Investigating the emergence of vertebrate cranial cartilage diversity

项目总结 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作用。取决于 在位置和发育阶段，体内的软骨组织以一系列表型形式存在，具有 不同的生理特性。在哺乳动物中，这种多样性在头部最引人注目，那里有透明的软骨 胚胎颅骨(暂时性)和颌骨关节(永久性)与组织学上不同的弹性软骨共存， 一种存在于外耳和喉部的永久性亚型。然而，几乎所有关于软骨发育的研究都集中在 在透明软骨上。这导致了对弹性软骨是如何指定的，以及更广泛地说是如何指定的理解不足 区分永久性和暂时性软骨的调控网络。单细胞RNA测序 斑马鱼脑神经脊衍生物的(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截然不同的软骨亚型 它位于鳃上，类似于哺乳动物的弹性软骨。通过对染色质的单细胞分析 对于可及性，我们已经确定了在透明或弹性软骨中特别可获得的推定的增强剂。主题 对这些区域的分析表明，GATA和Sox9基序在弹性带上有很强的共同富集性 软骨轨迹，尤其是GATA3在弹性软骨中选择性表达，并与 间充质的我。我提出了一种模型，在该模型中，GATA3激活特定于鳃弹性软骨的增强剂进行结合 并被Sox9激活，导致软骨发育过程中弹性和透明亚型的分化。在……里面 我的第一个目标是，我计划在体内验证弹性软骨特异的顺式调控网络，通过测试假定的 斑马鱼中的增强剂。然后，我测试了经过验证的增强剂是否可以驱动鼠标弹性中的特定表达 软骨，进一步建立了鱼鳃和哺乳动物弹性软骨之间的同源性。使用组合 在先进的遗传、转基因和基因组学工具中，我将测试GATA3修改Sox9增强子结合 弹性软骨中的活化，导致与透明软骨的分化。我的研究将审问 发育过程中软骨亚型分化的调控网络，尤其是 外耳弹性软骨缺损的特殊修复方法的研究进展 (小耳/厌食症)，出现在许多颅面综合征。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。我计划执行这项任务 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 我希望在老鼠遗传学方面获得互补的专业知识。我的培训计划是量身定做的，让我专注于 在生物信息学方面的培训，让我接触到我的基础研究的临床相关性。一个强有力的研究项目 结合我们高度活跃的干细胞中心内的职业发展和互动，将使我能够 实现我成为一名独立研究员的目标。

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.