GROWTH FACTORS AND PROTO-ONCOGENES IN CARDIAC ONTOGENY

申请人已经证明了Beta1类型的转化生长 因子(TGFbeta1)控制至少16个基因的表达 新生大鼠心肌细胞和IS中的组织限制性基因 调节自分泌或旁分泌因子的原型 机械负荷引起的心肌肥厚。诱导 与其他基因不同的是，骨骼α-肌动蛋白启动子由转化生长因子β启动 依赖于血清反应因子(SRF)。然而，它的作用是 在体内，特别是在个体发育过程中，转化生长因子β尚未被 下定决心。胚胎中含有核糖核酸的第一批细胞 TGFbeta1是心脏前体细胞，在心脏发育过程中， 器官发生、转化生长因子β和转化生长因子β样多肽在 一种极其复杂的时空模式。功能性 胚胎性癌细胞和非胚胎癌细胞的研究 哺乳动物表明，转化生长因子β超家族的成员可能 心脏器官发生建立的直接两个关键方面 心肌谱系与心内膜的转化 前体细胞分化为瓣膜形成间充质。尽管 丰富的关于转化生长因子β表达的描述性信息 在发展心肌方面，并在转化为 细胞或仅有远亲关系的物种中，没有 到目前为止，对转化生长因子β信号转导的遗传分析，或 更广泛地说，增长因素信号通过损失- 哺乳动物心脏发育过程中的功能突变。占主导地位- 负突变是达到这一目的的一种手段，但有一定的 理论和技术优势相辅相成 重组。此外，“二元”或“两阶段”的程序 转基因小鼠，最初由Frank Ruddle和Philip描述 莱德，承诺瞄准潜在的致命优势- 预先确定的发育和组织特异性的抑制物 窗户。本项目的具体目标是：(1)界定 SFR及其附属SRE结合蛋白在细胞周期调控中的作用 由TGFbeta和TGFbeta的其他成员发起的Ska启动子 超级大家庭。(2)建立显性-负性突变。 Ras和AP1是否是诱导SKA表达所必需的 在新生大鼠心肌中，或在ES的心肌发生过程中 细胞。(3)确定是否存在显性负性突变 RAS和AP1在体内导致发育性心脏缺陷，以及 为RAS和AP1信号转导的作用提供证据 心脏发育过程中的路径。(四)确定能力 激酶缺陷型转化生长因子β和激活素受体在脑内的作用 培养的心肌。ES细胞和转基因小鼠作为 转化生长因子β信号转导的显性抑制物。(5)目标 AP1、ras和转化生长因子β受体的显性抑制等位基因 转基因小鼠瓣膜间充质的转化生长因子β3或β2 顺式作用序列，以确定这些突变是否可以 造成发育缺陷的隔膜或形成 心脏瓣膜。这些调查将包括初步的 AP1在完整动物体内作用的遗传分析， RAS和TGFβ受体在心脏的建立中的作用 肌肉谱系、心肌基因转录与心脏 器官发生。

The applicant has demonstrated that type beta1 transforming growth factor (TGFbeta1) controls the expression of that at least 16 tissue-restricted genes in neonatal rat cardiac muscle cells and is the prototype for an autocrine or paracrine factor mediating ventricular hypertrophy triggered by mechanical load. Induction of the skeletal alpha-actin promoter by TGFbeta unlike other genes, is dependent on serum response factor (SRF). However, the role of TGFbeta in vivo, and during ontogeny in particular, has not been determined. The first cells of the embryo to contain RNA for TGFbeta1 are cardiac precursor cells, and, in the course of cardiac organogenesis, TGFbetas and TGFbeta-like peptides are expressed in a elaborately complex spatial and temporal pattern. Functional studies in embryonal carcinoma cells and in phyla other than mammals suggest that members of the TGFbeta superfamily might direct two key aspects of cardiac organogenesis establishment of the cardiac muscle lineage, and transformation of endocardial precursor cells into valve-forming mesenchyme. Despite the abundance of descriptive information concerning TGFbeta expression in developing myocardium, and encouraging results in transformed cells or in species that are related only distantly, there has not been to date a genetic analysis of TGFbeta signal transduction, or growth factor signalling more generally, by means of loss-of- function mutation in the developing mammalian heart. Dominant- negative mutations are one means to this end, with certain theoretical and technical advantages which complement homologous recombination. Furthermore, procedures for "binary" or "two-stage" transgenic mice, described initially by Frank Ruddle and Philip Leder, hold the promise of targeting potentially lethal dominant- inhibitors to predetermined development and tissue-specific windows. Specific Aims of the present project are to: (1) Define the role of SFR and accessory SRE-binding proteins in regulation of the SkA promoter by TGFbeta and other members of the TGFbeta superfamily. (2) Establish with dominant-negative mutations. whether ras and AP1 are necessary for induction of SkA expression in neonatal rat cardiac muscle, or during cardiac myogenesis in ES cells. (3) Determine whether the dominant negative mutations of ras and AP1 cause developmental cardiac defects in vivo, and provide evidence for the role of ras and AP1 signal transduction pathways in the developing heart. (4) Determine the ability of kinase-deficient TGFbeta and activin receptors to function in cultured cardiac muscle. ES cells, and transgenic mice as dominant-inhibitors of TGFbeta signal transduction. (5) Target dominant-inhibitory alleles of AP1, ras, and the TGFbeta receptor to valvular mesenchyme of transgenic mice, via TGFbeta3 or beta2 cis-acting sequences, to determine whether these mutations can cause development defects in septation or in formation of the cardiac valves. These investigations will comprise an initial genetic analysis of the role played in the intact animal by AP1, ras, and the TGFbeta receptor in establishment of the cardiac muscle lineage, cardiac muscle gene transcription, and cardiac organogenesis.