A comparative analysis of gene and protein expression in chronic and acute models of photoreceptor degeneration in adult zebrafish. A comparative analysis of gene and protein expression in chronic and acute models of photoreceptor degeneration in adult zebrafish

Adult zebrafish are capable of photoreceptor (PR) regeneration following acute phototoxic lesion (AL). We developed a chronic low light (CLL) exposure model that more accurately reflects chronic photoreceptor degeneration observed in many human retinal diseases. Here, we characterize the morphological and transcriptomic changes associated with acute and chronic models of PR degeneration at 8 time points over a 28-day window using immunohistochemistry and 3’mRNA-seq. We first observed a differential sensitivity of rod and cone PRs to CLL. Next, we found no evidence for Müller glia (MG) gliosis or regenerative cell-cycle reentry in the CLL model, which is in contrast to the robust gliosis and proliferative response from resident MG in the AL model. Differential responses of microglia between the models was also observed. Transcriptomic comparisons between the models revealed gene-specific networks of PR regeneration and degeneration, including genes that are activated under conditions of chronic PR stress. Finally, we showed that CLL is at least partially reversible, allowing for rod and cone outer segment outgrowth and replacement of rod cell nuclei via an apparent upregulation of the existing rod neurogenesis mechanism. Collectively, these data provide a direct comparison of the morphological and transcriptomic photoreceptor degeneration and regeneration models in zebrafish. Overall design: 3'mRNA sequencing of single adult zebrafish retinas harvested at 7 different time-points post chronic exposure to ~5,000 lux "low light" lesion, along with dark adapted 0hr controls (n=6).

成年斑马鱼在急性光毒性损伤（acute phototoxic lesion，AL）后具备光感受器（photoreceptor，PR）再生能力。本研究构建了慢性低光照（chronic low light，CLL）暴露模型，该模型更贴合多种人类视网膜疾病中观察到的慢性光感受器退行性病变过程。本研究通过免疫组织化学与3’端mRNA测序（3’mRNA-seq）技术，在28天周期内的8个时间点，对急性与慢性光感受器退行性病变模型的形态学及转录组学变化进行了表征。
本研究首先发现视杆与视锥光感受器对CLL的敏感性存在显著差异。随后观察到，CLL模型中未出现米勒胶质细胞（Müller glia，MG）胶质增生或再生性细胞周期再进入现象，这与AL模型中驻留型米勒胶质细胞产生显著胶质增生与增殖反应的结果形成鲜明对比。此外，两种模型中小胶质细胞的响应模式亦存在明显差异。
模型间的转录组学比较分析揭示了光感受器再生与退行性病变的基因特异性调控网络，其中包含在慢性光感受器应激条件下激活的基因。最后，本研究证实CLL诱导的病变至少可部分逆转：通过上调现有视杆神经发生机制，可促进视杆与视锥外节长出，并实现视杆细胞核的替换。
综上，本研究数据直接对比了斑马鱼光感受器退行性病变与再生模型的形态学及转录组学特征。
整体实验设计：对成年斑马鱼的单视网膜样本进行3’mRNA测序，样本采集于慢性暴露于约5000勒克斯"低光照"损伤后的7个不同时间点，同时设置暗适应0小时对照组（每组n=6）。