Developmental and genetic mechanisms of tissue identity and patterning

 描述（由申请人提供）：了解解剖变异的分子基础是生物学中的一项基本挑战。在某些情况下，控制人类解剖缺陷的基因反而是其他物种正常变异的基础;因此，对多样性的一般分子机制的全面理解有望对人类健康有更深入的了解。在某些品种的家鸽中，遗传作图和发育研究表明，控制肢型身份（前肢或后肢）和生长的两个基因的调节变化与脚上羽毛取代鳞片有关。皮肤的这些变化伴随着肌肉和肌腱图案的缺陷，从而证明这种表型不限于表皮附属物。在人类中，这些相同基因的突变会导致引人注目的肢体畸形，包括马蹄内翻足，Holt-Oram综合征和Liebenberg综合征。该项目旨在了解这些基因如何发挥作用来指定组织身份（例如，鳞片或羽毛），并调节四肢肌肉组织的模式。鸽子是追求这些目标的理想系统，因为它在单个物种中具有巨大的形态变异，从而促进了全基因组关联研究，传统的遗传作图和功能发育生物学。 该项目将追求三个不同的目标。目的1将确定与肢型身份转换相关的两个基因中的顺式调控突变。增强子突变将使用具有足羽的品种（“耳罩”）的全基因组重测序和大规模平行染色质免疫沉淀测序（ChIP-seq）的组合进行精细定位，以鉴定增强子活性。候选突变的影响将使用卵内报告基因试验进行检测。目的2将确定基因调控网络支配皮肤附件的命运。这一目标将采用胚胎组织移植实验和高通量RNA测序，以确定如何以及何时指定后肢皮肤的身份。目的3将确定肢体型基因表达的变化对肌肉和皮肤图案的功能影响。该目的将测试两个候选基因在F2互交中对后肢皮肤和肌肉错误图案化的贡献。此外，实验胚胎操作将确定这些基因的特定调控是否是必要的正常后肢肌肉模式和表皮附属物的命运。 总之，这些互补的遗传学、基因组学和发育方法将在创新的模型系统中确定惊人变异的分子基础，从而为理解特定基因在一般脊椎动物正常和疾病变异中的作用开辟新的途径。

 DESCRIPTION (provided by applicant): Understanding the molecular basis of anatomical variation is a fundamental challenge in biology. In some cases, the genes that control anatomical defects in humans instead underlie normal variation in other species; therefore, a comprehensive understanding of the general molecular mechanisms of diversity promises greater insights about human health. In certain breeds of domestic pigeon, genetic mapping and developmental studies show that regulatory changes in two genes that control limb-type identity (fore- or hindlimb) and outgrowth are associated with the replacement of scales by feathers on the feet. These changes in the skin are accompanied by defects in muscle and tendon patterning, thereby demonstrating that this phenotype is not limited to epidermal appendages. In humans, mutations in these same genes cause striking limb malformations, including clubfoot, Holt-Oram syndrome, and Liebenberg syndrome. This project seeks to understand how these genes function to specify tissue identity (e.g., scales or feathers) and regulate patterning of lim musculature. The pigeon is an ideal system in which to pursue these goals because it features tremendous morphological variation within a single species, thereby facilitating genome-wide association studies, traditional genetic mapping, and functional developmental biology. This project will pursue three separate aims. Aim 1 will identify cis-regulatory mutations in the two genes associated with transformations in limb-type identity. Enhancer mutations will be fine-mapped using a combination of whole-genome resequencing of breed with foot feathers ("muffs"), and massively parallel chromatin immunoprecipitation sequencing (ChIP-seq) to identify enhancer activity. The impact of candidate mutations will be tested using in ovo reporter assays. Aim 2 will determine the gene regulatory network governing skin appendage fate. This aim will employ embryonic tissue transplantation experiments and high-throughput RNA sequencing to determine how and when hindlimb skin identity is specified. Aim 3 will determine the functional impact of changes in limb-type gene expression on muscle and skin patterning. This aim will test the contributions of the two candidate genes to hindlimb skin and muscle mis- patterning in an F2 intercross. Additionally, experimental embryonic manipulations will determine whether specific regulation of these genes is necessary for normal hindlimb muscle pattern and epidermal appendage fate. Together, these complementary genetic, genomic, and developmental approaches will identify the molecular basis of astonishing variation in an innovative model system, thereby opening new avenues to understand the roles of specific genes in normal and disease variation among vertebrates in general.