GENETICS OF VERTEBRATE DIGITAL ARCH FORMATION

脊椎动物数码弓形成的遗传学

• 批准号: 2403590
• 负责人: JEFFREY W INNIS
• 金额: $ 10.89万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-05-01 至 2001-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2403590
• 关键词: SDS polyacrylamide gel electrophoresis; alleles; cartilage development; developmental genetics; early embryonic stage; gene expression; gene mutation; gene rearrangement; genetic mapping; genetic markers; histogenesis; in situ hybridization; laboratory mouse; limbs; mammalian embryology; northern blottings; nucleic acid sequence; polymerase chain reaction; pulsed field gel electrophoresis; radionuclides; single strand conformation polymorphism; skeletal disorder; southern blotting

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.

已经提出肢体软骨模式和形成结果 从三个基本过程中：l）从新生的凝结，2） 单元素的分叉或分支，3）分割 现有的凝结或内部部门。变化 在特定阶段的序列或过程类型将改变组织 凝结的凝结和随后的最终形态 骨骼元素（Shubin和Alberch，1986）。分子遗传学 这些过程和随后的区分事件尚不清楚。 HD型，是一种半主导，纯合致命突变，完全 位于与人同源的区域的小鼠6染色体上的渗透率 7p。 HD自发产生，导致表型相似 在人类中是单一的。高清杂合子显示所有或部分的一部分 纯合突变小鼠未能开发数字的第一位数字 通过四只脚，有前腕的缺陷， tarsals。纯合突变小鼠通常由于未知原因而在子宫内死亡 而生存到成年的几只小鼠是不育的。高清纯合子 沿着自动脚架内的两个轴有肢体缺陷，并且显着 在远端发展中比杂合子更受影响 前肢结构。我们建议高清的正常产物 等位基因对于哺乳动物形成的早期分支至关重要 Shubin和Alberch（1986）提出的数字拱门。 我们已经使用遗传建造了高清基因座的高分辨率遗传图 涉及超过1,500只小鼠的十字（Innis等，1995a，1995b）。 密切 已经确定了侧翼和几个非重组标记。 我们有 组装了一个基因组克隆的重叠，跨越了已知的非重组 标记和减数分裂断裂点，定义了近端边界 高清遗传间隔。我们建议识别高清并将这个突变体用作 探索负责早期哺乳动物数字的机制的工具 拱形组。 为了解决我们的假设，对突变的知识，分布 HD正常等位基因的基因表达的时间和一个 检查突变对AER和AER表达的影响 ZPA特异性基因表达是必不可少的先决条件。我们建议 组装跨越HD遗传间隔的基因组克隆和 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执行：1）骨骼染色和 受影响胚胎的序列组织学切片，以识别异常， 2）胚胎四肢与候选者的原位杂交 基因探针寻找时间的改变，水平的分布 表达和3）与特定于基因的探针的原位杂交 在偏振活性区和顶端的细胞中表达 外皮脊定义突变对细胞生长的影响 和图案形成。