DEVELOPMENT OF CARDIAC ASYMMETRY IN ZEBRAFISH

心脏和腹部左右不对称的发生 器官在脊椎动物胚胎发育中是一个基本的，但很差的 明白了，流程。正态不对称性发展中的误差给出 上升为各种形式的内翻肌，包括右心， 腹部内脏内翻，以及许多其他先天性 反常现象。该项目的长期目标有两个：定义 发育中的轴向中胚层在LR图案化和TO中的诱导作用 了解正确指定轴所需的信号。 我们将集中讨论不对称的最早的形态迹象，即 心脏导管向右环行。斑马鱼，Danio Rerio，有 被选为本项目的模型系统，因为它开发了 在透明胚胎的外部，每天产生数百个卵，并且 特别容易受到基因和细胞操作的影响。其中之一 脊椎动物信号活动的最好研究来源 器官发生是脊索，它沿着主干组织结构 身体轴。有趣的是，脊索或脊索突变的斑马鱼 最近发现邻近的背中线结构具有 与正常心脏循环相关的异常。通过研究这一点 轴向中胚层发育突变与误差的关系 在心脏不对称时，我们可以开始了解LR的调节 规格。 我们将从彻底描述突变体的表型开始， 找出可能导致异常的本地关系 心脏定位。然后我们将尝试挽救正常的心脏部位 在野生型和突变体之间的细胞移植实验中 突变胚胎。利用正常人囊胚期的祖细胞 胚胎，我们可以重新填充突变的缺陷细胞系，并 从而试图挽救心脏正常的左右位向。这些 实验将有助于识别具有重要信号的结构 在LR组织中的角色。然后，我们将研究 实验性消融发育中轴线特定细胞群 其他正常斑马鱼的中胚层。颠覆所需的结构 心脏的LR型会导致心脏的异常循环- 管子。最后，我们有兴趣了解轴的作用 这些突变体中的模式基因sonic hedgehog(Ssh)。嘘，就是 最近在雏鸟体内发现了不对称表达，并推测 在心脏的LR模式中扮演一个角色。就地整体安装 杂交法将用于确定Shh在 正常和突变的斑马鱼。该基因的作用将在 封闭抗体的存在及其异位表达 注射shh信使RNA的胚胎中的蛋白质产物。这个 异位表达对心脏LR型组织的影响 并研究了其对LR发展的影响。

The development of left-right (LR) asymmetry of the heart and abdominal organs during vertebrate embryogenesis is a fundamental, yet poorly understood, process. Errors in the development of normal asymmetry give rise to various forms of situs inversus, including dextrocardia, inversion of the abdominal viscera, and a host of other congenital anomalies. The long-term goal of this project is twofold: to define the inductive role of the developing axial mesoderm in LR patterning and to understand the signals required for proper specification of the axis. We will concentrate on the earliest morphologic sign of asymmetry, namely rightward looping of the heart-tube. The zebrafish, Danio rerio, has been chosen as the model system for this project because it develops externally in a transparent embryo, produces hundreds of eggs daily, and is particularly amenable to genetic and cellular manipulation. One of the best-studied sources of signaling activity for vertebrate organogenesis is the notochord, which organizes structures along the main body axis. Interestingly, zebrafish with mutations of the notochord or adjacent dorsal midline structures have recently been found to have associated abnormalities of normal cardiac looping. By studying this relationship between mutations of axial mesoderm development and errors in cardiac asymmetry, we can begin to understand the regulation of LR specification. We will begin by thoroughly characterizing the mutants phenotypically, to find local relationships that may be responsible for the abnormal cardiac orientation. We will then attempt to rescue normal cardiac situs in the mutants by cell transplantation experiments between wild-type and mutant embryos. Using progenitor cells from the blastula stage of normal embryos, we can repopulate the deficient cell line of the mutant and thereby attempt to rescue normal LR orientation of the heart. These experiments will help to identify structures with important signaling roles in LR organization. We will then examine the effects of experimental ablation of specific groups of cells of the developing axial mesoderm in otherwise normal zebrafish. Disrupting structures needed for LR patterning of the heart will lead to aberrant looping of the heart- tube. Finally, we are interested in understanding the role of the axial patterning gene, sonic hedgehog (ssh), in these mutants. Shh was recently found asymmetrically expressed in the chick and postulated to play a role in LR patterning of the heart. Whole mount in situ hybridization will be used to determine expression patterns of shh in normal and mutant zebrafish. The role of the gene will be tested in the presence of blocking antibody as well as by ectopic expression of its protein product in embryos injected with shh messenger RNA. The consequences of ectopic expression on LR organization of the heart will be examined and its influence on LR development studied.