INDENTIFICATION OF SIGNALING MOLECULES THAT INDUCE HEART FORMATION

(改编自申请人的摘要)本项目的总体目标是 了解心肌细胞分化程序是如何被激活的 在脊椎动物的发育过程中。在之前的工作中，调查人员已经 在前外侧板中发现了一种心脏诱导活性 原肠胚期鸟类胚胎的内胚层，它可以诱导细胞从 后部原始条纹(通常会导致额外的- 胚胎组织和血液)，以产生心肌细胞。 最近，研究人员扩展了这些研究，以证明 除了来自前内胚层的心脏诱导信号外， 骨形态发生蛋白在内胚层和外胚层中的表达 在心脏诱导中也起着重要的作用。结果表明， 脊椎动物心肌细胞的测定至少有两个阶段 从原始条纹中出现的细胞仍然是可塑性的 他们形成心脏的能力。那些与前部接触的中胚层细胞 内胚层似乎处于一个发育的领域，在这个领域中，细胞表现出 有成为心脏的潜力。BMP-2细胞所处的发育领域 展现出成为核心的潜力。BMP-2和/或BMP-4似乎是 能够诱导该场内的细胞启动心脏 差异化计划。因此，两个不同信号的组合是 诱导心肌细胞所必需的骨形态发生蛋白信号 胚胎的侧部区域与尚未形成的 识别前内胚层分泌的诱导心脏的信号 队形。这项提议的目标将是识别信号 由前内胚层细胞分泌，诱导形成 心肌细胞与骨形态发生蛋白信号协作。 通过使用消减杂交技术，研究人员已经 最近发现了一种信号分子，称为“新月”，它是一种 可能是Wnt抑制物，并且在前部特异表达 雏鸡心肌细胞诱导时的内胚层 胚胎。在雏鸡发育的早期，新月体在一个区域中表达 这精确地重叠了内皮心诱导的位置 活动就是谎言。在这项提案中，调查人员打算首先 调查新月在哪里是心脏诱发活动的前部 内胚层。如果是这样的话，调查人员将重点关注 了解情况是如何发生的，调查人员将重点放在 对了解该信号分子如何协同工作的理解 在各种检测系统中使用BMP信号诱导心脏形成 调查人员已经开发出，他们将采用另一种方法 从前内胚层分离心脏诱导活性的方法。 特定目标1将调查新月体，一种假定的抗WNT 前内胚层分泌的分子与BMP信号协同作用 诱导心肌细胞的形成。特定目标2，如果是新月 可以与BMP协同诱导心肌细胞的形成 信号，调查人员将确定新月是否显示出 在此诱导下具有抗Wnt活性。具体目标3将决定是否 缺乏新月体的基因工程小鼠表现出心脏缺陷 队形。具体目标4如果调查人员发现新月会未能 在体外或体内表现出任何诱导心脏的活性，他们将 开发一种用于识别心脏诱导的功能筛查 由前内胚层分泌的分子。

(Adapted from the Applicant's Abstract) The broad aim of this project is to understand how the cardiac myocyte differentiation program is activated during vertebrate development. In previous work the investigators have identified a cardiac-inducing activity in the anterior lateral plate endoderm of the gastrula stage avian embryos, which can induce cells from the posterior primitive streak (which normally gives rise to extra- embryonic tissues and to blood) to give rise to cardiac myocytes. Recently, the investigators have extended these studies to demonstrate that in addition to a cardiac inducing signal from the anterior endoderm, Bone Morphogenetic Proteins (BMPs) expressed in both endoderm and ectoderm also play an important role in heart induction. The result suggest that there are at least two stages in vertebrate cardiac myocyte determination Cells emerging from the primitive streak are still plastic with respect to their ability to form heart. Those mesodermal cells which contact anterior endoderm seem to lie in a developmental field in which cells display a potential to become heart. BMP-2 developmental field in which cells display a potential to become heart. BMP-2 and/or BMP-4 then appear to be capable of inducing cells within this field to initiated the cardiac differentiation program. Thus, a combination of two distinct signals is necessary for the induction of cardiac myocytes: a BMP signal present in the lateral regions of the embryo collaborates with an as yet to be identified signal secreted by the anterior endoderm to induce heart formation. The goal of this proposal will be to identify the signal secreted by cells in the anterior endoderm that induces the formation of cardiac myocytes in collaboration with a BMP signal. By employing subtractive hybridization technology the investigations have recently identified a signaling molecule, termed "crescent", which is a putative Wnt inhibitor, and is specifically expressed in the anterior endoderm at the time when cardiac myocytes are being induced in the chick embryos. During early chick development, crescent is expressed in a domain that precisely overlaps the location where the endodermal heart inducing activity lies. In this proposal the investigators intend to firstly investigate where crescent is the heart inducing activity in the anterior endoderm. If such is the case, the investigators will focus on understanding how this such is the case, the investigators will focus on understanding on understanding how this signaling molecule collaborates with BMP signals to induce heart formation in the various assay systems that the investigators have developed, they will employ an alternative approach to isolate the heart inducing activity in the anterior endoderm. Specific Aim 1 will investigate whether crescent, a putative anti-Wnt molecule secreted by the anterior endoderm collaborates with BMP signals to induce the formation of cardiac myocytes. Specific Aim 2 if crescent can induce the formation of cardiac myocytes in collaboration with a BMP signal, the investigators will determine whether crescent displays an anti-Wnt activity in this induction. Specific Aim 3 will determine whether mice genetically engineered to lack crescent display a defect in heart formation. Specific Aim 4 if the investigators find that crescent fails to display any heart inducing activity either in vitro or in vivo, they will develop develop a functional screen to identify the cardiac inducing molecule secreted by the anterior endoderm.