GENETICS OF VERTEBRATE DIGITAL ARCH FORMATION

已有研究提出肢体软骨图案化和形成的结果 来自三个基本过程：L)间充质的从头凝聚，2) 单个元素的分叉或分支，以及3)分段 通过萌芽或内部分裂而存在的冷凝作用。变化中的 特定阶段的流程顺序或类型将改变组织 软骨形成前的冷凝和随后的最终形态 骨骼元素(Shubin和Alberch，1986)。青枯病的分子遗传学研究 这些过程和随后的差异化事件并不被理解。 指指少指，HD是一种半显性、纯合子致死性突变 小鼠6号染色体上与人类同源区域的外显性 7便士。HD是自发发生的，导致一种类似于 人类中的单指畸形。HD杂合子表现为全部或部分缺失 纯合子突变小鼠无法发育出第一个手指 所有的脚都有四只脚，有前腕和前腕的缺陷 鞑靼人。纯合子突变小鼠通常在子宫中死亡，原因不明 少数存活到成年的小鼠是不育的。HD纯合子 在Autopod内沿两个轴有肢体缺陷，并且显著 在远端发育方面比杂合子受影响更大， 前肢结构。我们认为房屋署的正常产品 等位基因是哺乳动物形成早期分支的关键 由Shubin和Alberch(1986)提出的数字拱门。 我们已经使用Genetic构建了HD基因座的高分辨率遗传图谱 涉及1,500多只小鼠的杂交(Inris等人，1995a，1995b)。紧密地 侧翼标记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体的基因组克隆的重叠群 标记和定义近端边界的减数分裂断点- HD遗传间隔。我们建议鉴定HD并将该突变体用作 探索早期哺乳动物数字化的机制的工具 拱形结构。 为了接近我们的假设，突变的知识，分布 和HD正常等位基因的基因表达时间，以及一个 突变对AER-和AER基因表达影响的检测 ZPA特异的基因表达是必不可少的先决条件。我们建议 组装跨越HD遗传区间的基因组克隆的重叠群 以确定突变。评估突变对肢体的影响 形态和基因表达我们将执行：1)骨骼染色和 对受影响的胚胎进行连续的组织切片以识别异常， 2)胚胎肢体与候选基因的整体原位杂交 基因探针寻找时间、水平分布的变化 表达和，3)与基因特异性探针的原位杂交 在极化活动区和根尖细胞中表达 外胚层脊来确定突变对细胞生长的影响 和图案的形成。

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.