Coronal Suture Development in Health and Disease

冠状缝在健康和疾病方面的发展

• 批准号: 10491859
• 负责人: Greg Peter Holmes
• 金额: $ 78.52万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-20 至 2026-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10491859
• 关键词: Affect; Age; Apert syndrome; Biological Assay; Calvaria; Cells; Chotzen Syndrome; Complex; Craniosynostosis; Defect; Development; Disease; Embryo; Embryonic Development; Erinaceidae; FGFR2 gene; Frontal bone structure; Gene Expression; Gene Expression Profile; Genes; Genetic Transcription; Goals; Growth; Health; Histologic; Human; Human Pathology; Impairment; In Vitro; Joints; Knock-out; Label; Ligands; Location; Maintenance; Maps; Mesenchyme; Mesoderm; Modeling; Molecular; Mus; Mutate; Mutation; Neural Crest; Neurologic; Operative Surgical Procedures; Osteoblasts; Osteogenesis; Parietal bone structure; Pathogenesis; Pathway interactions; Perinatal; Phenotype; Population; Population Study; Process; Proliferating; Role; Site; Source; Surgical sutures; Syndrome; TWIST1 gene; Tamoxifen; Techniques; Time; Tissues; Wild Type Mouse; bone; bone repair; cell type; coronal suture; craniofacial development; cranium; early embryonic stage; experimental study; genetic manipulation; in vivo; inhibitor; insight; migration; mouse model; mutant; mutant mouse model; novel; osteogenic; osteoprogenitor cell; postnatal; premature; preservation; prevent; programs; receptor; single-cell RNA sequencing; skeletal stem cell; smoothened signaling pathway; spatiotemporal; stem cell niche; stem cell population; stem cells; suture fusion; transcriptome sequencing; transcriptomics

ABSTRACT Skull development requires the proper function of sutures, comprised of the edges of adjacent bones (osteogenic fronts, OFs) and intervening suture mesenchyme (SM). Suturogenesis is a complex and intriguing process, particularly for the coronal suture between the frontal and parietal bones of the calvaria. Coronal suturogenesis involves the migration of osteoprogenitors through ectocranial mesenchyme to the OFs at early embryonic stages and maintenance of the SM to preserve an open suture that develops a stem cell niche postnatally. Misregulation of suturogenesis is a significant source of human pathology, such as wider sutures or craniosynostosis (CRS), the premature fusion of sutures. CRS can adversely affect neurological development and requires corrective surgery. The coronal suture is the most frequently fused suture in syndromic CRS, which is generally thought to result from an imbalance between osteoprogenitor induction, proliferation, and differentiation. The cell populations involved are poorly defined in terms of their transcriptional signatures and spatial organization, hindering understanding of the effects of genes mutated in coronal CRS. Understanding the pathogenesis of CRS has positive implications for human health in providing insight toward treating dysgenesis. In this proposal we will analyze murine coronal suturogenesis in three increasingly focused Aims: 1) define the organization of transcriptionally-characterized cell populations; 2) establish the roles of a newly identified SM population; and 3) investigate the role of the hedgehog (HH) signaling pathway that when altered can result in CRS in humans and mice. For Aim 1, we will generate and integrate spatial transcriptomics and single-cell (sc)RNA-seq analyses between embryonic day (E)12.5-E18.5 in wild type (WT) mice and two models of syndromic coronal CRS. From this, we will generate a detailed 4D representation of the transcriptional signatures and spatial organization of cell populations and identify potential mechanisms of dysgenesis in coronal CRS. For Aim 2, by scRNA-seq analysis we have identified a discreet population within the SM that separates the overlapping frontal and parietal bones of the coronal suture. We will use targeted genetic manipulations to ablate and fate map this population, and perform lineage differentiation and calvarial defect assays. These experiments will determine the function of this SM population as a barrier to prevent fusion of adjacent bones and their potential contribution to suture stem cells. For Aim 3, we have identified a novel coronal suture phenotype in mutants of Hhip, a HH inhibitor highly expressed in the SM population studied in Aim 2. We will characterize the Hhip-/- phenotype and combine targeted genetic manipulations of the HH pathway in the SM population with RNA-seq analysis to comprehensively determine the roles of HH signaling at the level of both transcriptional programs and cell populations. We will fate map a potential osteoprogenitor population adjacent to the OFs and regulated by HH signaling. This project will significantly advance our understanding of coronal suturogenesis and provide insight into disease mechanisms affecting craniofacial development.

抽象的 头骨发育需要缝合线的适当功能，缝合线由相邻骨骼的边缘组成（成骨 前沿，OF）和介入缝线间充质（SM）。缝合发生是一个复杂而有趣的过程， 特别是对于颅骨额骨和顶骨之间的冠状缝合。冠状缝发生 涉及骨祖细胞在胚胎早期通过颅外间充质迁移至 OFs SM 的阶段和维护，以保留开放的缝合线，从而在出生后形成干细胞生态位。 缝合线发生的失调是人类病理学的一个重要根源，例如较宽的缝合线或 颅缝早闭（CRS），缝线过早融合。 CRS会对神经发育产生不利影响 并且需要进行矫正手术。冠状缝是综合征型 CRS 中最常见的融合缝合线， 一般认为是由于骨祖细胞诱导、增殖和骨祖细胞之间不平衡造成的。 差异化。所涉及的细胞群在转录特征方面的定义很差，并且 空间组织，阻碍了对冠状 CRS 中基因突变影响的理解。了解 CRS 的发病机制对于人类健康具有积极的影响，为治疗发育不全提供了见解。 在本提案中，我们将分析小鼠冠状缝发生的三个日益集中的目标：1）定义 转录特征细胞群的组织； 2）建立新确定的SM的角色 人口; 3) 研究刺猬 (HH) 信号通路的作用，该通路改变时会导致 人类和小鼠的 CRS。对于目标 1，我们将生成并整合空间转录组学和单细胞 (sc)野生型 (WT) 小鼠胚胎日 (E)12.5-E18.5 和两种模型之间的 RNA-seq 分析 综合征性冠状CRS。由此，我们将生成转录签名的详细 4D 表示 细胞群的空间组织和空间组织，并确定冠状 CRS 发育不全的潜在机制。为了 目标 2，通过 scRNA-seq 分析，我们确定了 SM 中的一个离散群体，该群体将 冠状缝的额骨和顶骨重叠。我们将使用有针对性的基因操作来消除 绘制该群体的命运图谱，并进行谱系分化和颅骨缺陷测定。这些实验 将确定该 SM 群体作为防止相邻骨骼及其融合的屏障的功能 对缝合干细胞的潜在贡献。对于目标 3，我们发现了一种新的冠状缝表型 Hhip 的突变体，一种 HH 抑制剂，在目标 2 中研究的 SM 群体中高度表达。我们将表征 Hhip-/- 表型，并将 SM 群体中 HH 途径的靶向遗传操作与 RNA-seq 分析可全面确定 HH 信号在转录水平上的作用 程序和细胞群。我们将绘制邻近 OF 的潜在骨祖细胞群的命运图， 受 HH 信号调节。该项目将显着增进我们对冠状缝发生的理解 并深入了解影响颅面发育的疾病机制。