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

描述（由申请人提供）：颅缝融合的正确模式和时间对于正常颅骨发育至关重要。骨缝形成缺陷导致畸形和变形，导致颅骨收缩并损害认知能力。我们的知识的机制基础，区分正常和异常缝发展是不完整的。斑马鱼是研究脊椎动物骨骼发育基本过程的一种强有力的遗传和实验模型。我们已经在斑马鱼中进行了正向遗传筛选，以识别与人类患者中发生的缺陷相关的缝线形成缺陷的成鱼。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（bmp 1a）和整合素<$10（itg <$10）作为缝线形成过程中的重要参与者。鉴定参与细胞外基质（ECM）蛋白的翻译后修饰和ECM与细胞之间发生的信号传导的基因表明ECM和ECM调节的细胞信号传导的变化可能包括异常缝合发育/闭合的许多遗传和环境原因的共享下游途径。本提案的目的是研究ECM翻译后修饰的作用，特别是Bmp 1蛋白酶在正常和病理性缝线形成过程中的作用。我们将通过完成三个目标来实现这些目标。在目的1中，我们将确定新的遗传调节缝线形成的突变体缺陷BMP 1A筛选BMP 1A功能的显性修饰。我们的初步数据表明，这是一个高生产力的战略。在目的2中，我们将表征受损的Bmp 1活性对斑马鱼中的缝线ECM蛋白的影响，并确定ECM蛋白的改变修饰是否也发生在人类患者的骨结合中。我们可以从已知的综合征和未知原因的骨性结合患者中手术切除受影响的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筛选中有效利用的能力。我们将专门使用bmp 1a突变鱼来鉴定改变体内Bmp 1活性的小分子。然后可以使用局部化学浸渍的微珠植入测定法测试影响在bmp 1a突变鱼中观察到的最早表型的分子（改变的鳍折叠）在晚发型表型（骨结合）中的治疗功效。斑马鱼模型的优势在于，它能够使用正向遗传学方法有效识别对缝合线形成重要的基因和途径。在斑马鱼中发现的影响骨缝的基因可以在哺乳动物的骨结合模型中进行研究。斑马鱼还可用于高通量体内筛选可抑制或增强缝线融合的小分子化合物。改变Bmp 1a突变斑马鱼有害表型的化合物可能对骨结合或BMP 1相关表型患者具有治疗价值。

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.