Identifying the role of dynamic ECM-derived forces in zebrafish semicircular canal morphogenesis

项目摘要 一个器官的形态决定了它的功能。简单的上皮组织如何产生复杂形式的成熟 器官是发育生物学中的一个基本问题，对疾病具有重要意义， 再生医学一个很好的例子是简单的胚胎上皮细胞的重塑， 内耳的半规管（SCC）。它们复杂的形状是感知平衡和加速度所必需的 并且在所有有颌脊椎动物中是保守的。SCC可以在先天性综合征中异常发展， LAMM、CHARGE和Alagille综合征，原因不明。健康的潜在机制 发展知之甚少。我的初步研究结果表明，局部合成的富含透明质酸 细胞外基质（ECM）在SCC的发展中是重要的。在这项研究中，我将确定这是如何实现的。 ECM有助于在斑马鱼（Aim 1）的SCC形态发生中产生机械力。我将使用下一代 单细胞测序，以鉴定参与活性ECM（Aim 2）的模式化的基因，并鉴定和 表征SCC发展中涉及的其他ECM组分的作用（Aim 3）。新型分子- 本研究揭示的力学原理将广泛适用于其他形态发生事件， 活跃的细胞外基质和拓扑上皮重塑此外，由于这些进程在许多国家受到破坏， 先天性疾病和耳部疾病，我的发现将提高我们对这些疾病的认识。 拟议的研究借鉴了1）我在发展背景下的定量细胞生物学培训，2）新的 在我的博士后研究中获得的技能，包括斑马鱼研究和发育器官的成像 在单细胞分辨率，和3）我的K99培训计划，以实现自动图像分析，在体内生物物理 测量、建模以及单细胞测序和分析。为了实现我的目标，我将接受指导 Sean Megason，Tim Mitchison，L. Mahadevan和Allon Klein，他们在斑马鱼方面的专业知识 开发，基因编辑，生物物理学，定量成像，建模和单细胞RNA测序将提供 我需要的培训，以开始我的独立研究计划。为了促进我的职业发展 在辅导的K99阶段，我将学习相关课程并保持两个基础课程的合作 和转化研究，在会议上介绍我的工作，发表我的研究，写赠款，并获得进一步的 管理和指导学生的经验，形成自己的指导风格。我将受益匪浅 从经验，跟踪记录和定期反馈，我的同事导师开始我自己的实验室。 我的长期职业目标是领导一个跨学科的研究计划，调查机械和 形态发生的分子基础，并利用我的见解，以推进转化研究。我做了 在实现这一目标方面取得了重大进展，包括研究经验、成功的合作和 出版物。K99奖将使我获得成功所需的额外培训和专业知识， 过渡到独立。

PROJECT SUMMARY The form of an organ defines its function. How simple epithelial tissues give rise to the complex forms of mature organs is a fundamental question in developmental biology with important implications in disease and regenerative medicine. A beautiful example is the remodeling of a simple embryonic epithelium to form the three semicircular canals (SCC) of the inner ear. Their intricate form is required for sensing balance and acceleration and is conserved across all jawed vertebrates. SCCs can develop abnormally in congenital syndromes such as LAMM, CHARGE and Alagille syndrome, for unknown reasons. The mechanisms underlying healthy development are poorly understood. My preliminary findings indicate that local synthesis of a hyaluronan-rich extracellular matrix (ECM) is important in SCC development. In the proposed research, I will determine how this ECM helps generate mechanical forces in SCC morphogenesis in zebrafish (Aim1). I will use next-generation single-cell sequencing to identify genes involved in the patterning of an active ECM (Aim2) and to identify and characterize the role of other ECM components involved in SCC development (Aim3). The novel molecular- mechanical principles revealed from this study will be widely applicable to other morphogenetic events involving an active ECM and topological epithelial remodeling. Moreover, as these processes are disrupted in many congenital diseases and ear disorders, my findings will improve our understanding of these diseases. The proposed research draws on 1) my training in quantitative cell biology in a development context, 2) the new skills acquired in my post-doctoral research, including zebrafish research and imaging of the developing organ at single-cell resolution, and 3) my K99 training plan to implement automated image analysis, in vivo biophysical measurements, modeling, and single-cell sequencing and analysis. To fulfil my aims, I will receive mentorship from Drs. Sean Megason, Tim Mitchison, L. Mahadevan and Allon Klein, whose combined expertise in zebrafish development, gene-editing, biophysics, quantitative imaging, modeling and single-cell RNA sequencing will give me the training I need to commence my independent research program. To facilitate my career development during the mentored K99 phase, I will take relevant courses and maintain collaborations for both fundamental and translational research, present my work at meetings, publish my research, write grants, and gain further experience in management and mentoring students to formulate my own mentoring style. I will greatly benefit from the experience, track record and regular feedback from my co-mentors to start my own laboratory. My long-term career goal is to head an inter-disciplinary research program to investigate the mechanical and molecular basis of morphogenesis and to use the insights I develop to advance translational research. I’ve made significant progress towards this goal in the form of research experience, successful collaborations and publications. The K99 award will give me access to the additional training and expertise I need to successfully transition to independence.