Genetic Mechanisms of Vertebrate Caudal Limb Field

描述（由申请人提供）：在特定前后位置建立脊椎动物肢体野的机制尚不清楚。到目前为止，工作已经确定了肢体生长的监管机构，但是，该领域几乎没有遗传工具，以确定上游监管机构的肢体领域规范之前，生长。在这项提案中，我们将使用一种新的，自发的，显性的小鼠突变，Polypodia（PPD），表现出异位尾肢作为一种新的方式来确定新的，上游的遗传途径，基础上创建的肢体领域。这是同类中唯一报道的小鼠突变体，并提供了丰富的见解。我们目前的SNP和STR作图数据已经排除了大部分基因组，包括但不限于Pitxl、Tbx 4、Tbx 5、Fgf 8和Fgf 10的染色体区域，已知参与早期肢芽生长的基因，以及Disorganization基因座和视黄酸稳态基因。我们将完成已经进行的广泛的遗传作图，并推导出Ppd染色体间隔的单倍型。单倍型将有利于突变胚胎基因分型之前，异位肢体的发展。我们将通过比较纯合子和杂合子Ppd小鼠的表型来测试Ppd是否是真正的显性基因，并且我们将使用纯合子突变体来检查极早期胚胎中的视黄酸稳态。为了评估菌株变异对遗传率和表型变异的贡献，我们将对优先A组小鼠表型组项目菌株进行额外的遗传杂交。这些后代也将用于完善遗传图谱。将测定异位肢体骨结构和Tbx 4/5基因表达。我们还将研究目前已知的Ppd小鼠肢体生长介质的表达。候选基因测序和表达分析将启动，并努力验证候选突变使用遗传杂交和卵内电穿孔。Ppd的鉴定具有广泛的应用前景。该突变体将揭示后肢场规范的新的遗传和细胞机制。Ppd的知识将有助于我们探索人类寄生双胞胎和异位肢体病例之间的差异，以及被认为是小鼠Disorganization（Ds）基因同源物的病例或人类Ppd病例之间的差异。本研究将有助于研究前肢和后肢之间肢体起始机制的差异，并将促进Ppd在低等脊椎动物中进化保守性的研究。公共卫生：这项工作将有广泛的意义，我们了解胚胎发育，以及基础上非常常见的人类出生缺陷，涉及尾部结构和四肢。

DESCRIPTION (provided by applicant): The mechanisms responsible for establishing vertebrate limb fields in specific anterior-posterior locations are unknown. Work so far has identified regulators of limb outgrowth; however, the field has few genetic tools to identify upstream regulators of limb field specification prior to outgrowth. In this proposal we will use a new, spontaneous, dominant mouse mutant, Polypodia (Ppd) that exhibits ectopic caudal limbs as a novel way to identify new, upstream genetic pathways that underlie the creation of limb fields. This is the only reported mouse mutant of its kind, and offers a wealth of insight. Our current SNP and STR mapping data has excluded much of the genome including, but not limited to, the chromosomal regions of Pitxl, Tbx4, Tbx5, Fgf8, and Fgf10, genes known to be involved in early limb bud outgrowth, as well as the Disorganization locus and retinoic acid homeostasis genes. We will finish the already extensive genetic mapping we have performed and derive a haplotype for the Ppd chromosomal interval. The haplotype will facilitate mutant embryo genotyping prior to ectopic limb development. We will test whether Ppd is a true dominant by comparing the phenotypes of homozygous and heterozygous Ppd mice, and we will use homozygous mutants to examine retinoic acid homeostasis in very early embryos. To assess the contribution of strain variation to penetrance and phenotypic variation we will perform additional genetic crosses to priority Group A Mouse Phenome Project strains. These progeny also will be used for refining the genetic map. Ectopic limb bone structure and Tbx4/5 gene expression will be determined. We also will examine expression of currently known mediators of limb outgrowth in Ppd mice. Candidate gene sequencing and expression analysis will be initiated, and efforts to validate candidate mutations using genetic crosses and in ovo electroporation are presented. The identification of Ppd will have broad applications. This mutant will reveal new genetic and cellular mechanisms of hindlimb field specification. Knowledge of Ppd will help us to explore differences between human cases of parasitic twinning and ectopic limbs, as well as cases considered homologs of the mouse Disorganization (Ds) gene or cases of human Ppd. This research will enable the study of differences in limb initiation mechanisms between the forelimb and hindlimbs, and will promote study of the evolutionary conservation of Ppd in lower vertebrates. Public Health: This work will have broad significance to our understanding of embryonic development as well as the basis for very common human birth defects involving caudal structures and limbs.