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

• 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
• 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

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.

摘要 转录因子SIX 1或其辅因子EYA 1的变体是已知的潜在遗传原因， 鳃-耳-肾综合征（BOR）是一种常染色体显性遗传疾病，可导致听力损失和肾脏损害。 缺陷最近，一项临床研究报告了携带SIX 1 BOR变体的个体中的颅缝早闭（CS）， 包括预测导致单倍不足的5'变异体（p.Q11X和p.Q22X）;这些数据表明 CS可能是BOR中未诊断的缺陷。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。作为颅骨骨祖细胞的缺陷 出生前和/或出生后的骨细胞（OPC）可能通过增加颅缝中的骨沉积而导致CS， 在这个应用程序中，我计划解决关于Six 1功能的一个主要知识缺口：它在 颅骨的发育检测Six 1缺失引起的骨发育变化， 单倍不足，我将使用µCT图像定量分析头部形态，并使用 组织学分析（目的1）。为了验证Six 1及其辅助因子是否在规范和区分中起作用， 在OPCs中，我将使用RNAscope和qPCR（目的1）评估体内基因表达，并使用神经元 嵴源性间充质前体和OPCs（Aim 2）。最后，我将进行单细胞RNA-seq， RNA显微镜鉴定眶上弓间充质中的细胞群（这产生了 顶骨和额骨）受Six 1丢失和单倍不足影响（目的3）。结果从这个 应用将改变Six 1作为颅基板、神经和肌肉转录因子的功能范式 通过提供第一个直接证据，将其与正常颅骨发育和CS发病机制联系起来。 该应用将建立Six 1-het小鼠作为颅面疾病的新模型，并将有助于阐明 Six 1变体导致CS的机制。它还将提供软技能、资金和 我的下一个职业发展阶段所需要的合作。最后，这次培训将使我能够把我广泛的 Six 1基因在下颌骨和耳发育中转录功能研究进展 research.因此，我的研究可能最终被证明对CS患者的诊断和护理至关重要。