Loss of Stim2 in zebrafish induces retinal gene expression cellular changes resembling glaucoma characteristics [scRNA-seq]

Calcium is involved in vision processes in retina and is implicated in various pathologies including glaucoma. Rods are protected against prolonged lowering of intracellular calcium ion concentrations by Store Operated Calcium Entry (SOCE). We showed zebrafish lacking SOCE calcium sensor Stim2 had problem with vision as indicated by behavior tests, staining of retina and downregulation of genes related to light perception. In this work we aimed to understand the mechanism responsible for the vision problems in stim2 zebrafish knockout. scRNA-sequencing of neuronal origin cells from brains of 5 dpf larvae identified 27 clusters. Differently expressed genes were detected in ten clusters including amacrine and GABAergic retinal interneurons and GABAergic optic tectum cells. In five clusters the proportion of cells in stim2 KO fish versus control was significantly decreased including GABAergic diencephalon and optic tectum, and was increased in amacrine and GABAergic retinal interneurons. Transmission Electron Microscopy in stim2 KO fish revealed decrease in width of inner plexiform layer (IPL), ganglion cells and their dendrites numbers what is characteristic for glaucoma. Analysis of cell density in the inner nucleus layer, which includes amacrine cells among others, showed a significant decrease in the number of GABAergic neurons.The area of cristae in photoreceptor mitochondria was statistically lower in stim2 KO than in control retinas. Tg(HuC:GCaMP5G) zebrafish line and double knockout for stim2 expressing GCaMP5G under the same promoter [(stim2a;stim2b)-/- ; Tg(HuC:GCaMP5G)] were used. Single-cell suspensions were obtained by enzymatic and mechanical digestion of 5 days post fertilization (dpf) larva heads without eyes isolated from two fish lines: stim2 KO (2 replicates - 30 larvae per each sample) and Tg (control, 2 replicates- 30 larvae per each sample). The cells were sorted based on GCaMP5G fluorescence to obtain the cells of neuronal origin.