HEART DEVELOPMENT AND GENETICS

本项目的目标是了解 心血管发育。这种方法结合了遗传学、分子生物学 生物学和胚胎学。我们已经证明了斑马鱼的心脏和 脉管系统易于遗传解剖，可由 透明胚胎中的胚胎学操作，类似于 哺乳动物通过心管期。在两年的时间里 目前，我们已经完成了最大的基因筛查 从未进行过的心血管突变。我们已经鉴定出124个隐性 致命的突变，特别是破坏心血管发育。 例如，我们有缺乏心内膜或瓣膜的突变株，这些突变株 心脏太小或太大，而那些由 单室房间。此外，通过单细胞示踪剂注射，我们有 发现了胚泡中心脏区域的位置，并确定了 同时产生心肌和心内膜的细胞。具体目标1是 完成不同突变的互补分析 将突变映射到我们的斑马鱼基因组图上。这是第一步 突变基因的克隆。具体目标2是分析 胚胎心场和心脏祖细胞的谱系树。在……里面 特别是，我们的证据表明，有一种共同的祖细胞 在腹缘胚泡中，有心肌、心内膜、内皮、 和血液，我们需要通过定义亚类来检验这一假设。 我们有证据表明胚泡的心脏区域在空间上 至少部分是由不同的Homebox基因Tinman决定的，而我们 我将通过结合细胞的异位过度表达来评估这篇论文 追踪。具体目标3集中在我们发现的两个突变上 对血管系统的图案化有明显的兴趣。每个人 扰动一段特定内皮细胞的发生：(A)闭锁 废除心内膜(可能还有一些头部内皮细胞)。我们的 假说是它在心内膜祖细胞 形成迁移；(B)堵塞阻碍该区域内的船只组装 在那里两个背主动脉合并成一个单一的主动脉。许多 这种突变体的属性类似于人类疾病的缩水 大动脉。我们的主要问题是这些缺陷是否存在于内皮细胞中 在细胞或微环境中。 突变为整个社区提供了一种资源 心血管科学家。它们定义了血管发育的决定。 我们希望他们能让人们更容易接触到高等教育的形成 排序复杂的器官，如心脏，并可用于定义 相互作用的基因。最早的心场识别是一种 向定义最早的分子本质迈出的第一步 心脏祖细胞。尤其是内皮细胞的突变，提供了 首次有证据表明内皮细胞组装中存在离散的区域图案。 在医学术语中，突变为关键的单一步骤提供了指标 心脏装配，在一些常见的情况下可能会出错 先天性和成人心脏病。最终，这些将导致 心脏血管形成相关新基因的克隆和鉴定 这是心血管疾病倾向的基础。

The goal of this project is to understand the unitary steps of cardiovascular development. The approach combines genetics, molecular biology, and embryology. We have shown that the zebrafish heart and vasculature are amenable to genetic dissection, accessible to embryological manipulation in a transparent embryo, and analogous to the mammalian through the heart tube stage. During the two years of the current grant, we have completed the largest genetic screen for cardiovascular mutations ever undertaken. We have identified 124 recessive lethal mutations which specifically disrupt cardiovascular development. For example, we have mutants which lack endocardium or valves, those which have hearts that are too small or too large, and those constituted of a single chamber. In addition, by single-cell tracer injection, we have discovered the location of a heart field in the blastula and identified cells that give rise to both myocardium and endocardium. Specific Aim 1 is to complete the complementation analysis of the different mutations and to map the mutations onto our zebrafish genome map. This is the first step towards cloning of the mutant genes. Specific Aim 2 is to analyze the embryonic heart field and lineage tree of cardiac progenitors. In particular, our evidence suggests that there is a common progenitor cell in the ventral-marginal blastula for myocardium, endocardium, endothelium, and blood and we need to test this hypothesis by defining the sublineages. We have evidence that the heart field of the blastula is spatially determined, at least in part, by the divergent homebox gene tinman, and we will assess this thesis by ectopic overexpression combined with cell tracking. Specific Aim 3 focuses upon the two mutations we discovered that are of clear-cut interest to the patterning of the vasculature. Each perturbs genesis of a specific stretch of endothelium: (a) cloche abolishes the endocardium (and possibly some head endothelium). Our hypothesis is that it blocks endocardial progenitors during their formation ow migration; (b) gridlock blocks vessel assembly in the region where the two dorsal aortae merge to become a single aorta. Many attributes of this mutant resemble the human disease coarctation of the aorta. Our primary question is whether the defects are in the endothelial cells or in the microenvironment. The mutations provide a resource for the entire community of cardiovascular scientists. They define decisions in vascular development. We hope that they can render more accessible the fashioning of higher order complex organs such as the heart, and can be used to define interacting genes. The identification of the earliest heart field is a first step towards definition of the molecular nature of the earliest cardiac progenitors. The endothelial mutations, in particular, provide the first evidence for discrete regional patterning in endothelial assembly. In medical terms, the mutations provide indices to the key unitary steps of cardiac assembly, ones which may go awry in some of the common congenital and adult heart diseases. Ultimately, these will lead to the cloning of the new genes relevant to fashioning cardiovasculature and which underlie propensities to cardiovascular illness.