Anatomical atlas and transgenic toolkit for late skull formation in zebrafish

描述(申请人提供)：成人头骨的最终形态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。许多常见的人类先天颅骨缺陷都是在这些发育事件中产生的。人类患者的治疗方案远非完美，改进需要对胚胎后颅骨形成的生物学有更全面的了解。然而，由于其复杂的发育和相对较晚的发生，这些临床上相关的颅骨发育阶段在实验生物体中较难获得。斑马鱼在骨骼形成方面表现出与哺乳动物的基本相似之处，包括头盖骨穹隆和颅缝的形成。虽然后来的头骨和缝线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，这使得斑马鱼成为一个理想的系统来加深我们对这些复杂事件的理解。通过一系列相互关联的目标，我们建议建立并向社区提供工具，为使用斑马鱼检查头骨和缝合线的形成奠定基础。我们将首先构建一个在线的、互动的正常头骨发育图集，包括头盖骨穹隆形成的各个阶段。地图集的基础将是高分辨率计算机断层扫描(Micro-CT)生成的图像，这些图像将经过注释并可供下载。此外，还将拍摄到转基因斑马鱼在不同发育阶段的软骨细胞和成骨细胞等关键细胞群中表达荧光团的图像。对于转基因技术，我们将优化最近开发的固定和清除大(&gt；1 mm)组织样本的方法，并使用多功能变焦宏观共焦显微镜。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，还将允许对特定结构进行高分辨率成像。用于成像研究的转基因品系也将作为使用phiC31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，同时保留组织特有的表达模式；该试剂(鱼系和质粒)将可供社区使用。最后，这项应用中的两个实验室都在进行持续的基因筛查，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 对于具有临床相关表型的突变型，我们将应用上述成像方法来描述颅骨发育过程中的形态和基因表达缺陷。通过综合制作一份全面的图谱和一套转基因和遗传工具，我们将极大地促进斑马鱼在头骨发育研究中的使用，并极大地促进与哺乳动物的比较研究，这将改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.