Transcriptome profiling of zebrafish optic fissure fusion

Incomplete fusion of the optic fissure leads to ocular coloboma, a congenital eye defect that affects up to 7.5 per 10,000 births and accounts for up to 10 percent of childhood blindness. The molecular and cellular mechanisms that facilitate optic fissure fusion remain elusive. We have profiled global gene expression during optic fissure morphogenesis by transcriptome analysis of tissue dissected from the margins of the zebrafish optic fissure and the opposing dorsal retina before (32 hours post fertilisation, hpf), during (48 hpf) and after (56 hpf) optic fissure fusion. Differential expression analysis between optic fissure and dorsal retinal tissue resulted in the detection of several known and novel developmental genes. The expression of selected genes was validated by qRT-PCR analysis and localisation investigated using in situ hybridisation. We discuss significantly overrepresented functional ontology categories in the context of optic fissure morphogenesis and highlight interesting transcripts from hierarchical clustering for subsequent analysis. We have identified netrin1a (ntn1a) as highly differentially expressed across optic fissure fusion, with a resultant ocular coloboma phenotype following morpholino antisense translation-blocking knockdown and downstream disruption of atoh7 expression. To support the identification of candidate genes in human studies, we have generated an online open-access resource for fast and simple quantitative querying of the gene expression data. Our study represents the first comprehensive analysis of the zebrafish optic fissure transcriptome and provides a valuable resource to facilitate our understanding of the complex aetiology of ocular coloboma. We used RNA-seq to identify differentially expressed genes across optic fissure fusion in zebrafish. Samples were isolated retinal regions including the optic fissure (OF) region and control dorsal retina (DR) at timepoints; prefusion (32 hpf), fusing (48 hpf) and post fusion (56 hpf). 5 biological replicates.

视神经裂闭合不全可引发眼缺损（ocular coloboma），该先天性眼部缺陷的发病率可达每万名新生儿7.5例，且高达儿童失明病因的10%。目前，介导视神经裂闭合的分子与细胞机制仍不明晰。我们针对斑马鱼视神经裂边缘及对侧背侧视网膜的解剖分离组织开展转录组分析，以此解析视神经裂形态发生过程中的全局基因表达谱，采样时间点涵盖视神经裂闭合前（受精后32小时，hpf）、闭合中（48 hpf）以及闭合后（56 hpf）三个阶段。通过对视神经裂组织与背侧视网膜组织进行差异表达分析，我们检测到多个已知发育基因与全新的发育基因。我们采用定量实时聚合酶链反应（qRT-PCR）验证了部分候选基因的表达水平，并利用原位杂交（in situ hybridisation）技术探究了这些基因的组织定位情况。我们围绕视神经裂形态发生过程中显著富集的功能本体论（functional ontology）类别展开讨论，并从层级聚类结果中筛选出值得后续研究的转录本。我们发现netrin1a（ntn1a）在视神经裂闭合过程中呈现高度差异表达，通过翻译阻断型吗啉代反义寡核苷酸敲低netrin1a的表达后，可诱导眼缺损表型，并引发atoh7表达的下游紊乱。为助力人类相关研究中的候选基因筛选工作，我们搭建了在线开放资源平台，可实现对基因表达数据的快速、简便定量查询。本研究是首个针对斑马鱼视神经裂转录组的全面系统性分析，可为我们理解眼缺损复杂的发病机制提供宝贵的研究资源。我们通过RNA测序技术，鉴定出斑马鱼视神经裂闭合过程中的差异表达基因。实验样本为分离的视网膜区域，包括视神经裂（optic fissure, OF）区域与对照背侧视网膜（dorsal retina, DR），采样时间点分别为闭合前（32 hpf）、闭合中（48 hpf）与闭合后（56 hpf），每组设置5个生物学重复。