Regulation of neuroectoderm morphogenesis by Nodal signaling programs

摘要 胚胎发育是一系列严格控制的事件，将单个细胞构建成复杂的有机体。一个 早期胚胎发育的关键里程碑是原肠形成，这时身体计划首先由行为决定 数以千计的单个细胞。然而，这些细胞如何沟通以确保适当的形态发生并不是 完全理解。中胚层和神经外胚层(NE，未来神经管)组织形态发生的驱动 通过会聚和伸展(C&E)，当细胞插入同时使身体变窄和延长时 轴，并将神经折叠在一起，以确保适当的神经管关闭。在这些高度中断的地方 保守的细胞运动可能导致神经管缺陷，影响全球许多怀孕。节点是一个 一种保守的信号通路，在原肠发育过程中很活跃，最为人所知的是它在 脊椎动物的中胚层和内胚层规格。然而，人们对Nodal在其中的作用知之甚少 C&E的形态发生独立于中胚层的形成，特别是在东北地区。通过利用斑马鱼体外和 在活体模型中，我将专门研究Nodal在形态发生中的作用，而不是更好地了解它 在组织规范过程中起作用。为了研究结节在NE形态发生中的作用，我可以 利用斑马鱼体外胚泡胚外植体、幼稚的胚胎簇独立分析NE延伸 形成所有三个生殖层并经历C&E形态发生的细胞，以响应外源Nodal信号。 外植体中结节信号的早期高峰促进了中胚层的延伸和规范。然而， 我们实验室的初步数据表明，Nodal信号的相对延迟的峰值促进了NE- 独立于其在中胚层规范中的作用的特定的C&E形态发生。这表明， 结节的时间调控调控NE特有的形态发生。基于我充满希望的初步数据，我 假设Nodal信号激活一种新的、受时间调节的NE特异性转录程序 这推动了C&E的形态发生独立于中胚层规格。为了检验这一假设，我提出了3个 目的：目的：Aim 1将研究颞结信号动力学在去甲肾上腺素特异性形态发生中的作用。 光遗传结节受体精确操纵胚胎外植体中的信号。在《目标2》中，我将恢复 利用创新的转基因品系在结节缺陷斑马鱼胚胎NE内特异地发出结节信号 确定Nodal在体内是否能在没有中胚层的情况下驱动NE延伸。最后，在《目标3》中我将 对受时间调控的候选基因进行过度表达和敲除，以确定它们是否 外植体和体内NE特异性延伸的驱动因素。该提案将定义基础知识 关于结节在NE特异性形态发生中的主要作用，其中的中断可能有助于神经管 缺陷普及率和严重程度。

Summary Embryonic development is a series of tightly regulated events that build a single cell into a complex organism. A key milestone in early embryonic development is gastrulation, when the body plan is first shaped by the behaviors of thousands of individual cells. However, how these cells communicate to ensure proper morphogenesis is not completely understood. Mesoderm and neuroectoderm (NE, future neural tube) tissue morphogenesis is driven by convergence and extension (C&E), when cells intercalate to simultaneously narrow and elongate the body axis and bring the neural folds together to ensure proper neural tube closure. Disruptions in these highly conserved cell movements can result in neural tube defects that affect many pregnancies worldwide. Nodal is a well conserved signaling pathway that is active during gastrulation and most well-known for the role it plays in mesoderm and endoderm specification in vertebrates. However, little is understood about the role of Nodal in C&E morphogenesis independent of mesoderm formation, particularly in NE. By utilizing zebrafish ex vivo and in vivo models, I will study the role of Nodal specifically in morphogenesis independent of its better-known function during tissue specification. To examine the role of Nodal underlying NE morphogenesis, I can independently analyze NE extension using ex vivo zebrafish blastoderm explants, naïve clusters of embryonic cells that form all three germ layers and undergo C&E morphogenesis in response to exogenous Nodal signaling. An early peak of Nodal signaling in explants promotes mesoderm extension and specification. However, preliminary data from our lab demonstrated that a relatively delayed peak of Nodal signaling promotes NE- specific C&E morphogenesis that is independent of its role in mesoderm specification. This suggests that the temporal regulation of Nodal regulates NE-specific morphogenesis. Based on my promising preliminary data, I hypothesize that Nodal signaling activates a novel, temporally regulated, NE-specific transcriptional program that drives C&E morphogenesis independent of mesoderm specification. To test this hypothesis, I propose 3 aims: Aim 1 will investigate the role of temporal Nodal signaling dynamics in NE-specific morphogenesis using optogenetic Nodal receptors to precisely manipulate signaling in embryonic explants. In Aim 2, I will restore Nodal signaling specifically within the NE of Nodal-deficient zebrafish embryos using innovative transgenic lines to determine whether Nodal can drive in vivo NE extension in the absence of mesoderm. Finally, in Aim 3 I will perform over expression and knock-down of temporally regulated candidate genes to determine if they are drivers of NE-specific extension both explants and in vivo. This proposal will define the foundational knowledge on the primary role of Nodal in NE-specific morphogenesis, disruptions in which may contribute to neural tube defect prevalence and severity.