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.

圆锥干缺陷占先天性缺陷的很大一部分 心脏畸形，现在存在几种动物模型 证明该发育途径的可重复异常。在 在某些情况下，单个突变基因是导致异常的原因 表型。 Splotch 小鼠代表了持久性动物模型 动脉干及相关异常，其中具体 已鉴定出遗传缺陷。 Splotch 鼠标是 Pax-3基因突变。 Pax-3 是发育关键类别的成员 转录因子。该提案的目的是确定 心脏发育过程中 Pax-3 调节的分子途径。 特定转录因子调控基因的鉴定 仍然是发育遗传学领域的主要绊脚石， 并且尚未鉴定出 Pax-3 调控的靶基因。为了 识别目标基因，我提出了一种双重方法，需要 基因组内存在高亲和力 Pax-3 结合位点 正常但非 Pax-3 中的序列和推定靶基因表达 缺乏的老鼠。 Pax-3 配对识别的特定 DNA 序列 域将从基因组片段库中选择。基因组 将鉴定与这些片段相对应的克隆。这些基因组 然后，将使用源自完全克隆的探针进一步筛选克隆。 不同的方法。差异显示PCR将用于鉴定cDNA Pax-3 缺陷的正常同窝伴侣中特异表达的片段 小鼠在圆锥干之前达到 Pax-3 表达峰值 分隔。通过这种双重方法鉴定的基因组克隆将代表 Pax-3 监管的优秀候选者。直接的进一步证据 通过展示以下内容来提供调节：1) 特异性、高亲和力 Pax-3 蛋白与启动子序列的结合，2) Pax-3 的能力 反式激活含有候选启动子的报告构建体 区域，3) 适当的空间和时间表达模式。 将详细检查特定候选基因 Nf1。这个基因是 负责 I 型神经纤维瘤病。小鼠被设计为缺乏 功能性 Nf1 等位基因一致显示出特定的异常 表型上与斑点不同的圆锥干发育 老鼠。 Pax-3 和 Nf1 可能有助于形成所需的平行通路 心脏发育正常。然而，现有证据表明 Nf1 位于 Pax-3 的下游。如果是这样，NF1 表达应该改变 斑点小鼠和培育出两种基因均缺陷的纯合小鼠 应该类似于 Splotch 纯合子胚胎。 因此，鉴定受特定基因调控的基因的一般方案 提出了发育过程中的转录因子。的顺序为 导致圆锥干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 几位知名且忠诚的导师的指导，他们已经了解并 支持候选人，该提案将提供基础 向独立过渡所必需的，并作为催化剂 研究。