Dissecting the role of Six1 and its co-factors during calvarial bone and suture development

剖析 Six1 及其辅助因子在颅骨和缝线发育过程中的作用

基本信息

批准号：
10664478
负责人：
Andre L P Tavares
金额：
$ 16万
依托单位：
UNIVERSITY OF DELAWARE
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-08-01 至 2028-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10664478
关键词：
Address Adult Affect Animal Model Area Birth Bone Development Brain Injuries Branchio-Oto-Renal Syndrome Calvaria Caring Cartilage Cell Differentiation process Cell Lineage Cell physiology Cells Cephalic Clinical Research Collaborations Complex Congenital Abnormality Craniosynostosis Data Defect Deposition Development Diagnosis Disease Dose Embryo Embryonic Development Foundations Frontal bone structure Funding Future Gene Expression Genetic Genetic Transcription Head Heterozygote Histologic Homeostasis Human Image In Vitro Individual Intracranial Hypertension Joint structure of suture of skull Joints K-Series Research Career Programs Kidney Knowledge Lead Left Legal patent Link Live Birth Maintenance Mandible Mesenchymal Mesenchyme Modeling Morphogenesis Morphology Mus Muscle National Institute of Dental and Craniofacial Research Neural Crest Osteoblasts Osteogenesis Parietal bone structure Pathogenesis Patients Pattern Physiologic Ossification Population Reporting Repression Research Role Shapes Specific qualifier value Surgical sutures Testing Time Tissues Training Transgenic Animals Variant Work autosome bone career clinically relevant cofactor craniofacial craniofacial development cranium cytochemistry dosage experimental study hearing impairment in vivo mineralization neural osteogenic osteoprogenitor cell postnatal precursor cell premature single-cell RNA sequencing skills transcription factor

项目摘要

ABSTRACT Variants in the transcription factor SIX1 or its co-factor EYA1 are known underlying genetic causes of Branchio-oto-renal syndrome (BOR), an autosomal dominant disease that results in hearing loss and kidney defects. Recently, a clinical study reported craniosynostosis (CS) in individuals carrying SIX1 BOR variants, including 5’ variants (p.Q11X and p.Q22X) that are predicted to lead to haploinsufficiency; these data suggest that CS may be an undiagnosed defect in BOR. If left untreated, CS can be associated with distortion of skull shape, increased intracranial pressure, and/or brain damage. As defects in the calvarial bone osteoprogenitor cells (OPC) before and/or after birth may lead to CS via increased bone deposition in the cranial sutures, in this application, I plan to address a major knowledge gap regarding Six1 function: What is its role in the development of the calvarial bones? To detect changes in bone development caused by Six1 loss and haploinsufficiency, I will quantitatively analyze head morphology using µCT images and tissue formation using histological analyses (Aim 1). To verify if Six1 and its co-factors have a role the specification and differentiation of OPCs, I will assess gene expression in vivo using RNAscope and qPCR (Aim 1) and in vitro using neural crest-derived mesenchymal precursors and OPCs (Aim 2). Lastly, I will perform single cell RNA-seq and RNAscope to identify cell populations in the supraorbital arch mesenchyme (that gives rise to the rudiments for parietal and frontal bones) that are affected by Six1 loss and haploinsufficiency (Aim 3). Results from this application will shift the paradigm of Six1 function as a cranial placode, neural and muscle transcriptional factor by providing the first direct evidence linking it to normal calvarial development and the pathogenesis of CS. This application will establish Six1-het mice as a new model for craniofacial disease, and will help elucidate the mechanisms by which Six1 variants lead to CS. It will also provide training in soft skills, funding and collaborations required for my next career step. Finally, this training will allow me to bring my extensive knowledge of Six1 transcriptional function in mandible and otic development to a new area of clinically relevant research. Consequently, my research may ultimately prove crucial for CS patient diagnosis and care.

抽象的转录因子 SIX1 或其辅助因子 EYA1 的变异是已知的潜在遗传原因鳃-耳-肾综合征 (BOR)，一种常染色体显性遗传疾病，可导致听力损失和肾脏损害最近，一项临床研究报告了携带 SIX1 BOR 变异的个体存在颅缝早闭 (CS)，这些数据表明，包括预计会导致单倍体不足的 5' 变体（p.Q11X 和 p.Q22X）； CS 可能是 BOR 中未确诊的缺陷，如果不及时治疗，CS 可能与颅骨变形有关。形状、颅内压升高和/或脑损伤。出生前和/或出生后的细胞（OPC）可能会通过颅缝骨沉积增加而导致 CS，在这个应用程序中，我计划解决有关 Six1 功能的主要知识差距：它在检测因 Six1 缺失和导致的骨骼发育变化？单倍体不足，我将使用 µCT 图像定量分析头部形态，并使用组织学分析（目标 1）验证 Six1 及其辅助因子是否具有规范和分化作用。对于 OPC，我将使用 RNAscope 和 qPCR 评估体内基因表达（目标 1），并使用神经网络评估体外基因表达最后，我将进行单细胞 RNA-seq 和 RNAscope 用于识别眶上弓间充质中的细胞群（这产生了顶骨和额骨）受到 Six1 丢失和单倍体不足的影响（目标 3）。应用将改变 Six1 作为颅板、神经和肌肉转录因子的功能范式提供第一个将其与正常颅骨发育和 CS 发病机制联系起来的直接证据。该应用将建立 Six1-het 小鼠作为颅面疾病的新模型，并将有助于阐明 Six1 变体通向 CS 的机制它还将提供软技能、资金和技术方面的培训。最后，这次培训将使我能够发挥我的广泛作用。对下颌骨和耳发育中 Six1 转录功能的了解进入临床相关的新领域经过研究，我的研究最终可能对 CS 患者的诊断和护理至关重要。