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

摘要 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 布兰奇奥-耳-肾综合征(BOR)，一种常染色体显性遗传病，导致听力损失和肾脏 缺陷。最近，一项临床研究报告了携带SIX1 Bor变异体的个体患有颅缝早闭(CS)。 包括预测会导致单倍体不足的5‘变异体(p.Q11X和p.Q22X)；这些数据表明 CS可能是BOR的一种未诊断的缺陷。如果不进行治疗，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/或出生后的细胞(OPC)可能通过增加颅缝中的骨沉积而导致CS 这个应用程序，我计划解决关于SIX1功能的一个主要知识缺口：它在 头盖骨的发育？检测SIX1缺失和SIX1缺失引起的骨骼发育变化 单倍体功能不全，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)。为了验证SIX1及其辅助因子是否具有规范和区分的作用 对于OPC，我将使用RNAScope和qPCR(AIM 1)在体内评估基因表达，并使用神经网络在体外评估基因表达 CREST来源的间充质前体和OPC(目标2)。最后，我将进行单细胞RNA-seq和 RNAScope识别眶上弓间充质中的细胞群(这产生了 顶骨和额骨)，受SIX1缺失和单倍体功能不全影响(目标3)。由此产生的结果 应用将改变SIX1作为颅骨胎盘、神经和肌肉转录因子的功能范式 通过提供将其与正常颅骨发育和CS的发病机制联系起来的第一个直接证据。 这项应用将建立SIX1-HET小鼠作为头面部疾病的新模型，并将有助于阐明 SIX1变异导致CS的机制。它还将提供软技能、资金和 我的下一步事业需要合作。最后，这次培训将让我把我广泛的 对SIX1转录功能的认识在下颌骨和耳部发育到临床相关的新领域 研究。因此，我的研究最终可能被证明对CS患者的诊断和护理至关重要。

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.