Anatomical atlas and transgenic toolkit for late skull formation in zebrafish

描述（由申请人提供）：成人颅骨的最终形式是通过一系列复杂的形态发生事件和生长实现的，主要是在胚胎后发育期间。许多常见的人类颅骨先天性缺陷都是在这些发育事件中形成的。人类患者的治疗方案远非完美，改进需要更全面地了解胚胎后颅骨形成的生物学基础。然而，由于其复杂的发展和相对较晚的出现，这些临床相关的阶段，在头骨发育已不太容易在实验生物。斑马鱼在骨骼发生方面与哺乳动物基本相似，包括头骨拱顶和颅缝的形成。虽然后来的头骨和骨缝形成事件在斑马鱼中的研究相对较少，但它们仍然可以进行操作和成像，使斑马鱼成为进一步了解这些复杂事件的理想系统。通过一组相互关联的目标，我们建议建立并提供给社区的工具，这将奠定基础，使用斑马鱼检查头骨和缝合形成。我们将首先构建一个正常颅骨发育的在线交互式图谱，包括颅骨穹窿形成的各个阶段。地图集的基础将是高分辨率计算机断层扫描（微型CT）生成的图像，这些图像将附有注释并可供下载。这些将由转基因斑马鱼在关键细胞群体中表达荧光团的图像来补充，例如处于不同发育阶段的软骨细胞和成骨细胞。对于转基因，我们将优化最近开发的用于固定和清除大（>1 mM）组织样品的方法，并使用多功能变焦宏观共聚焦显微镜。这种方法将允许创建较低分辨率的数据集，从中我们可以生成整个头骨中基因表达的三维重建，并且还将允许特定结构的高分辨率成像。用于成像研究的转基因系也将作为使用phiC 31重组酶的转基因系统的基础，以允许在基因组环境中替换转基因编码序列，同时保留组织特异性表达模式;试剂（鱼线和质粒）将可供社区使用。最后，本申请中的两个实验室都在进行基因筛查，以确定导致青少年或成人头骨缺陷的突变。使用一组精选的 具有临床相关表型的突变体，我们将应用上述成像方法来描述颅骨发育过程中形态和基因表达的缺陷。通过综合地图集和一套转基因和遗传工具的组合生成，我们将大大推进斑马鱼在头骨发育研究中的使用，并大大促进与哺乳动物的比较研究，从而推进人类患者的治疗选择。

DESCRIPTION (provided by applicant): The final form of the adult skull is achieved through a complex series of morphogenetic events and growth, largely during post-embryonic development. Many common human congenital defects in the skull have their foundation in these developmental events. The treatment options in human patients are far from perfect, and improvements demand a more complete understanding of the biology underlying post-embryonic skull formation. However, by their complex development and relatively late occurrence, these clinically relevant stages in skull development have been less accessible in experimental organisms. The zebrafish displays fundamental similarity in skeletogenesis to mammals, including in formation of the vault of the skull and the cranial sutures. Although the later events of skull and suture formation have been relatively less well studied in zebrafish, they are nonetheless accessible for manipulations and imaging, making the zebrafish an ideal system to further our understanding of these complex events. Through a set of interconnected Aims, we propose to establish and make available to the community tools that will lay the foundation for the use of zebrafish to examine skull and suture formation. We will first construct an online, interactive atlas of normal skull development, encompassing the stages during which the vault of the skull is forming. The foundation of the atlas will be images generated by high-resolution computed tomography (micro-CT), which will be annotated and available for download. These will be complemented by images of transgenic zebrafish expressing fluorophores in critical cell populations, such as chondrocytes and osteoblasts at different stages of development. For the transgenics, we will optimize recently developed methods for fixation and clearing of large (>1 mM) tissue samples and use a versatile zoom macro-confocal scope. This approach will allow creation of lower resolution data sets from which we can generate three-dimensional reconstructions of gene expression in an entire skull, and will also allow high resolution imaging of specific structures. The transgenic lines used for the imaging studies will also serve as the basis for a transgenic system, using phiC31 recombinase, to allow replacement of the transgene coding sequences in genomic context while preserving tissue-specific expression patterns; the reagents (fish lines and plasmids) will available to the community. Finally, both of the laboratories in this application are engaged in ongoing genetic screens to identify mutations causing defects in the juvenile or adult skull. Using a select set of mutants with clinically relevant phenotypes, we will apply the imaging approaches above to describe the defects in morphology and gene expression during skull development. Through the combined generation of a comprehensive atlas and a set of transgenic and genetic tools, we will substantially advance the use of zebrafish in the study of skull development, and greatly facilitate comparative studies with mammals that will advance treatment options in human patients.