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 颅骨发育需要缝合线的正确功能，由相邻骨骼的边缘组成（成骨 正面，Ofs）和干预缝合缝合体（SM）。 Suturogeny是一个复杂而有趣的过程， 特别是对于瓦尔瓦里亚的额叶骨头和顶骨之间的冠状缝合线。冠状动脉发生 在早期胚胎中，涉及骨基因生成剂通过外颅间质迁移到OFS SM的阶段和维护，以保留出现后出现干细胞生态位的开放缝合线。 神经发生的正调是人类病理学的重要来源，例如更宽的缝合线或 颅骨突变（CRS），缝合线的过早融合。 CRS会对神经系统发展产生不利影响 并且需要纠正手术。冠状缝合线是综合症CR中最常见的缝合线， 通常认为是由于破骨剂诱导，增殖和 分化。所涉及的细胞群体的转录特征和 空间组织，阻碍了对冠状CR中突变基因的影响的理解。了解 CRS的发病机理对人类健康具有积极的影响，以提供治疗失调的洞察力。 在该提案中，我们将分析三个越来越重点的目的的鼠冠状动脉缝合发生：1）定义 转录特征的细胞群体的组织； 2）确定新鉴定的SM的角色 人口; 3）研究刺猬（HH）信号通路的作用 人类和老鼠的CR。对于AIM 1，我们将生成和集成空间转录组和单细胞 （SC）胚胎日（E）12.5-E18.5之间的RNA-seq分析在野生型（WT）小鼠中 综合征冠状动脉CR。由此，我们将生成转录签名的详细4D表示 以及细胞群体的空间组织，并确定冠状动脉CR中失去障碍的潜在机制。为了 AIM 2，通过SCRNA-SEQ分析，我们已经确定了SM内的谨慎群体 冠状缝合线的额叶和顶骨重叠。我们将使用有针对性的遗传操纵来消融 命运映射该人群，并执行谱系分化和钙缺损测定法。这些实验 将确定SM人群的功能，以防止相邻骨头融合及其 对缝合干细胞的潜在贡献。对于AIM 3，我们已经确定了一种新型的冠状缝合表型 Hop的突变体是在AIM 2中研究的SM种群中高度表达的HH抑制剂。我们将表征 HHIP - / - 表型和组合SM种群中HH途径的靶向遗传操作 RNA-seq分析以全面确定HH信号在两个转录水平上的作用 程序和细胞种群。我们将命运与OFS相邻的潜在骨基因生成剂量绘制 由HH信号调节。该项目将大大提高我们对冠状动脉发生的理解 并洞悉影响颅面发育的疾病机制。