Mechanisms of Craniosynostosis in Bent Bone Dysplasia Syndrome

弯曲骨发育不良综合征颅缝早闭的机制

• 批准号: 10536399
• 负责人: Audrey Nickle
• 金额: $ 4.76万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10536399
• 关键词: ATAC-seq; Affect; Alleles; Animal Model; Atlases; Binding; Birth; Calvaria; Cell Lineage; Cell Nucleus; Cell membrane; Cells; ChIP-seq; Chromatin; Congenital Abnormality; Connective Tissue Cells; Craniosynostosis; Cre driver; Cultured Cells; Data; Data Set; Dense Connective Tissue; Development; Developmental Bone Diseases; Disease; Dura Mater; Embryo; Enhancers; Equilibrium; Fibroblast Growth Factor Receptor 2; Fibroblasts; Frontal bone structure; Gene Expression; Gene Expression Regulation; Gene Targeting; Genes; Genetic; Genomics; Histologic; Immunofluorescence Immunologic; In Situ Hybridization; In Vitro; Individual; Joints; Knock-in; Knowledge; Ligaments; Maintenance; Mediating; Membrane; Mesoderm; Missense Mutation; Molecular; Mus; Mutation; Neural Crest Cell; Nuclear; Operative Surgical Procedures; Osteoblasts; Osteogenesis; Pathogenesis; Pathway interactions; Pattern; Periosteum; Phenotype; Play; Population; Prevalence; Regulation; Role; Signal Transduction; Structure; Surgical sutures; Symptoms; Syndrome; TP53 gene; Testing; Therapeutic; Variant; WNT Signaling Pathway; XCL1 gene; base; bone; chromatin remodeling; coronal suture; craniofacial; cranium; differential expression; disease mechanisms study; gene repression; in silico; infancy; member; mouse model; mutant; novel; osteogenic; postnatal; premature; prevent; progenitor; promoter; single-cell RNA sequencing; skeletal disorder; stem cells; transcriptome sequencing

PROJECT SUMMARY Craniosynostosis, or the premature fusion of one or more suture joints in the skull, is the second most common congenital craniofacial birth defect with a prevalence of around 1/2500 births. Mutations to Fibroblast growth factor receptor 2 (FGFR2) are common genetic causes behind craniosynostosis syndromes, including the FGFR2M391R and the FGFR2Y381D variants which cause Bent Bone Dysplasia Syndrome (BBDS). Our lab has demonstrated that these mutations affect osteoblast fate via relocalization of Fgfr2 from the plasma membrane to the nucleus in vitro, but lack of an animal model has prevented the connection of these genetic and molecular changes to specific symptoms of BBDS such as craniosynostosis. We have developed a mouse model harboring a Cre-inducible knock-in of the Fgfr2M391R allele. Activating this mutation in neural crest cells (NCCs) via the Wnt1-Cre driver leads to progressive postnatal fusion of the frontal, coronal, sagittal, and lambdoid sutures. Both the sagittal and lambdoid sutures consist entirely of mesoderm-derived bone, however activation of the Fgfr2M391R allele via the Mesp1-Cre driver does not induct pan-suture craniosynostosis. Genetic lineage tracing of the embryonic mouse calvaria shows that NCCs give rise to the frontal bones, as well as the majority of non- osteogenic dense connective tissue at the sutures. This suggests that these previously uncharacterized non- osteogenic sutural cells are regulated by Fgfr2 and mechanistically involved in suture development and maintenance, as well as the pathogenesis of craniosynostosis in BBDS. Using a combination of genetic lineage tracing and single genomics, I will determine how expression of the Fgfr2M391R variant alters cell identity and contributions of dense connective tissues in the postnatal sutures. Among these cells are a population of recently observed osteofibrous progenitor cells which have yet to be fully characterized but are likely crucial to maintenance of postnatal suture patency. I will better resolve this population of cells and identify the role of Fgfr2 in maintaining the balance between their differentiation into osteoblasts vs. fibroblasts. I will also expand upon the knowledge of the nuclear role of mutant Fgfr2M391R and how it alters gene regulation within sutural cells to induce craniosynostosis. Due to its previously established role in altering chromatin remodeling and gene expression, I predict that Fgfr2M391R alters chromatin accessibility at additional gene targets. These may include members of the p53 and Wnt signaling pathways, which I find to be differentially expressed in postnatal sutures of our Wnt1-Cre; Fgfr2M391R/+ mouse model and have a known role in calvaria development and craniosynostosis. Using single nuclei ATAC-sequencing, I will determine how the Fgfr2M391R variant alters chromatin accessibility, cross referencing this dataset with differentially expressed genes from our bulk and single cell RNA-seq datasets. This will identify the function of nuclear Fgfr2 and novel signaling crosstalk necessary for regulating osteofibrous cells in the calvarial sutures. Completion of this study will advance our understanding of the connection between cavarial dense connective tissues, suture development, and the pathogenesis of craniosynostosis.

项目摘要 颅缝早闭，或一个或多个骨缝关节在头骨过早融合，是第二常见的 先天性颅面出生缺陷，患病率约为1/2500。成纤维细胞生长的突变 因子受体2（FGFR 2）是颅缝早闭综合征背后的常见遗传原因，包括 FGFR 2 M391 R和FGFR 2 Y381 D变异体引起弯曲骨发育不良综合征（BBDS）。我们的实验室 表明这些突变通过Fgfr 2从质膜的重新定位影响成骨细胞的命运 在体外细胞核，但缺乏动物模型，阻止了这些遗传和分子的连接， BBDS特定症状的变化，如颅缝早闭。我们开发了一种小鼠模型， Cre诱导的Fgfr 2 M391 R等位基因敲入。激活神经嵴细胞（NCC）中的这种突变， Wnt 1-Cre驱动器导致出生后额、冠、矢状和矢状缝的进行性融合。两 矢状缝和齿状缝完全由中胚层来源的骨组成，然而， Fgfr 2 M391 R等位基因通过Mesp 1-Cre驱动器不诱导全缝颅缝早闭。遗传谱系追踪 胚胎小鼠颅骨的研究表明，NCC产生了额骨，以及大多数非- 骨缝处的成骨致密结缔组织。这表明，这些以前没有特征的非- 成骨缝细胞受Fgfr 2调节，并在机制上参与缝的发育， 维持，以及BBDS中颅缝早闭的发病机制。利用遗传谱系 追踪和单基因组学，我将确定Fgfr 2 M391 R变体的表达如何改变细胞的身份， 致密结缔组织在出生后缝合中的作用。在这些细胞中， 观察到的骨纤维祖细胞尚未完全表征，但可能对 产后缝合通畅性的维持。我将更好地解决这一群体的细胞，并确定Fgfr 2的作用， 维持成骨细胞和成纤维细胞之间的平衡。我还将扩展 了解突变体Fgfr 2 M391 R的核作用以及它如何改变缝合细胞内的基因调控， 引起颅缝早闭。由于其先前确定的改变染色质重塑和基因表达的作用， 表达，我预测Fgfr 2 M391 R改变染色质可及性在其他基因的目标。这些可以包括 p53和Wnt信号通路的成员，我发现在出生后的缝合中差异表达 在我们的Wnt 1-Cre; Fgfr 2 M391 R/+小鼠模型中，具有已知的在颅骨发育和颅缝早闭中的作用。 使用单核ATAC测序，我将确定Fgfr 2 M391 R变体如何改变染色质可及性， 将该数据集与来自我们的批量和单细胞RNA-seq数据集的差异表达基因交叉引用。 这将确定核Fgfr 2的功能和调节骨纤维化所必需的新的信号串扰。 颅骨缝中的细胞这项研究的完成将促进我们对 颅窝致密结缔组织、骨缝发育和颅缝早闭的发病机制。