Cranial suture formation in the zebrafish

描述(由申请人提供)：颅缝早闭是一个严重的健康问题，发生在超过1/2500的活产儿中。目前的手术治疗是不完善的，更好地了解颅缝形成的分子、遗传和细胞基础将导致改进的治疗策略。我们对缝合形成的遗传规律的了解在很大程度上来自于对人类突变的鉴定，这些突变导致了颅缝融合或缝合形成的其他缺陷。特别是，Twist1基因单倍体不足导致颅缝融合，转录因子RUNX2延迟缝合和不完全缝合的事实表明，颅骨和缝合线的形成过程对调控成骨细胞分化的基因剂量敏感。斑马鱼已被证明是研究骨骼发生的一个有价值的模型系统，其过程与哺乳动物的过程基本相似。我们提出的实验将通过三个特定的目标推进斑马鱼用于头盖骨和缝合形成的研究。首先，我们将通过茜素红骨染色分析正常颅顶生长的过程，并生成正常颅骨生长的定量形态描述。我们还将通过基因表达模式的分析和在不同分化阶段的成骨细胞中表达荧光标记基因的转基因株的使用，在分子水平上表征颅骨和缝合的生长。在第二个目标中，我们将通过确定神经脊对缝合和颅骨的动态贡献，使用基因标记对神经脊细胞及其后代进行不可磨灭的标记，来检验这一假设，即神经脊细胞的瞬时群体在缝合图案形成中发挥重要作用。最后，我们将使用在前两个目的中获得的信息来指导我们进一步描述斑马鱼osterix基因的突变，该突变在头骨和缝合形成中显示出显著的缺陷。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；我们对头骨和骨缝形成的正常过程的描述将提供必要的知识基础，以确定突变表型的基础。我们的工作还将增加斑马鱼作为一个强大的模型系统的实用性，有助于我们了解颅突融合和其他颅骨发育缺陷，并对脊椎动物共有的颅骨和缝合形成的细胞和分子过程有更深入的了解。 公共卫生相关性：颅缝形成中的颅缝融合和其他缺陷是一个重大的健康问题，目前的手术治疗还不完善。为了更好地理解导致异常缝合发育的过程，我们将斑马鱼描述为一种新的颅骨和缝合形成的遗传模型系统。

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.