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.

抽象的 转录因子Six1或其共同因素EYA1中的变体是已知的基本遗传原因 分支 - 局部肾脏综合征（BOR），一种常染色体显性疾病，导致听力损失和肾脏 缺陷。最近，一项临床研究报道了携带61个BOR变体的个体颅突变（CS）， 包括5'变体（P.Q11X和P.Q22X），预计会导致单倍不足；这些数据暗示 该CS可能是BOR中的一个单位缺陷。如果未治疗，CS可以与头骨的失真相关联 形状，颅内压增加和/或脑损伤。如钙典写的骨骨基因生激素的缺陷 细胞（OPC）在出生之前和/或之后可能通过颅骨缝合线中的骨沉积增加而导致CS 我计划解决有关Six1功能的主要知识差距：它在该功能中的作用是什么 颅骨的发育？检测由六1损失引起的骨骼发育变化 单倍不足，我将使用µCT图像和组织形成进行定量分析头部形态 组织学分析（AIM 1）。验证Six1及其共同因素是否具有指定和分化的作用 在OPC中，我将使用RNASCOPE和QPCR（AIM 1）在体内评估基因表达（AIM 1），并在体外使用中性 波峰衍生的间质前体和OPC（AIM 2）。最后，我将执行单细胞RNA-seq和 rnascope鉴定上骨上质源中的细胞群体（这引起了基础 受六1损失和单倍不足的影响的顶骨和额骨）（AIM 3）。结果 应用将移动Six1的范式作为颅骨，神经和肌肉转录因子的功能 通过提供第一个直接证据，将其与正常的颅骨发育和CS的发病机理联系起来。 该应用将建立61助小鼠作为颅面疾病的新模型，并有助于阐明 Six1变体导致CS的机制。它还将提供软技能，资金和资金的培训 我的下一个职业步骤需要合作。最后，这项培训将使我能够带来广泛的 在下颌骨和耳效中了解六1个转录功能的知识 研究。因此，我的研究最终可能对CS患者诊断和护理至关重要。