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The regulatory role of Bone morphogenetic protein 1 in suture patency and fusion

骨形态发生蛋白1在缝线通畅和融合中的调节作用

基本信息

批准号：
8733243
负责人：
Matthew P Harris
金额：
$ 35万
依托单位：
BOSTON CHILDREN'S HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-09-19 至 2014-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8733243
关键词：
Address Adult Affect Alleles Biochemical Biological Assay Bone Morphogenetic Proteins Chemicals Congenital abnormal Synostosis Craniosynostosis Data Defect Development Disease Dysmorphology Educational process of instructing Etiology Experimental Models Extracellular Matrix Extracellular Matrix Proteins Fishes Generations Genes Genetic Genetic Models Genetic Screening Goals Human Impaired cognition Integrins Joint structure of suture of skull Knowledge Lead Live Birth Maintenance Maps Mental Retardation Methods Microspheres Modeling Modification Molecular Genetics Mus Mutation Organism Participant Pathologic Pathology Pathway interactions Patients Pattern Peptide Hydrolases Phenotype Post-Translational Protein Processing Post-Translational Regulation Process Regulation Role Signal Transduction Skeletal Development Surgical sutures Testing Therapeutic Time Tissues Treatment Efficacy Vertebrates Welding Zebrafish base constriction cranium design implantation improved in vivo insight malformation mouse model mutant new therapeutic target novel null mutation procollagen C-endopeptidase public health relevance research study skeletal small molecule suture fusion treatment strategy

项目摘要

DESCRIPTION (provided by applicant): The correct patterning and timing of the fusion of cranial sutures is essential for normal skull development. Defects in suture formation lead to malformations and deformations, which cause constriction of the cranium and can impair cognition. Our knowledge of the mechanistic bases that distinguish normal from abnormal suture development is incomplete. The zebrafish is a powerful genetic and experimental model for exploring fundamental processes in vertebrate skeletal development. We have performed forward genetic screens in zebrafish to identify adult fish with defects in suture formation that are relevant to defects occurring in human patients. To efficiently map and clone the responsible genes, we developed new massively parallel sequencing and analytic strategies. This enabled us to identify bone morphogenetic protein 1a (bmp1a) and integrin ¿10 (itg¿10) as important participants during suture formation. Identifying genes involved in the post- translational modification of extracellular matrix (ECM) proteins and in the signaling that occurs between the ECM and the cell suggests changes in ECM and ECM-regulated cell signaling may comprise a shared downstream pathway for many genetic and environmental causes of abnormal suture development/closure. The goal of this proposal is to investigate the role of ECM post-translational modification and, specifically, the role of Bmp1 protease during normal and pathological suture formation. We will achieve these goals by completing 3 Aims. In Aim 1 we will identify new genetic regulators of suture formation by using mutants deficient in bmp1a to screen for dominant modifiers of Bmp1a function. Our preliminary data indicate this is a highly productive strategy. In Aim 2 we will characterize the effects of impaired Bmp1 activity on suture ECM proteins in zebrafish and determine whether altered modification of ECM proteins also occurs in synostoses from human patients. We have access to surgically excised affected and adjacent normal suture tissue from patients with known syndromic and unknown causes of synostosis. In Aim 3 we will capitalize on the zebrafish model's ability to be effectively utilizedin small molecule screens. We will specifically use bmp1a mutant fish to identify small molecules that alter Bmp1 activity in vivo. Molecules that affect the earliest phenotype observed in bmp1a mutant fish (altered finfolds) can then be tested for therapeutic efficacy in the late onset-phenotype (synostosis) using localized chemical- impregnated microbead implantation assays. The strength of the zebrafish model is that it enables the efficient identification of genes and pathways important for suture formation using forward genetic methods. Genes found to affect sutures in the zebrafish can then be studied in mammalian models of synostosis. Zebrafish are also useful for high-throughput in vivo screens for small molecule compounds that can inhibit or potentiate suture fusion. Compounds that alter deleterious phenotypes in zebrafish with Bmp1a mutations may have therapeutic value in patients with synostosis or with BMP1-associated phenotypes.

描述（由申请人提供）：颅缝融合的正确模式和时间对正常颅骨发育至关重要。缝线形成的缺陷会导致畸形和变形，从而导致颅骨收缩并损害认知能力。我们对区分正常和异常缝线发育的机制基础的了解是不完整的。斑马鱼是一个强大的遗传和实验模型，用于探索脊椎动物骨骼发育的基本过程。我们在斑马鱼中进行了前向遗传筛选，以识别与人类患者发生的缺陷相关的缝线形成缺陷的成年鱼。为了有效地定位和克隆相关基因，我们开发了新的大规模并行测序和分析策略。这使我们能够确定骨形态发生蛋白1a （bmp1a）和整合素¿10 （itg¿10）是缝合形成过程中的重要参与者。鉴定参与细胞外基质（ECM）蛋白翻译后修饰和ECM与细胞之间信号传递的基因表明，ECM和ECM调节的细胞信号传递的变化可能包含一个共享的下游途径，这是导致异常缝线发育/闭合的许多遗传和环境原因。本提案的目的是研究ECM翻译后修饰的作用，特别是Bmp1蛋白酶在正常和病理缝合形成中的作用。我们将通过完成三个目标来实现这些目标。在Aim 1中，我们将通过使用bmp1a缺陷突变体筛选bmp1a功能的显性修饰因子来鉴定新的缝线形成的遗传调节因子。我们的初步数据表明，这是一个高效的策略。在Aim 2中，我们将描述Bmp1活性受损对斑马鱼缝合线ECM蛋白的影响，并确定ECM蛋白修饰的改变是否也发生在人类滑膜紧闭患者中。我们可以从已知综合征和未知原因的结膜紧闭患者那里获得手术切除的受影响和邻近的正常缝合组织。在目标3中，我们将利用斑马鱼模型在小分子屏幕中有效利用的能力。我们将专门使用bmp1a突变鱼来鉴定体内改变Bmp1活性的小分子。在bmp1a突变鱼中观察到的影响早期表型的分子（鳍折叠改变），然后可以使用局部化学浸渍微珠植入试验来测试对晚期发病表型（关节断裂）的治疗效果。斑马鱼模型的优势在于，它能够使用正向遗传方法有效地识别对缝线形成重要的基因和途径。在斑马鱼身上发现的影响缝合线的基因，可以在哺乳动物的关节闭锁模型中进行研究。斑马鱼也可用于高通量体内筛选可抑制或增强缝合线融合的小分子化合物。改变具有Bmp1a突变的斑马鱼有害表型的化合物可能对关节闭锁或bmp1相关表型的患者具有治疗价值。