Abstract
Purpose :
Following ablation of photoreceptors by intense light lesion, the zebrafish is capable of complete regeneration due to the ability of their Müller glia (MG) to re-enter the cell cycle, creating progenitors which differentiate into new photoreceptors. The purpose of this study was to determine if 3’mRNA sequencing, paired with a detailed morphological and immunohistochemical (IHC) analysis, is an effective and unbiased method to investigate the regenerating zebrafish retina.
Methods :
Adult albino zebrafish were subjected to three days of intense phototoxic lesion to destroy photoreceptors. Tissue was collected at 8 timepoints during the regeneration process. Right eyes were designated for IHC with subsequent mean fluorescence intensity analysis using ImageJ. Left eyes were designated for RNA isolation, cDNA library preparation, 3’mRNA sequencing, and bioinformatics. Differential gene expression analysis was performed at each time point and PCA and a time series analysis was performed across the entire dataset.
Results :
Early in the damage response, photoreceptors were destroyed as evidenced by morphological and gene expression losses of the opsins. A dynamic response by immune cells cleared the photoreceptor debris as GFAP-positive and reactive MG re-entered the cell cycle to produce pools of progenitors. Finally, our data highlight a distinct window of time between 5 and 10 days post lesion as a transition from progenitor proliferation/migration to differentiation into new photoreceptors. PCA analysis of the top 200 significantly differentially regulated genes demonstrated distinct clustering of each of the time points over two experimental replicates, confirming the method’s precision. And transcript analysis largely mimicked morphological and protein changes at each time point, suggesting that this database can be utilized to search for novel genetic players in the regenerative process.
Conclusions :
3’mRNA sequencing presents an economical alternative to traditional RNA sequencing for investigations of regenerative biology in the zebrafish retina, with minor limitations. These transcriptomic data, paired with detailed IHC quantitative and morphological analysis, present a powerful method to observe the stem cell processes in a model capable of a robust regeneration response.
This is a 2021 ARVO Annual Meeting abstract.