Department of Molecular Biosciences, Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, Texas, United States.
Department of Molecular Biosciences, Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, Texas, United States Howard Hughes Medical Institute, The University of Texas at Austin, Austin, Texas, United States.
Invest Ophthalmol Vis Sci. 2014 Jun 26;55(7):4585-92. doi: 10.1167/iovs.14-14888.
To identify the mutation and cell biological underpinnings of photoreceptor defects in zebrafish au5 mutants.
Whole genome sequencing and SNP mapping were used to determine the genomic interval that harbors the au5 mutation. A candidate mutation was cloned and sequenced, and mRNA rescue used to validate that the affected gene was correctly identified. In situ hybridization, immunohistochemistry, and confocal imaging were used to determine the effects on photoreceptor development and maintenance in mutant retinae, and to determine if ciliogenesis or cilia-dependent development was affected in mutant embryos. Expression of tagged proteins and high-speed in vivo confocal imaging was used to quantify intraflagellar transport (IFT) and IFT particle localization within multiciliated cells of the Xenopus epidermis.
The au5 mutants possess a nonsense mutation in cluap1, which encodes a component of the IFT machinery. Photoreceptor defects result from degeneration of photoreceptors, and defects in ciliogenesis precede degeneration. Cilia in the olfactory pit are absent, and left-right heart positioning is aberrant, consistent with a role for cluap1 during ciliogenesis and cilia-dependent development. High-speed in vivo imaging demonstrates that cluap1 undergoes IFT and that it moves along the cilium bidirectionally, with similar localization and kinetics as IFT20, an IFT-B complex component.
We identified a novel mutation in cluap1 and determined that photoreceptor maintenance is dependent on cluap1. Imaging data support a model in which cluap1 is a component of the IFT-B complex, and cilia formation requires cluap1 function. These data may provide new insights into the mechanism of photoreceptor degeneration in retinal ciliopathies.
鉴定斑马鱼 au5 突变体中感光器缺陷的突变和细胞生物学基础。
全基因组测序和 SNP 图谱用于确定携带 au5 突变的基因组间隔。克隆和测序候选突变体，并使用 mRNA 拯救来验证受影响的基因是否被正确识别。原位杂交、免疫组织化学和共聚焦成像用于确定突变视网膜中感光器发育和维持的影响，并确定突变胚胎中的纤毛发生或依赖纤毛的发育是否受到影响。表达标记蛋白和高速体内共聚焦成像用于定量多纤毛细胞中内鞭毛运输 (IFT) 和 IFT 颗粒在 Xenopus 表皮中的定位。
au5 突变体在 cluap1 中具有无义突变，该基因编码 IFT 机制的一个组成部分。感光器缺陷是由感光器退化引起的，纤毛发生缺陷先于退化。嗅窝中的纤毛缺失，左右心脏定位异常，提示 cluap1 在纤毛发生和依赖纤毛的发育过程中发挥作用。高速体内成像表明 cluap1 经历 IFT 并且它沿着纤毛双向移动，与 IFT20（IFT-B 复合物成分）的定位和动力学相似。
我们在 cluap1 中发现了一个新的突变，并确定了感光器的维持依赖于 cluap1。成像数据支持这样的模型，即 cluap1 是 IFT-B 复合物的一个组成部分，纤毛形成需要 cluap1 功能。这些数据可能为视网膜纤毛病变中感光器退化的机制提供新的见解。
