标，我们已经开发和验证了新的技术，包括在鸡的肢体中进行体内增强子分析的报告试验；使用超声波(超声波)在发育中的肢体的有限区域异位表达基因；以及体内荧光素酶报告试验，允许对BMP信号进行定量。虽然我的实验室成员在实现这些目标所需的技术和实验设计方面非常熟练，但我们也聘请了专家，我们定期与他们会面，帮助解决问题和解释数据。这一建议的目标的实现对理解人类肢体畸形具有重要意义。这些畸形在人类活产中是最高的。了解肢体正常发育的基本机制将有助于深入了解肢体畸形发生的原因。这是通过再生的生物和医学方法预防和/或治疗它们的必要步骤。

DESCRIPTION (provided by applicant): The long-term goal of this research is to understand how the zone of polarizing activity (ZPA) functions in space and developmental time to control anterior/posterior (A/P) limb patterning. The chick and mouse developing limbs provide a system where a combination of experimental manipulations can be combined with molecular and genetic approaches to understand how patterned structures are specified, determined and realized. During the past decade, the effector molecules of limb bud signaling centers were defined. However, how the signaling centers are established and the precise targets of the effector molecules are poorly understood. This is especially true for the ZPA, acting through the secreted protein Sonic hedgehog (SHH). There are three aims that cover ZPA function from its initiation, through signaling to target cells, to the function of the interdigits as signaling centers downstream of SHH. Using experimental and molecular techniques, we will: determine the minimal sequence(s) of the chick and mouse conserved Lmbr1 intron 5 region that are necessary for Shh expression in the ZPA; use the oligozeugodactyly mutant limb as a model system to analyze the effects of SHH signaling on target gene expressions and development of limb phenotypes; and test the role of the interdigits as signaling centers functioning through Tbx genes and BMP signaling. To accomplish these aims, we have developed and validated new techniques that include a reporter assay for in vivo enhancer analysis in the chick limb; the use of ultrasound (sonoporation) to express genes ectopically in limited areas of the developing limb; and an in vivo Luciferase reporter assay that permits quantitation of BMP signaling. While the members of my laboratory are highly accomplished in the techniques and experimental designs needed to carry out these aims, we also have engaged experts, with whom we meet regularly, to assist in trouble shooting and data interpretation. Accomplishment of the aims of this proposal has significant implications for understanding human limb malformations. These malformations are among the highest among human live births. Understanding the fundamental mechanisms of normal limb development will lead to insights into why limb malformations occur. This is a necessary step toward preventing and/or curing them through regenerative biological and medical approaches.