GROWTH FACTORS AND PROTO-ONCOGENES IN CARDIAC ONTOGENY

申请人已经证明，β 1型转化生长 因子（TGF β 1）控制着至少16个 组织限制性基因在新生大鼠心肌细胞中的表达， 自分泌或旁分泌因子介导 机械负荷引起的心室肥大。 诱导 与其他基因不同，TGF β启动的骨架α-肌动蛋白启动子， 依赖于血清反应因子（SRF）。 然而， TGF β在体内，特别是在个体发育过程中， 测定 胚胎中第一个含有RNA的细胞 TGF β 1是心脏前体细胞，在心脏的过程中， 器官发生、TGF β和TGF β样肽在 一种极其复杂的时空模式 功能 在胚胎癌细胞和除 哺乳动物认为TGF β超家族成员可能 直接心脏器官发生的两个关键方面， 心肌谱系和内皮细胞的转化 前体细胞转化为瓣膜形成间充质。 尽管 关于TGF β表达的丰富描述性信息 在心肌发育中， 细胞或物种，只是远亲，没有 到目前为止对TGF β信号转导进行了遗传分析，或 生长因子信号传导更一般地，通过损失- 哺乳动物心脏发育中的功能突变。 占优势的- 负突变是达到这一目的的一种手段， 理论和技术优势互补， 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 莱德，保持对潜在致命优势的目标的承诺- 预定发育和组织特异性的抑制剂 Windows. 本项目的具体目标是：（1）定义 SFR和辅助SRE结合蛋白在调节 SkA启动子通过TGF β和TGF β的其他成员 超家族 (2)建立显性负突变。 ras和AP 1是否是诱导SkA表达所必需的 在新生大鼠心肌中，或在ES的心肌发生期间， 细胞 (3)确定是否显性负突变 ras和AP 1在体内引起发育性心脏缺陷， 为ras和AP 1信号转导的作用提供证据 发育中的心脏。 (4)确定的能力 激酶缺陷型TGF β和激活素受体在 培养的心肌 ES细胞和转基因小鼠作为 TGF β信号转导的显性抑制剂。 (5)目标 AP 1、ras和TGF β受体的显性抑制等位基因 转基因小鼠的瓣膜间质，通过TGF β 3或β 2 顺式作用序列，以确定这些突变是否可以 在分隔或形成的过程中引起发育缺陷 心脏瓣膜 这些调查将包括初步的 对AP 1在完整动物中所起作用的遗传分析， ras和TGF β受体在建立心脏 肌谱系、心肌基因转录和心脏 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.