Cranial suture formation in the zebrafish

描述（由申请人提供）：颅缝早闭是一个重大的健康问题，发生在超过1/2500活产。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。我们对骨缝形成的遗传调控的理解很大程度上来自于对导致颅缝早闭或其他骨缝形成缺陷的人类突变的鉴定。特别是，事实上，单倍不足TWIST 1导致颅缝早闭，和转录因子RUNX 2延迟和不完全的缝合关闭，表明颅骨和缝形成的过程是敏感的基因调节成骨细胞分化的剂量。斑马鱼已被证明是骨骼发生研究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标推进斑马鱼在头骨和骨缝形成研究中的应用。首先，我们将通过茜素红骨染色来分析颅顶生长的正常过程，并生成正常颅骨生长的定量形态测量描述。我们还将通过分析基因表达模式和使用在成骨细胞分化的不同阶段表达荧光标记基因的转基因系，在分子水平上表征颅骨和骨缝的生长。在第二个目标中，我们将测试的假设，即一个短暂的人口神经嵴细胞发挥了重要作用，在图案化的缝合线，通过确定的动态贡献的神经嵴的缝合线和头骨，使用遗传标记，不可磨灭地标记神经嵴细胞及其后代。最后，我们将使用前两个目的中获得的信息来指导我们进一步表征斑马鱼osterix基因的突变体，该突变体在头骨和缝合形成中显示出惊人的缺陷。我们的目标是最终通过正向遗传学来确定其他斑马鱼突变体;我们对头骨和缝线形成的正常过程的表征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实用性，有助于我们理解颅缝早闭和其他缺陷，在以后的头骨发育，并产生更大的见解头骨和骨缝形成的细胞和分子过程中共享的脊椎动物。 公共卫生相关性：颅缝早闭和其他颅缝形成缺陷是一个严重的健康问题，目前的手术治疗是不完善的。为了更好地了解导致缝线异常发育的过程，我们将把斑马鱼描述为头骨和缝线形成的新型遗传模型系统。

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.