GENETICS OF VERTEBRATE DIGITAL ARCH FORMATION

已经提出，肢体软骨的形成和形成导致 从三个基本过程：l）间充质的从头凝聚，2） 单个元素的分叉或分支，以及3） 通过萌芽或内部分裂而存在的凝聚。变化 特定阶段的流程顺序或类型将改变组织 软骨形成前的凝聚和随后的最终形态 骨骼元素（Shubin和Alberch，1986）。的分子遗传学 这些过程和随后的分化事件不被理解。 缺指（趾）畸形，Hd，是一种半显性，纯合子致死突变， 位于小鼠6号染色体上与人类同源的区域 七便士。 HD自发产生，并导致类似于 人类的单指畸形Hd杂合子表现为缺失全部或部分 第一个数字，而纯合子突变小鼠未能开发数字1 所有脚上都有四个，前腕骨有缺陷， 跗骨纯合子突变小鼠通常死于子宫内，原因不明 少数存活到成年的老鼠是不育的。HD纯合子 在自动足内沿沿着有肢体缺陷， 在远中端的发育中， 前肢结构我们建议，Hd的正规积 等位基因是关键的早期分支形成的哺乳动物 Shubin和Alberch（1986）提出的数字拱门。 我们已经建立了一个高分辨率的遗传图谱的Hd基因座使用遗传 涉及超过1，500只小鼠的杂交（Innis等，1995 a，1995 b）。 密切 侧翼和几个非重组标记已被鉴定。 我们有 组装了跨越已知非重组的基因组克隆的重叠群， 标记和减数分裂断点定义的近端边界-的 遗传间隔。我们建议鉴定Hd并将该突变体用作 一种探索早期哺乳动物数字化机制的工具 拱形结构 为了接近我们的假设，了解突变，分布 和Hd的正常等位基因的基因表达的时间，以及 检测突变对AER-1表达的影响， ZPA特异性基因表达是必要的先决条件。我们建议 组装跨越Hd遗传区间的基因组克隆的重叠群， 来识别突变评估突变对肢体的影响 形态学和基因表达，我们将进行：1）骨骼染色， 对受影响的胚胎进行连续组织切片以识别异常， 2)胚胎肢体的整体原位杂交 基因探针来寻找时间、水平分布的改变， 表达和3）与基因特异性探针的原位杂交 表达于极化活动区和顶端细胞 外胚层嵴，以确定突变对细胞生长的影响 和模式形成。

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.