Investigating the emergence of vertebrate cranial cartilage diversity

研究脊椎动物颅软骨多样性的出现

• 批准号: 10421047
• 负责人: Mathi Thiruppathy
• 金额: $ 4.76万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10421047
• 关键词: Address; Adult; Anabolism; Basic Science; Binding; Bioinformatics; Biological Assay; Biology; Biomechanics; Cartilage; Cells; Cephalic; Chondrocytes; Chromatin; Clinical; Connective Tissue; Craniocerebral Trauma; Defect; Development; Ear; Elastic Cartilage; Elastin; Embryo; Enhancers; Epigenetic Process; Epiglottis structure; External Ear; Filament; Fishes; Future; GATA3 gene; Gene Expression; Gene Transfer Techniques; Generations; Genes; Genetic; Genomics; Gills; Goals; Head; Histologic; Human; Hyaline Cartilage; Imaging Device; In Situ; Joints; Larynx; Light; Location; Logic; Mammals; Mentorship; Mesenchymal Stem Cells; Mesenchyme; Minority; Modeling; Mus; Neural Crest; Nose; Patients; Phenotype; Physiological; Property; Reporter; Research; Research Personnel; Research Project Grants; Role; Specific qualifier value; Study models; Surface; Syndrome; Techniques; Temporomandibular Joint; Testing; Tissues; Training; Transgenic Mice; Transgenic Organisms; Validation; Vertebrates; Zebrafish; bone; capsule; career development; cartilage development; craniofacial; cranium; experimental study; gene conservation; gene regulatory network; genomic tools; in vivo; in vivo evaluation; microtia; morphogens; mouse genetics; mutant; novel; programs; promoter; reconstruction; repaired; selective expression; single cell analysis; single cell sequencing; single-cell RNA sequencing; stem cells; therapy development; transcriptome; zebrafish development

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.

项目摘要 软骨是一种专门的结缔组织，在脊椎动物的许多部分中具有结构作用。取决于 在位置和发育阶段，体内的软骨组织以多种表型形式存在 不同的生理特性。在哺乳动物中，这种多样性在头部最引人注目，透明软骨 胚胎颅骨（瞬态）和下颌关节（永久）与组织学不同的软骨共存， 在外耳和喉中发现的永久性亚型。然而，几乎所有关于软骨发展重点的研究 在透明软骨上。这导致对如何指定弹性软骨的了解不足，更普遍地 分化永久性和瞬态软骨的监管网络。单细胞RNA测序 斑马鱼中颅神经rest（CNCC）衍生物的（scrnaseq）揭示了软骨的意外多样性 幼虫和成人头。通过原位验证，我发现一个与透明软骨高度不同的软骨亚型 这本身定位在ills上，类似于哺乳动物的弹性软骨。通过染色质的单细胞分析 可访问性，我们已经确定了在透明或弹性软骨中特别可以使用的推定增强剂。主题 对这些区域的分析揭示了沿弹性的GATA和SOX9基序的强大共同点 软骨轨迹，尤其是GATA3在弹性软骨中显示选择性表达和相关性 间充质。我提出了一个模型，其中GATA3素数提高了特定于g弹性软骨的结合模型 和Sox9的激活，导致软骨发育过程中弹性和透明亚型的差异。在 我的第一个目的是，我计划通过测试推定的体内验证弹性软骨特异性的顺式调节网络 斑马鱼的增强剂。然后，我测试了经过验证的增强剂是否可以在鼠标弹性中驱动特定的表达 软骨，进一步建立鱼g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SOX9增强子结合 和弹性软骨的激活，导致与透明软骨的差异。我的研究会审问 在开发过程中，软骨亚型差异的基础监管网络，特别是 与开发疗法有关外耳弹性软骨缺陷的特异性疗法的相关性 （Microtia/anotia）发生在许多颅面综合征中。更广泛地，我的项目将为 了解瞬态和永久软骨亚型之间的差异。我打算执行此操作 在加州大学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 我获得小鼠遗传学方面的补充专业知识。我的培训计划是为我专注的量身定制的 对生物信息学的培训，使我接触了我的基础研究的临床相关性。一个强大的研究项目 结合我们高度动态的干细胞中心中的职业发展和互动，将使我能够 实现成为一名独立研究人员的目标。