Cranial suture formation in the zebrafish

描述（由申请人提供）：颅缝闭锁是一种重要的健康问题，发生在超过1/2500的活产婴儿中。目前的外科治疗是不完善的，更好地了解颅缝线形成的分子、遗传和细胞基础将导致改进的治疗策略。我们对缝线形成的遗传调控的理解在很大程度上来自于对导致颅缝闭合或缝线形成中其他缺陷的人类突变的鉴定。特别是，TWIST1单倍体功能不全导致颅缝闭合，转录因子RUNX2导致缝合延迟和不完全闭合，这表明颅骨和缝合形成过程对调节成骨细胞分化的基因的剂量很敏感。斑马鱼已被证明是研究骨骼形成的一个有价值的模型系统，与哺乳动物的过程有很大的相似之处。我们提出的实验将通过三个特定的目的来推进斑马鱼在颅骨和缝合线形成研究中的应用。首先，我们将通过茜素红骨染色分析正常颅骨穹窿生长过程，并生成正常颅骨生长的定量形态学描述。我们还将通过分析不同分化阶段成骨细胞的基因表达模式和使用表达荧光标记基因的转基因细胞系，在分子水平上表征颅骨和缝合线的生长。在第二个目标中，我们将通过确定神经嵴对缝合线和颅骨的动态贡献，使用遗传标记来不可磨灭地标记神经嵴细胞及其后代，来验证神经嵴细胞的瞬时群体在缝合线图案中发挥重要作用的假设。最后，我们将利用在前两个目标中获得的信息来指导我们进一步表征斑马鱼骨基因的突变体，该突变体在颅骨和缝合线形成方面表现出明显的缺陷。我们的最终目标是通过正向遗传学鉴定更多的斑马鱼突变体；我们对颅骨和缝合形成的正常过程的描述将为确定突变表型的基础提供必要的知识基础。我们的工作也将增加斑马鱼作为一个强大的模型系统的效用，有助于我们理解颅缝闭合和其他后期颅骨发育缺陷，并对脊椎动物颅骨和缝合形成的细胞和分子过程有更深入的了解。

DESCRIPTION (provided by applicant): Craniosynostosis is a significant health problem, occurring in more than 1/2500 live births. Current surgical treatments are imperfect, and a better understanding of the molecular, genetic, and cellular basis of cranial suture formation would lead to improved treatment strategies. Our understanding of the genetic regulation of suture formation has come in large part from identification of human mutations leading either to craniosynostosis or other defects in suture formation. In particular, the fact that haploinsufficiency for TWIST1 leads to craniosynostosis, and for the transcription factor RUNX2 to delayed and incomplete suture closure, demonstrates that the processes of skull and suture formation are sensitive to dosage of genes regulating osteoblast differentiation. The zebrafish has proven a valuable model system for the study of skeletogenesis, with substantial parallels to the processes in mammals. Our proposed experiments will advance the use of zebrafish for the study of skull bone and suture formation through three Specific Aims. First, we will analyze the normal process of cranial vault growth by Alizarin Red bone staining, and generate a quantitative morphometric description of normal skull growth. We will also characterize skull and suture growth at a molecular level, through analysis of gene expression patterns and the use of transgenic lines that express fluorescent marker genes in osteoblasts at different stages of differentiation. In the second aim, we will test the hypothesis that a transient population of neural crest cells play an important role in patterning the sutures by determining the dynamic contribution of neural crest to the sutures and skull bones, using genetic labeling to indelibly mark neural crest cells and their descendents. Finally, we will use the information obtained in the first two aims to guide us in further characterizing a mutant in the zebrafish osterix gene, which displays striking defects in skull and suture formation. We aim eventually to identify additional zebrafish mutants through forward genetics; our characterization of the normal processes of skull and suture formation will provide the necessary foundation of knowledge to determine the basis of the mutant phenotypes. Our work will also increase the utility of zebrafish as a powerful model system to contribute to our understanding of craniosynostosis and other defects in later skull development, and yield greater insights into the cellular and molecular processes of skull and suture formation shared among vertebrates. PUBLIC HEALTH RELEVANCE: Craniosynostosis and other defects in formation of cranial sutures represent a significant health problem, and current surgical treatments are imperfect. To better understand the processes leading to abnormal suture development, we will be characterizing the zebrafish as a novel genetic model system for skull bone and suture formation.