Genetic Mechanisms of Vertebrate Caudal Limb Field

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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.