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

项目总结 器官的形状决定了它的功能。简单的上皮组织是如何产生复杂形式的成熟的 器官是发育生物学中的一个基本问题，具有重要的疾病和生物学意义 再生医学。一个很好的例子是一个简单的胚胎上皮的重塑，形成了三个 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 在所有有颌骨的脊椎动物中都是保守的。SCCS可在先天性综合征中异常发育，如 Lamm，Charge和Alagille综合征，原因不明。健康背后的机制 人们对发展知之甚少。我的初步发现表明，局部合成富含透明质酸的 细胞外基质(ECM)在鳞状细胞癌的发生发展中起重要作用。在拟议的研究中，我将确定这一点 ECM有助于在斑马鱼的SCC形态发生中产生机械力(Aim1)。我会用下一代 单细胞测序以确定参与活性ECM(AIM2)图案化的基因，并识别和 描述参与SCC开发的其他ECM组件的作用(Aim3)。这种新型的分子-- 这项研究揭示的力学原理将广泛适用于其他形态发生事件，包括 活跃的细胞外基质和拓扑性上皮重塑。此外，由于这些进程在许多情况下被扰乱 先天性疾病和耳疾，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)我在发育背景下的定量细胞生物学方面的培训，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 在单细胞分辨率下，以及3)我的K99培训计划，以实施体内生物物理自动图像分析 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 肖恩·梅格森、蒂姆·米奇森、L.Mahadean和Allon Klein博士，他们在斑马鱼方面的专业知识结合在一起 开发、基因编辑、生物物理学、定量成像、建模和单细胞RNA测序将给出 我需要开始我的独立研究计划所需的培训。为了促进我的职业发展 在指导的K99阶段，我将学习相关课程并保持两个基础课程的协作 和翻译研究，在会议上展示我的工作，发表我的研究，写赠款，并进一步获得 在管理和辅导学生方面的经验形成了自己的辅导风格。我会受益匪浅的 根据我的经验、往绩和同事的定期反馈，我创办了自己的实验室。 我的长期职业目标是领导一个跨学科的研究项目，研究机械和 形态发生的分子基础，并利用我开发的见解来推进翻译研究。我已经做出了 在这一目标方面取得重大进展，表现为研究经验、成功的合作和 出版物。K99大奖将让我获得成功所需的额外培训和专业知识 向独立过渡。

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.