A Developmental Craniofacial Atlas for the Zebrafish

描述(由申请人提供)：本申请涉及广泛的挑战领域(06)使能技术，以及特定的挑战主题06-DE-102颅面发育结构和分子图谱。我们建议构建斑马鱼头面部骨骼的发育图谱，斑马鱼是一种广泛使用的模式生物，用于了解颅骨发育的保守特征和人类疾病，如腭裂。该地图集将为希望发现斑马鱼头面部骨骼的复杂解剖结构如何发育的科学家提供关键资源。它将成为了解目前可供研究的许多斑马鱼头面部突变体的缺陷以及通过全基因组倡议产生的新突变体的关键参考。这也将有助于理解斑马鱼头骨与其他生物(如老鼠)的发育。该地图集将以网络为基础，在FaceBase和ZFIN上免费提供，FaceBase是一个迅速成为了解头面部发育的标准的网络资源，ZFIN是斑马鱼发育和遗传学的特定信息学资源。它将包括一系列的发育阶段，从受精后1天的胚胎开始，此时骨骼原基已经存在，但骨骼和软骨尚未形成。它将从选定的阶段持续到3周大的幼年阶段，那时头骨的几乎所有元素都存在，形状与成人大致相同，但尺寸很小。它将是三维的和互动的：用户将能够虚拟地解剖头骨-调用感兴趣的元素的特定子集，旋转图像并从任何角度观察一组元素，在任何平面上剖切结构，以及检查结构在不同发育阶段的变化。一项关键的创新是，地图集将完全基于表达转基因荧光蛋白的活的、发育中的有机体的图像。这些标记将由关键的骨骼调节基因的启动子驱动，包括在所有颅面间充质中表达的fl1，以及在这种间充质的一个子集中表达的dlx5a，该间充质仅限于头骨的腹侧元素，包括下颌骨。在骨骼形态发生和生长阶段，形成骨骼的细胞将通过软骨的Sox10和foxp2a以及骨骼的Osterix等标志物的表达来特异性地揭示。表达这些标记之一的鱼也将被荧光染料(如茜素红)进行重要的反染色，以揭示矿化的骨骼。该图集将为每个阶段提供由共焦显微镜制作的高倍率、直通聚焦、双色图像堆叠。这种成像充分利用了目前可用的许多转基因结构、斑马鱼的光学清晰度，以及斑马鱼相对较小的体积和构建头骨的相对较少的细胞数量。这些属性结合在一起，在整个开发过程中提供了无与伦比的特异性和细节的细胞级分辨率。该图集还将包括，特别是在发育后期，由光学投影断层摄影术(OPT)制作的较低放大倍数的中分辨率图像。用户将能够以与共焦堆栈相同的方式交互地操纵这些OPT堆栈，并将允许更好地3-d理解骷髅组织和发展的更多全球特征。 公共卫生意义：该项目将制作的斑马鱼头面部图谱将为动物模型中的骨骼解剖学提供资源，这对理解人类颅面疾病(如腭裂)具有重要价值。该资源将为了解斑马鱼的正常形态发育以及诊断颅面部突变表型的性质提供参考。

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (06) Enabling Technologies, and specific Challenge Topic 06-DE-102 Structural and Molecular Atlases of Craniofacial Development. We propose to construct a developmental atlas for the craniofacial skeleton of zebrafish, a widely used model organism for understanding conserved features of development of the skull, and human disorders such as cleft palate. The atlas will provide a key resource for scientists wishing to discover how the complex anatomy of the zebrafish craniofacial skeleton develops. It will serve as a critical reference for learning the defects of the many zebrafish craniofacial mutants now available for study, as well as new mutants now being generated through genome-wide initiatives. It also will be useful for understanding development of the zebrafish skull compared with other organisms, such as mouse. The atlas will be web-based and freely available on FaceBase, a web resource rapidly becoming the standard for understanding craniofacial development, and on ZFIN, the specific informatics resource for zebrafish development and genetics. It will include a series of developmental stages beginning with the 1-day postfertilization embryo when the skeletal primordia are present, but bones and cartilages have not yet formed. It will continue with selected stages through the 3-week-old juvenile stage when nearly all of the elements of the skull are present, and shaped approximately as in the adult, but of miniature size. It will be 3-dimensional and interactive: The user will be able to virtually dissect the skull - calling up particular subsets of elements of interest, to rotate the image and observe a set of elements from any point of view, to section through the structure in any plane, and to examine how the structure changes at different developmental stages. A key innovation is that the atlas will be based entirely on images taken of the living, developing organism expressing transgenic fluorescent proteins. These markers will be driven by the promoters of key skeletal regulatory genes, including fli1, expressed in all craniofacial mesenchyme, and dlx5a, expressed in a subset of this mesenchyme that is restricted to the ventral elements of the skull, including the lower jaw. During stages of skeletal morphogenesis and growth, the cells making the skeleton will be revealed specifically by expression of markers including sox10 and foxp2a for cartilage, and osterix for bone. The fish expressing one of these markers also will be vitally counterstained with a fluorescent dye such as Alizarin red to reveal the mineralized bone. The atlas will feature, for every stage, high magnification, through-focus, 2-color image stacks made by confocal microscopy. Such imaging takes full advantage of the many currently available transgenic constructs, the optical clarity of the zebrafish, and of its relatively small size and the relatively small number of cells that build the skull. These attributes combine to provide cell-level resolution with unsurpassed specificity and detail throughout an extended period of development. The atlas will also include, especially for the later developmental stages, medium-resolution images at lower magnification made by optical projection tomography (OPT). The user will be able to interactively manipulate these OPT stacks in the same way as for the confocal stacks, and will allow better 3-d understanding of the more global features of skull organization and development. PUBLIC HEALTH RELEVANCE: The zebrafish craniofacial atlas that will come from this project will provide a resource for skeletal anatomy in an animal model that is valuable for understanding human craniofacial disorders such as cleft palate. The resource will provide for understanding normal morphological developmental in zebrafish as well as being a reference for diagnosing the nature of craniofacial mutant phenotypes.