Reduced representation bisulfite sequencing (RRBS) of the retina of crumbs 2a (crb2a m289/m289) zebrafish model of Leber congenital amaurosis and retinitis pigmentosa.. Reduced representation bisulfite sequencing (RRBS) of the retina of crumbs 2a (crb2a m289/m289) zebrafish model of Leber congenital amaurosis and retinitis pigmentosa.

The crumbs cell polarity complex plays a crucial role in apical-basal epithelial polarity. When human CRB1 is mutated, it results in autosomal recessive Leber congenital amaurosis and retinitis pigmentosa, with no established genotype-phenotype correlation. Using the oko meduzym289/m289 (crb2a-/-) zebrafish model, we performed integrative transcriptomic and methylomic analysis to identify dysregulated genes and pathways. We reveal delayed retinal cell type specification confirmed in patient-derived retinal organoids, with disruption to cell cycle modulation and epigenetic transcriptional control. Hence, using reduced representation bisulphite sequencing (RRBS) we explored differential DNA methylation, identifying hypermethylated pathways involving biological adhesion, Hippo and transforming growth factor beta (TGFbeta) signalling. Functional epigenetic modules (FEM) were highlighted through the integration of RNA-seq and RRBS, confirming cell cycle involvement and disturbance of TGFbeta, BMP, Hippo and SMAD protein signal transduction. Taken together our work provides insights for epigenetic regulation in early retinal development and considerations for future therapeutic development. Overall design: We used RRBS to identify differentially methylated regions/genes between the zebrafish model of CRB, crb2a m289. Samples were isolated retinal regions of the dorsal retina at 56 hpf; crb2a-/- and control wildtype. Biological replicates were taken.

Crumbs细胞极性复合物（Crumbs cell polarity complex）在上皮细胞顶-基极性建立中发挥关键作用。当人类CRB1基因发生突变时，会引发常染色体隐性遗传性Leber先天性黑矇（Leber congenital amaurosis）与色素性视网膜炎（retinitis pigmentosa），目前尚未明确其基因型-表型相关性。本研究利用oko meduzy m289/m289（crb2a-/-）斑马鱼模型，开展整合转录组与甲基化组分析，以筛选失调基因及异常通路。我们发现视网膜细胞类型特化延迟，该现象在患者来源的视网膜类器官中得到验证，同时伴随细胞周期调控紊乱与表观遗传转录控制失衡。为此，我们通过简化代表性亚硫酸氢盐测序（reduced representation bisulphite sequencing, RRBS）探究差异DNA甲基化情况，鉴定出涉及生物黏附、Hippo通路及转化生长因子β（transforming growth factor beta, TGFβ）信号转导的高甲基化通路。结合RNA测序与RRBS数据的整合分析，我们识别出功能表观遗传模块（functional epigenetic modules, FEM），进一步证实细胞周期参与紊乱以及TGFβ、骨形态发生蛋白（BMP）、Hippo及SMAD蛋白信号转导通路失衡。综上，本研究为早期视网膜发育中的表观遗传调控机制提供了新见解，也为未来治疗手段开发提供了参考依据。整体实验设计：我们采用RRBS技术，在CRB相关斑马鱼模型（crb2a m289）与野生型对照之间筛选差异甲基化区域/基因。样本取自受精后56小时（56 hpf）的斑马鱼背侧视网膜分离区域，并设置了生物学重复。