Screen for mutations affecting skull and suture formation in zebrafish

描述(申请人提供)：颅缝的关闭是颅骨形态发生过程中的一个关键过程。在扁平骨的边缘，也就是调节骨形成的中心，纤维缝合线将单独的骨骼结合在一起。随着大脑的生长，缝合线允许个体骨骼进一步生长和移动；一旦脑颅完全发育成熟，缝合线就会在人类成年早期融合。重要的是，缝合线的通畅与大脑发育密切相关。过早缝合，即颅缝早闭，会导致头部形状异常，并与颅内压升高、脑血流受损、视力和听力受损以及智力残疾有关。尽管它们在颅骨生长和脑发育中发挥着重要作用，但缝合形成的分子和细胞机制，它们如何与颅骨相互作用来调节生长，以及诱导缝合融合的信号还不是很清楚。我们目前对缝合形成规律的大部分知识来自于对导致颅突融合或其他缝合形成缺陷的许多人类突变中的少数几个的鉴定。在这里，我建议利用斑马鱼系统的力量来进行缝合形成的遗传和活体成像研究。在斑马鱼中，头骨和缝线的形成是相对较晚的事件，在文献中几乎没有关于这些过程的报道。虽然几个大规模的突变筛选已经揭示了发育长达6天的头面部骨骼图案化的关键途径，但在~4-6周的以后的头骨和缝合形成过程还没有在任何大规模筛选中进行测试。斑马鱼头骨和骨缝形成后期具有特定缺陷的突变体尚未被鉴定，因此，我们建议通过遗传筛选来分离和鉴定此类突变体。首先，我们将对1000个ENU诱变的基因组进行正向遗传筛查，检查6周时ENU诱变雄鱼的F3后代，这是野生型鱼类完成头骨闭合的时间。在正在进行的330个基因组的初步筛选中，我们已经确定了12个突变体，它们在头骨和缝合形成以及骨骼发育的其他后期方面存在特定缺陷。根据这些数字，我们预计总共鉴定出30个特定的突变体。其次，我们将利用我们实验室现有的大量转基因品系来进一步表征突变的表型，根据受影响的细胞类型和过程对它们进行分类。最后，如果不是全部的话，我们将映射出大部分的突变体 到&lt；3 Mb的间隔。根据表型特征，将克隆8-10个突变体的子集来鉴定突变基因。结合起来，拟议的实验将建立一组具有良好特征的斑马鱼突变，影响后来骨骼发育的许多方面，特别是那些在头骨和缝合形成方面具有非常特殊缺陷的斑马鱼，从而建立一个强大的模型系统，用于研究这些与临床相关的头骨形成的后期方面。重要的是，目前颅缝融合和其他颅骨缺陷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。

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.