GENETICS OF VERTEBRATE DIGITAL ARCH FORMATION

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It has been proposed that limb cartilage patterning and formation results from three basic processes: l) de novo condensation of mesenchyme, 2) bifurcation or branching of single elements, and 3) segmentation of existing condensations by budding or internal division. Variation in sequence or type of process at specific stages will alter the organization of prechondrogenic condensations and the subsequent final morphology of skeletal elements (Shubin and Alberch, 1986). The molecular genetics of these processes and subsequent differentiative events are not understood. Hypodactyly, Hd, is a semidominant, homozygous lethal mutation with full penetrance located on mouse chromosome 6 in a region homologous to human 7p. Hd arose spontaneously and results in a phenotype similar to monodactyly in humans. Hd heterozygotes show absence of all or part of the first digit while homozygous mutant mice fail to develop digits one through four on all feet and have defects of the anterior carpals and tarsals. Homozygous mutant mice usually die in utero for unknown reasons and the few mice that survive to adulthood are infertile. Hd homozygotes have limb defects along two axes within the autopod and are significantly more affected than the heterozygote in their development of distal, anterior limb structures. We propose that the normal product of the Hd allele is critical for early branching in the formation of the mammalian digital arch as proposed by Shubin and Alberch (1986). We have built a high-resolution genetic map of the Hd locus using genetic crosses involving over 1,500 mice (Innis et al., 1995a, 1995b). Closely flanking and several nonrecombinant markers have been identified. We have assembled a contig of genomic clones spanning the known nonrecombinant markers and the meiotic breakpoint defining the proximal boundary of-the Hd genetic interval. We propose to identify Hd and to use this mutant as a tool to explore the mechanisms responsible for early mammalian digital arch formation. To approach our hypothesis, knowledge of the mutation, the distribution and timing of gene expression of the normal allele of Hd, and an examination of the effect of the mutation on the expression of AER- and ZPA-specific gene expression are essential prerequisites. We propose to assemble a contig of genomic clones spanning the Hd genetic interval and to identify the mutation. To assess the effect of the mutation on limb morphology and gene expression we will perform: 1) skeletal staining and serial histological sectioning of affected embryos to identify anomalies, 2) whole mount in situ hybridization of embryonic limbs with candidate gene probes to look for alterations in timing, distribution of level of expression and, 3) in situ hybridization with probes specific for genes expressed in cells of the zone of polarizing activity and the apical ectodermal ridge to define the effect of the mutation on cellular growth and pattern formation.