Screen for mutations affecting skull and suture formation in zebrafish

筛选影响斑马鱼头骨和缝合线形成的突变

• 批准号: 8546656
• 负责人: Shannon Fisher
• 金额: $ 40万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-17 至 2018-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8546656
• 关键词: Adolescent; Adult; Affect; Animal Model; Animals; Biological Assay; Biological Models; Biological Process; Biology; Bone Matrix; Brain; Cartilage; Cataloging; Catalogs; Cell Differentiation process; Cell physiology; Cells; Cephalic; Cerebrovascular Circulation; Chondrocytes; Clinical; Collection; Communities; Craniosynostosis; Defect; Development; Disease; Elements; Ethylnitrosourea; Event; Fertilization; Fishes; Gene Expression; Genes; Genetic; Genetic Complementation Test; Genetic Screening; Genome; Growth; Growth and Development function; Head; Health; Hearing; High Prevalence; Human; Image; Individual; Intracranial Hypertension; Joint structure of suture of skull; Joints; Knowledge; Lead; Life; Literature; Live Birth; Maps; Meiosis; Metabolism; Molecular; Molecular Genetics; Morphogenesis; Movement; Mutate; Mutation; Operative Surgical Procedures; Organ; Osteoblasts; Osteoclasts; Osteogenesis; Pathway interactions; Pattern; Phenotype; Prevention; Process; Psyche structure; Regulation; Resources; Role; Shapes; Signal Transduction; Skeletal Development; Skeleton; Staging; Staining method; Stains; Surgical sutures; System; Time; Transgenes; Transgenic Organisms; Vision; Zebrafish; base; bone; cell type; clinically relevant; craniofacial; cranium; disability; emerging adult; flat bone; gene cloning; in vivo; male; mutant; next generation sequencing; offspring; premature; public health relevance; research study; skeletal; skeletal abnormality; suture fusion

DESCRIPTION (provided by applicant): The closure of cranial sutures is a key process during skull morphogenesis. At the edges of the flat bones, which are centers of regulated bone formation, fibrous sutures unite the individual bones. The sutures allow for further growth and movement of individual bones as the brain grows; once the neurocranium is fully grown, the sutures fuse, in early adulthood in humans. Importantly, patency of the sutures and brain development are intimately connected. Premature closure of the sutures, craniosynostosis, results in abnormal head shape and is associated with increased intracranial pressure, impaired cerebral blood flow, impaired vision and hearing, and mental disabilities. Despite their prominent roles in skull growth and brain development, the molecular and cellular mechanisms by which sutures form, how they interact with the skull bones to regulate growth, and the signals that induce suture fusion are not well understood. Much of our current knowledge of the regulation of suture formation comes from the identification of only a few of the many human mutations leading either to craniosynostosis or other defects in suture formation. Here I propose to harness the power of the zebrafish system for genetic and live imaging studies of suture formation. Skull and suture formation are relatively late events in the zebrafish, and there is almost nothing in the literature about these processes. While several large-scale mutant screens have revealed key pathways in patterning of the craniofacial skeleton up to six days of development, the later processes of skull and suture formation, at ~4-6 weeks, have not been assayed in any large-scale screen. Zebrafish mutants with specific defects in late skull and suture formation have not been identified; therefore, we propose a genetic screen to isolate and characterize such mutants. First, we will conduct a forward genetic screen of 1000 ENU-mutagenized genomes by examining F3 offspring of ENU- mutagenized males at 6 weeks, the time when skull closure is being completed in wild-type fish. In an ongoing pilot screen of 330 genomes, we have identified 12 mutants with specific defects in skull and suture formation and other late aspects of skeletal development. Based on these numbers, we anticipate identifying a total of ~30 specific mutants. Second, we will take advantage of a large collection of transgenic lines available in our lab to characterize the mutant phenotypes further, classifying them according to cell types and processes affected. Finally, we will map most if not all of the mutants to intervals of <3 Mb. Based on the phenotypic characterization, a subset of 8-10 mutants will be cloned to identify the mutated gene. Combined, the proposed experiments will establish a collection of well-characterized zebrafish mutations affecting many aspects of later skeletal development, in particular those with very specific defects in skull and suture formation, thereby establishing zebrafish as a powerful model system for the study of these clinically relevant later aspects of skull formation. Importantly, current surgical treatments for craniosynostosis and other skull defects are limited, and a detailed understanding of the underlying biology will lead t better approaches for treatment and prevention.

描述（由申请方提供）：颅缝闭合是颅骨形态发生过程中的关键过程。在扁骨的边缘，也就是有规律的骨形成的中心，纤维缝合线将各个骨连接起来。随着大脑的生长，缝合线允许个体骨骼进一步生长和移动;一旦脑颅完全生长，缝合线就会融合，在人类成年早期。重要的是，缝合线的通畅性和大脑发育密切相关。缝线过早闭合，即颅缝早闭，导致头部形状异常，并与颅内压升高、脑血流受损、视力和听力受损以及精神残疾相关。尽管它们在颅骨生长和大脑发育中发挥着重要作用，但缝形成的分子和细胞机制，它们如何与颅骨相互作用以调节生长，以及诱导缝融合的信号还没有得到很好的理解。我们目前对骨缝形成调控的认识，大部分来自于对导致颅缝早闭或骨缝形成其他缺陷的许多人类突变中的少数几种的鉴定。在这里，我建议利用的权力，斑马鱼系统的遗传和实时成像研究缝线的形成。在斑马鱼中，头骨和骨缝的形成是相对较晚的事件，并且在文献中几乎没有关于这些过程的内容。虽然几个大规模的突变体筛选已经揭示了长达6天发育的颅面骨骼模式化的关键途径，但在约4-6周时的头骨和缝形成的后期过程尚未在任何大规模筛选中进行分析。在晚期头骨和骨缝形成中具有特定缺陷的斑马鱼突变体尚未确定，因此，我们提出了一种遗传筛选来分离和表征此类突变体。首先，我们将通过检查6周时ENU诱变雄性的F3后代对1000个ENU诱变基因组进行正向遗传筛选，6周时野生型鱼的颅骨闭合完成。在对330个基因组进行的试验性筛选中，我们已经确定了12个在头骨和骨缝形成以及骨骼发育的其他后期方面具有特定缺陷的突变体。根据这些数字，我们预计将确定总共约30个特定的突变体。其次，我们将利用实验室中大量可用的转基因系来进一步表征突变表型，并根据受影响的细胞类型和过程对其进行分类。最后，我们将绘制大部分的突变体， 到<3 Mb的间隔。基于表型表征，将克隆8-10个突变体的子集以鉴定突变基因。结合起来，拟议的实验将建立一个收集的特征良好的斑马鱼突变影响许多方面的后来的骨骼发育，特别是那些非常具体的缺陷，在头骨和缝合形成，从而建立斑马鱼作为一个强大的模型系统，这些临床相关的头骨形成的后期方面的研究。重要的是，目前对颅缝早闭和其他颅骨缺损的手术治疗是有限的，对潜在生物学的详细了解将导致更好的治疗和预防方法。