Investigating the emergence of vertebrate cranial cartilage diversity

项目摘要 软骨是一种特殊的结缔组织，在脊椎动物身体的许多部位都有结构性作用。取决 在位置和发育阶段上，体内的软骨组织以一系列表型形式存在， 不同的生理特性。在哺乳动物中，这种多样性在头部最为显著， 胚胎头骨（暂时性）和颌关节（永久性）的组织学上不同的弹性软骨共存， 一种在外耳和喉中发现的永久亚型。然而，几乎所有关于软骨发育的研究都集中在 透明软骨上这导致了一个穷人的理解如何弹性软骨是指定的，更普遍的 区分永久性和暂时性软骨的调节网络。单细胞rna测序 斑马鱼颅神经嵴（CNCC）衍生物的scRNAseq研究揭示了斑马鱼软骨的意外多样性。 幼虫和成虫的头部通过原位验证，我发现了一种与透明软骨高度不同的软骨亚型 位于鳃部类似于哺乳动物的弹性软骨通过染色质的单细胞分析 可接近性，我们已经确定了在透明或弹性软骨中特异性可接近的推定增强子。基序 对这些区域的分析揭示了加塔和Sox 9基序的强烈共富集，特别是沿着弹性区。 软骨轨迹，特别是Gata3在弹性软骨中显示选择性表达， 间充质我提出了一个模型，其中Gata3引发了对鳃弹性软骨特异性的增强子， 和Sox 9的激活，导致软骨发育过程中弹性和透明亚型的分化。在 我的第一个目标是，我计划通过测试假定的 斑马鱼的增强剂。然后，我测试验证的增强子是否可以驱动小鼠弹性蛋白的特异性表达， 软骨，进一步建立鱼鳃和哺乳动物弹性软骨之间的同源性。使用组合 尖端的遗传，转基因和基因组学工具，我将测试Gata3修改Sox 9增强子结合 和弹性软骨的激活，导致与透明软骨的分歧。我的研究将审问 在发育过程中，软骨亚型分化的调控网络，特别是 与开发外耳弹性软骨缺损特异性修复疗法的相关性 （小耳/无耳），发生在许多颅面综合征。更广泛地说，我的项目将为 了解暂时性和永久性软骨亚型之间的差异。我计划执行这个 在南加州大学Gage Crump博士的指导下，以斑马鱼为重点的项目，其他合作者允许 让我获得小鼠遗传学方面的互补专业知识。我的培训计划是为我量身定制的， 生物信息学的培训，让我接触到我的基础研究的临床相关性。强大的研究项目 结合我们高度动态的干细胞中心内的职业发展和互动， 实现我成为独立研究员的目标。

PROJECT SUMMARY Cartilage is a specialized connective tissue with structural roles in many parts of the vertebrate body. Depending on location and developmental stages, cartilaginous tissues in the body exist in a range of phenotypic forms with different physiological properties. In mammals, this diversity is most striking in the head, where hyaline cartilages of the embryonic skull (transient) and jaw joints (permanent) coexist with histologically distinct elastic cartilage, a permanent subtype found in the external ear and larynx. Yet nearly all studies of cartilage development focus on hyaline cartilage. This has led to a poor understanding of how elastic cartilage is specified, and more generally of regulatory networks that differentiate permanent and transient cartilages. Single-cell RNA sequencing (scRNAseq) of cranial neural crest (CNCC) derivatives in zebrafish reveals unexpected diversity of cartilages in the larval and adult head. Through in situ validation, I find a cartilage subtype highly distinct from hyaline cartilage that localizes to the gills and resembles mammalian elastic cartilage. Through single-cell analysis of chromatin accessibility, we have identified putative enhancers specifically accessible in hyaline or elastic cartilage. Motif analysis of these regions reveals a strong co-enrichment of Gata and Sox9 motifs specifically along the elastic cartilage trajectory, with Gata3 in particular showing selective expression in elastic cartilage and associated mesenchyme. I propose a model in which Gata3 primes enhancers specific to gill elastic cartilage for binding and activation by Sox9, leading to divergence of elastic and hyaline subtypes during cartilage development. In my first aim, I plan to validate elastic cartilage-specific cis-regulatory networks in vivo by testing putative enhancers in zebrafish. I then test whether validated enhancers can drive specific expression in mouse elastic cartilage, further establishing homology between fish gill and mammalian elastic cartilage. Using a combination of cutting-edge genetic, transgenic, and genomics tools, I will test that Gata3 modifies Sox9 enhancer binding and activation in elastic cartilage, leading to divergence from hyaline cartilage. My research will interrogate regulatory networks underlying cartilage subtype divergence during development, with particular relevance to developing therapies for specific repair of elastic cartilage defects of the external ear (microtia/anotia) that occur in many craniofacial syndromes. More broadly, my project will pave the way for understanding the divergence between transient and permanent cartilage subtypes. I plan to carry out this zebrafish-focused project under the mentorship of Dr. Gage Crump at USC, with additional collaborators allowing me to gain complementary expertise in mouse genetics. My training plan is tailored to provide me focused training in bioinformatics and expose me to the clinical correlates of my basic research. A strong research project combined with career development and interactions within our highly dynamic stem cell center will allow me to achieve my goal of becoming an independent researcher.