MOLECULAR MECHANISMS OF CARDIAC CONOTRUNCAL DEVELOPMENT

心脏干发育的分子机制

• 批准号: 2211450
• 负责人: Jonathan A. Epstein
• 金额: $ 7.76万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-09-30 至 1996-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2211450
• 关键词: DNA binding protein; Mus musculus; RNase protection assay; congenital heart disorder; developmental genetics; disease /disorder model; gene expression; gene targeting; genetic manipulation; genetic promoter element; genetic regulation; genetic strain; genome; histogenesis; messenger RNA; molecular genetics; northern blottings; open reading frames; polymerase chain reaction; reporter genes; transcription factor

Conotruncal defects represent a significant proportion of congenital cardiac malformations, and several animal models now exist that demonstrate reproducible abnormalities of this developmental pathway. In some cases, a single mutant gene is responsible for the abnormal phenotype. The Splotch mouse represents an animal model for persistent truncus arteriosus and related abnormalities, in which the specific genetic defect has been identified. The Splotch mouse is the result of a mutated Pax-3 gene. Pax-3 is a member of a developmentally critical class of transcription factors. The aim of this proposal is to identify molecular pathways regulated by Pax-3 during cardiac development. The identification of genes regulated by specific transcription factors remains a major stumbling block in the field of developmental genetics, and no target genes regulated by Pax-3 have been identified. In order to identify target genes, I propose a dual approach that requires both the presence of a high affinity Pax-3 binding site within the genomic sequence, and putative target gene expression in normal but not Pax-3 deficient mice. Specific DNA sequences recognized by the Pax-3 paired domain will be selected from amongst a pool of genomic fragments. Genomic clones corresponding to these fragments will be identified. These genomic clones will then be further screened with probes derived from a completely different approach. Differential display PCR will be used to identify cDNA fragments specifically expressed in normal litter mates of Pax-3 deficient mice at the time of peak Pax-3 expression just prior to conotruncal septation. Genomic clones identified by this dual approach will represent excellent candidates for regulation by Pax-3. Further evidence for direct regulation will be provided by demonstrating: 1) specific, high affinity binding of Pax-3 protein to promoter sequences, 2) the ability of Pax-3 to trans activate a reporter construct containing the candidate promoter region, and 3) appropriate spatial and temporal expression patterns. A specific candidate gene, Nf1, will be examined in detail. This gene is responsible for type I neurofibromatosis. Mice engineered to lack a functional Nf1 allele uniformly display a specific abnormality of conotruncal development that is phenotypically different from the Splotch mouse. Pax-3 and Nf1 may contribute to parallel pathways required for normal cardiac development. However, available evidence suggests that Nf1 lies downstream of Pax-3. If so, NF1 expression should be altered in Splotch mice, and mice bred to be homozygous deficient for both genes should resemble the Splotch homozygote embryos. Thus, a general scheme for identifying genes regulated by a specific transcription factor during development is proposed. The sequence of molecular events leading to conotruncal development will be clarified by using two mouse models in which the particular mutant gene is known. Under the guidance of several renowned and devoted mentors who already know and support the candidate, this proposal will provide the foundation necessary, and serve as a catalyst, for the transition to independent research.

共鸣缺陷代表了很大比例的先天性 心脏畸形，现在存在几种动物模型 证明了这种发育途径的可再现异常。在 在某些情况下，单个突变基因负责异常 表型。斑点鼠标代表持久的动物模型 动脉truncus and相关异常，其中特异性 遗传缺陷已被鉴定出来。斑点鼠标是a的结果 突变的PAX-3基因。 PAX-3是发展上关键阶级的成员 转录因子。该提议的目的是确定 心脏发育过程中由PAX-3调节的分子途径。 鉴定由特定转录因子调节的基因 仍然是发展遗传学领域的主要绊脚石， 并且尚未确定由PAX-3调节的靶基因。为了 识别靶基因，我提出了一种双重方法，既需要 基因组内的高亲和力PAX-3结合位点的存在 序列和假定的靶基因表达在正常但不在PAX-3中 不足的小鼠。 PAX-3配对识别的特定DNA序列 将从基因组片段池中选择域。基因组 将确定与这些片段相对应的克隆。这些基因组 然后，将用从一个完全衍生的探针进一步筛选克隆 不同的方法。差分显示PCR将用于识别cDNA 在PAX-3的正常垃圾伴侣中专门表达的片段不足 峰值pax-3表达时的小鼠在圆锥形之前 分隔。这种双重方法确定的基因组克隆将代表 PAX-3进行调节的优秀候选者。直接的进一步证据 法规将通过演示提供：1）特定的高亲和力 PAX-3蛋白与启动子序列的结合，2）PAX-3与 反式激活包含候选启动子的记者构造 区域以及3）适当的空间和时间表达模式。 将详细检查特定的候选基因NF1。这个基因是 负责I型神经纤维瘤病。小鼠设计为缺乏 功能性NF1等位基因均匀地显示出特定的异常 在表型上与斑点不同的综合发展 老鼠。 PAX-3和NF1可能有助于平行途径 正常心脏发育。但是，现有证据表明NF1 PAX-3的下游。如果是这样，应在NF1表达式中改变 斑点小鼠和繁殖的小鼠对这两个基因都是纯合的纯合子 应该类似于斑点纯合子胚胎。 因此，一种鉴定受特定调节基因的一般方案 提出了开发过程中的转录因子。序列 导致共鸣发育的分子事件将通过 使用两个已知特定突变基因的小鼠模型。在下面 已经知道和 支持候选人，该建议将为候选人提供基金会 必要并充当催化剂，以过渡到独立 研究。