Physical, cellular, and molecular control of tissue fission and fusion

项目摘要 组织分裂和融合在胚胎发育过程中形成了功能器官。它们中的异常 发育过程可导致先天性出生缺陷和综合征，如腭裂、Meckel-Gruber综合征、 和永久性动脉干。在重塑过程中，细胞必须产生力，重新配置细胞接触，并 与周围环境互动。细胞和分子调控机制的基础机制 人们对潜在的组织分裂和融合知之甚少。机械感觉器官的形成称为 斑马鱼中的神经肥大为破译这些过程提供了一个理想的模型。在神经肥大期间 形态发生，前神经肥大从迁移的后外侧线原基分裂出来，后来与 皮肤打开皮肤细胞中的三细胞连接；因此，我可以在这方面研究组织分裂和融合 形态发生事件。在这个模型中，我的初步发现表明，细胞和 原基分裂和神经肥大与皮肤融合的组织。在拟议的研究中，我将应用国家- 最先进的体内生物物理测量以确定RhoA介导的肌球蛋白是否驱动神经肥大 沉积(目标1)。然后，我将使用一种新的蛋白质耗尽方法，提供空间和时间控制 测试细胞-细胞和细胞-细胞外基质(ECM)黏附是否介导神经肥大的萌发(目标2)和 研究神经肥大与皮肤融合的机制(目标3)。物理、分子和细胞 这项研究揭示的原理将广泛适用于涉及组织分裂和 融合的同时保持单细胞的完整性。此外，这项研究的结果将改善我们的 对组织分裂融合异常所致先天出生缺陷的认识及对策 来纠正这些缺陷。为了完成拟议的研究，我将结合我在细胞力学方面的技能 作为研究生发展起来的分析；在我早期的斑马鱼博士后培训中获得的新技能 遗传学、分子生物学和高分辨率实时成像；以及在K99期间拟议的技术培训 实施体内生物物理测量的阶段，包括测量体内细胞-细胞外基质应力和细胞-细胞 粘合张力。当我开始自己的实验室时，我将得到Holger Knaut博士、Daniele Panozzo博士和Anna- Katerina Hadjantonakis、Jeremy Nance、Carsten Grashoff和Johannes Stegmaier。他们不仅会提供 他们在斑马鱼遗传学、生物物理学、定量成像和建模方面的科学专长，以及他们的 在指导学生、资质、出版、建立科学合作方面的宝贵经验， 和实验室管理。我的长期职业目标是领导一个研究实验室，揭开基因， 胚胎形态发生中细胞和组织力学的生物物理、细胞和分子调控。我有过 通过研究经验、成功的合作和出版，朝着这一目标取得了重大进展。 然而，我坚信，在K99期间提出的额外技术和职业培训指导了 阶段是我成功过渡到独立的必经之路。

Project Summary Tissue fission and fusion give forms to functional organs during embryonic development. Abnormalities in these processes can lead to congenital birth defects and syndromes such as cleft palate, Meckel-Gruber syndrome, and persistent truncus arteriosus. During remodeling, cells must generate force, reconfigure cell contacts, and interact with their surroundings. The mechanisms underlying the cellular and molecular regulation mechanisms underlying tissue fission and fusion are poorly understood. The formation of mechano-sensory organs called neuromasts in zebrafish provides an ideal model in which to decipher these processes. During neuromast morphogenesis, the pro-neuromast splits from the migrating posterior lateral line primordium and later fuses with skin to open tricellular junctions in skin cells; hence, I can study both tissue fission and fusion in this morphogenetic event. In this model, my preliminary findings suggest a mechanical ‘tug of war’ between cells and tissues in primordium splitting and neuromast fusion with the skin. In the proposed research, I will apply state- of-the-art in vivo biophysical measurements to determine whether RhoA-mediated actomyosin drives neuromast deposition (Aim 1). I will then use a novel protein depletion approach that offers spatial and temporal control to test whether cell-cell and cell-extracellular matrix (ECM) adhesions mediate neuromast budding (Aim 2) and investigate the mechanism by which neuromasts fuse with skin (Aim 3). The physical, molecular and cellular principles revealed in this study will be widely applicable to morphogenetic events involving tissue fission and fusion while maintaining the integrity of single cells. Moreover, findings from this study will improve our understanding of congenital birth defects due to abnormal tissue fission and fusion and further inform strategies to correct these defects. To accomplish the proposed research, I will combine my skills in cell mechanics analyses developed as a graduate student; new skills acquired in my early postdoctoral training in zebrafish genetics, molecular biology and high-resolution live imaging; and the proposed technical training during the K99 phase to implement in vivo biophysical measurements, including measuring in vivo cell-ECM stress and cell-cell adhesion tension. As I start my own lab, I will be mentored by Drs. Holger Knaut, Daniele Panozzo, Anna- Katerina Hadjantonakis, Jeremy Nance, Carsten Grashoff, and Johannes Stegmaier. They will offer not only their scientific expertise in zebrafish genetics, biophysics, quantitative imaging, and modeling but also with their valuable experience in mentoring students, grantsmanship, publication, establishing scientific collaborations, and lab management. My long-term career goal is to head a research laboratory and uncover the genetic, biophysical, cellular, and molecular regulation of cell and tissue mechanics in embryonic morphogenesis. I have made significant progress toward this goal with research experience, successful collaborations, and publications. However, I firmly believe that the additional technical and career training proposed during the K99 mentored phase is necessary for me to successfully transition to my independence.