Retinoid Signaling in Neural Patterning in Zebrafish

描述(申请人提供)：组织内信号梯度的形成是动物发育的一个基本方面。分级信号的功能是指定具有不同身份的细胞，使它们根据信号浓度遵循独立的发育程序。在发育中的脊椎动物中枢神经系统中，来自胚胎中胚层的信号形成了表面上看是同质的神经上皮的主轴，指定了不同的神经元类型及其相互连接，形成了一个功能神经网络。特别是在后脑，这转化为菱形脑之间的边界，沿着前后(A-P)轴产生一系列7个节段。然而，令人惊讶的是，人们对脊椎动物早期A-P模式的分子机制知之甚少，我们正在利用斑马鱼的遗传和胚胎优势来解决这个问题。在后脑，由维生素A衍生物维甲酸(RA)及其核受体(RARs)介导的信号与神经模式有关，但我们的初步研究表明，简单的RA信号的A-P梯度不足以解释整个过程。这项拟议研究的长期目标是了解脊椎动物后脑发育过程中A-P模式的细胞和分子基础。2个基本假设指导着拟议的研究。首先，我们假设cyp26家族的酶对RA的局部降解是其对后脑影响分级的关键。其次，我们假设RAR表达的限制性区域决定了后脑和邻近的颅骨中胚层对RA的组织特异性反应。目的1是利用驱动GFP的RA反应元件追踪活体胚胎对RA的反应，以确定后脑对RA的反应范围和浓度依赖关系。转基因斑马鱼的光学清晰度和可用性使其特别适合于这项研究。目的2是通过破坏cyp26的内源性表达和/或RA自身的自身调节，分析RA降解在信号转导中的作用。目的3利用斑马鱼特有的突变体、吗啉和药理拮抗剂的组合，重点研究单个RARs的作用。在这里，我们首先将重点放在中胚层和发育中的神经管之间需要RARG的特定组织相互作用上，并将使用RAR缺陷胚胎的细胞移植来获得神经模式的生化基础。

DESCRIPTION (provided by applicant): The formation of signaling gradients within tissues is a fundamental aspect of animal development. Graded signals function to specify cells with different identities, causing them to follow independent, developmental programs depending on signal concentration. In the developing vertebrate central nervous system, signals from the embryonic mesoderm pattern the primary axes of an apparently homogeneous neuroepithelium, specifying different neuronal types and their interconnections to form a functional neural network. In the hindbrain in particular, this translates into boundaries between rhombomeres, producing a series of 7 segments along the anterior-posterior (A-P) axis. Surprisingly little is known, however, about the molecular mechanisms that control early A-P patterning in vertebrates and we are addressing this issue using the genetic and embryological advantages of zebrafish. In the hindbrain, signaling mediated by the vitamin A derivative, retinoic acid (RA) and its nuclear receptors (RARs) has been implicated in neural patterning, but our preliminary studies show that a simple A-P gradient of RA signaling is insufficient to explain the entire process. The long-term goal of the proposed research is to understand the cellular and molecular basis of A-P patterning during vertebrate hindbrain development. 2 primary hypotheses guide the proposed research. First, we hypothesize that local degradation of RA by enzymes of the cyp26 family is the key to its graded effects on the hindbrain. Secondly, we hypothesize that restricted domains of RAR expression determine tissue-specific responses to RA in the hindbrain and in adjacent cranial mesoderm. Aim 1 is to follow the response to RA in living embryos using an RA-response element driving GFP, to define the range and concentration-dependence on RA in the hindbrain. The optical clarity and availability of transgenic zebrafish make it uniquely suited for this study. Aim 2 is to analyze the functions of RA degradation on signaling, by disrupting the endogenous expression of cyp26s and/or their autoregulation by RA itself. Aim 3 focuses on the roles of individual RARs using a combination of mutants, morpholinos and pharmacological antagonists uniquely available in zebrafish. Here we will focus initially on a particular tissue interaction that requires RARg, between the mesoderm and developing neural tube, and will use cell transplantation in RAR-deficient embryos to get to the biochemical basis for neural patterning.