Comprehensive molecular profiling of regenerated retinal neurons in the adult zebrafish

Unlike mammals, zebrafish possess a remarkable capacity to regenerate ablated retinal neurons. In response to cellular injury and death, Müller glia dedifferentiate into a stem cell state, and each gives rise to a single Müller glia-derived progenitor. Progenitors then proliferate and differentiate into to replace the ablated retinal neurons. This study aimed to determine if the injury context influences the quantity of regenerated neurons and to comprehensively identify the cellular and molecular characteristics and diversity of newly born regenerated neurons during regenerative neurogenesis. To definitely label Muller glia-derived regenerated neurons, we utilized a genetic lineage tracing line, Tg(mmp9:creERt2;Ola.actb2:loxp-dsRed-loxp-eGFP). By integrating this approach with scRNAseq and morphological profiling, we characterize quantity, molecular and morphological identity, and diversity of regenerated neurons across two distinct injury models, an intense light photolytic lesion or an intraocular injection of the excitotoxin, NMDA which procedures selective damage to photoreceptors or inner retinal neurons, respectively. Our data show that while regenerated neurons are biased toward the cell type originally ablated, both lesion modes consistently generate other cell types. This suggests a robust intrinsic neurogenic program present among Muller glia-derived progenitors. Transcriptome analysis demonstrates a high degree of similarity between regenerated and endogenous populations, indicating successful restoration of homeostatic molecular signitures. Finally, subtype analysis of amacrine cells show generation of diverse subtype and successful reestablishment of their neurochemical and morphological identities equivalent to their original counterparts. These findings demonstrate that regenerative neurogenesis in the zebrafish retina can faithfully reconstitute the specialized cellular diversity and molecular and structural complexity of the mature retina. Overall design: The transgenic line, Tg(mmp9:creERt2;Ola.actb2:loxp-dsRed-loxp-eGFP), was utilized to perform lineage tracing of Müller glia-derived, regenerated neurons. Animals were subjected either to an intense light photolytic lesion or an intraocular injection of the excitotoxin, NMDA. Regenerated, eGFP+ retinal neurons at 14 days post lesion were collected via FACS. eGFP+ cells were loaded into the 10x Genomics Chromium Single Cell System and libraries were generated using V4 chemistry following the manufacturer's instructions. Libraries were sequenced on the Illumina NovaSeq platform.