Epithelial/Mesenchymal Interactions in Limb Patterning

描述（由申请人提供）：本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上控制前/后（A/P）肢体模式。小鸡和老鼠的肢体发育提供了一个系统，在这个系统中，实验操作的结合可以与分子和遗传方法相结合，以了解如何指定、确定和实现模式结构。在过去的十年中，肢体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分子的精确靶标尚不清楚。对于通过分泌蛋白Sonic hedgehog （SHH）发挥作用的ZPA来说尤其如此。从ZPA的启动，到向靶细胞发出信号，再到趾间作为SHH下游信号中心的功能，ZPA的功能有三个目标。利用实验和分子技术，我们将：确定鸡和小鼠保守的Lmbr1内含子5区域的最小序列(s)，这是Shh在ZPA中表达所必需的；以少合子活性突变肢体为模型系统，分析SHH信号传导对目标基因表达和肢体表型发育的影响；并通过Tbx基因和BMP信号传导测试指间区作为信号中心的作用。为了实现这些目标，我们开发并验证了新技术，包括用于鸡肢体体内增强子分析的报告基因试验；利用超声波在发育肢体的有限区域特异表达基因；以及允许BMP信号定量的体内荧光素酶报告基因测定。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取得了很高的成就，但我们也聘请了专家，我们定期与他们会面，以协助解决问题和解释数据。实现这一建议的目标对理解人类肢体畸形具有重要意义。这些畸形在人类活产儿中是最高的。了解正常肢体发育的基本机制将有助于了解肢体畸形发生的原因。这是通过再生生物和医学方法预防和/或治愈它们的必要步骤。

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.