Genetic Mechanisms of Vertebrate Caudal Limb Field

脊椎动物尾肢区的遗传机制

• 批准号: 7074923
• 负责人: JEFFREY W INNIS
• 金额: $ 17.96万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-04-15 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7074923
• 关键词: animal breeding; biotechnology; congenital skeletal disorder; developmental genetics; electroporation; embryogenesis; gene mutation; genetic library; genetic mapping; genetic strain; laboratory mouse; leg; limbs; mammalian embryology; mutant; nucleic acid repetitive sequence; single nucleotide polymorphism

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.

描述（由申请人提供）：尚不清楚在特定前后位置建立脊椎动物肢体场的机制。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来识别肢体现场规范的上游调节剂在出现之前。在此提案中，我们将使用一种新的，自发的，显性的小鼠突变体，息肉（PPD），该突变体（PPD）表现出异位尾四肢，作为一种新的方式，用于识别肢体田地创造的新的上游遗传途径。这是唯一报告的老鼠突变体，并提供了丰富的见解。我们目前的SNP和STR映射数据排除了大部分基因组，包括但不限于Pitxl，TBX4，TBX4，TBX5，FGF8和FGF10的染色体区域，这些基因已知与早期肢体芽的生长有关，以及混乱的基因座和视网膜甲基甲基抗体基因。我们将完成我们所执行的已广泛基因映射，并为PPD染色体间隔提供单倍型。单倍型将在异位肢体发育之前促进突变的胚胎基因分型。我们将通过比较纯合和杂合PPD小鼠的表型来测试PPD是否是真正的主导性，并且我们将使用纯合突变体在非常早期的胚胎中检查视黄酸稳态。为了评估应变变异对外渗和表型变异的贡献，我们将对优先级A组A型遗传杂交进行小鼠现象项目菌株。这些后代还将用于完善遗传图。将确定异位肢体结构和TBX4/5基因表达。我们还将检查PPD小鼠中肢体生长的当前已知介体的表达。将开始候选基因测序和表达分析，并提出了使用遗传杂交和OVO电穿孔验证候选突变的努力。 PPD的识别​​将具有广泛的应用。该突变体将揭示后肢场规范的新遗传和细胞机制。对PPD的了解将有助于我们探索人类寄生双胞胎和异位四肢的差异，以及被认为是小鼠混乱（DS）基因或人类PPD病例的病例。这项研究将使前肢和后肢之间的肢体启动机制差异进行研究，并将促进对下脊椎动物中PPD的进化保护的研究。公共卫生：这项工作将对我们对胚胎发展的理解以及涉及尾端结构和四肢的非常常见的人类出生缺陷的基础具有广泛的意义。