Intercellular Signalling Pathways Regulate Zebrafish Hindbrain Development

我们的实验室使用斑马鱼后脑发育作为模型来理解细胞如何经历复杂的形态发生重排来形成头部内的组织。后脑是中枢神经系统的一个短暂的分段部分，位于脊髓的前部和中脑的后部。后脑细胞亚群从神经管脱落。这些被称为颅神经嵴的细胞随后迁移形成颌骨软骨。留在后脑内的细胞必须经历同样复杂的细胞形状变化，以使第四脑室膨胀，这是一个作为次级神经循环系统的腔体。颌骨软骨和后脑室的形成都需要细胞形成精确的细胞极性，经历细胞形状的变化，并表达细胞行为所必需的精确的一组基因。这些过程是如何调控的？我们最近发现了两个关键的细胞信号通路成分，分别调节颚软骨形成和脑室形态发生。项目一：骨形态发生蛋白3 （Bone Morphogenetic Protein 3, BMP3）对神经嵴细胞的调控。神经嵴细胞从后脑背侧剥离，向外侧迁移，分化形成颌骨软骨细胞。我们使用Crispr-Cas9生成了斑马鱼bmp3突变体，该突变体对颌骨形成产生了深刻的改变。我们最初的目标是生成一条Smad3报告线，以确定Bmp3信号传导的精确时空参数。然后，我们将进行共聚焦成像来研究软骨内细胞的重排以及这在bmp3突变体中是如何改变的。最后，我们将对bmp3突变体进行转录组学分析，以确定该途径控制软骨发育的分子机制。项目二：Taz对脑室形态发生的调控。脑室系统由一系列充满液体的腔体组成，这些腔体起着次级神经循环系统的作用。当组织沿中线分离并随后通过脑脊液的分泌使腔膨胀时，后脑室形成。为了了解Hippo信号在神经发育中的作用，我们产生了taz突变斑马鱼，发现这种突变体的脑室明显变小，并且在中线分离方面存在缺陷。Taz蛋白通过Wnt信号通路的串扰得到稳定，两种信号通路协调调节位于后脑节段边界的细胞的极性。我们提出一个模型，其中Taz调节后脑菱形球边界细胞的身份。为了验证这个模型，我们计划在Hippo激酶级联中创建额外的突变体，并对后脑节段边界细胞中的基因表达进行系统分析。最后，我们将确定taz依赖性转录本如何在调节后脑边界细胞身份和脑室形成中发挥作用。

Introduction: Our laboratory uses zebrafish hindbrain development as a model for understanding how cells undergo complex morphogenetic rearrangements to shape tissues within the head. The hindbrain is a transiently segmented portion of the central nervous system that lies anterior to the spinal cord and posterior to the midbrain. A subpopulation of hindbrain cells delaminate from the neural tube. These cells, known as cranial neural crest, subsequently migrate to create the jaw cartilage elements. Cells that remain within the hindbrain must undergo equally complex changes in cell shape to inflate the fourth brain ventricle, a cavity that functions as a secondary neural circulatory system. Both jaw cartilage and hindbrain ventricle formation require cells to develop precise cell polarity, undergo cell shape changes, and express the precise set of genes necessary for cellular behaviour. How are such processes regulated? We have recently identified two key cell signalling pathway components that regulate jaw cartilage formation and brain ventricle morphogenesis, respectively. Project 1: Regulation of neural crest cells by Bone Morphogenetic Protein 3 (BMP3). Neural crest cells delaminate from the dorsal hindbrain and migrate laterally  and differentiate to form the jaw cartilage elements. We generated a zebrafish bmp3 mutant using Crispr-Cas9 and this mutant displays profound alterations to jaw formation. Our initial goal will be to generate a Smad3 reporter line to determine the precise temporal and spatial parameters of Bmp3 signalling. We will then perform confocal imaging to study the rearrangement of cells within cartilage and how this is altered in bmp3 mutants. Finally, we will undertake transcriptomic analyses of bmp3 mutants to define the molecular mechanisms by which this pathway controls cartilage development. Project 2: Regulation of brain ventricle morphogenesis by Taz. The brain ventricular system consists of a series of fluid-filled cavities that function as a secondary neural circulatory system. The hindbrain ventricle forms as tissues separate along the midline and subsequently inflate the cavity via secretion of cerebrospinal fluid. In an effort to understand Hippo signalling in neural development, we generated taz mutant zebrafish and found that such mutants display a significantly smaller brain ventricle with a defect in midline separation. Taz protein is stabilized by crosstalk from the Wnt signalling pathway and the two pathways coordinately regulate polarity of cells located at hindbrain segment boundaries. We propose a model in which Taz regulates hindbrain rhombomere boundary cell identity. To test this model, we plan to create additional mutants in the Hippo kinase cascade and perform a systematic analysis of gene expression in hindbrain segment boundary cells. Finally, we will determine how Taz-dependent transcripts function in regulating hindbrain boundary cell identity and brain ventricle formation.