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中发挥作用，以控制前/后（A/P）肢体模式。鸡和小鼠的四肢发育提供了一个系统，在该系统中，实验操作的组合可以与分子和遗传方法相结合，以了解图案化结构是如何指定，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确定。然而，如何建立信号中心和效应分子的精确靶标知之甚少。这对于ZPA来说尤其如此，它通过分泌蛋白Sonic hedgehog（SHH）起作用。有三个目标涵盖ZPA功能，从它的起始，通过信号传导到靶细胞，到作为SHH下游信号中心的趾间功能。我们将利用实验和分子生物学技术，确定Shh在ZPA中表达所必需的鸡和小鼠Lmbr 1内含子5保守区的最小序列，并以寡指突变肢体为模型系统，分析SHH信号传导对靶基因表达和肢体表型发育的影响;并测试趾间作为通过Tbx基因和BMP信号传导起作用的信号传导中心的作用。为了实现这些目标，我们已经开发和验证了新的技术，包括在鸡肢体的体内增强子分析的报告分析;使用超声波（声致孔）异位表达基因在有限的地区的发展肢体;和在体内荧光素酶报告分析，允许定量的BMP信号。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都非常精通，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。实现这一目标的建议有重大意义的理解人类肢体畸形。这些畸形是人类活产婴儿中最严重的。了解正常肢体发育的基本机制将有助于深入了解肢体畸形发生的原因。这是通过再生生物学和医学方法预防和/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.