Quantifying the effects of lack of Stim2 on neuronal origin cells at single cell resolution

Stromal interaction molecule (STIM) proteins reside on the endoplasmic reticulum (ER) membrane and regulate Ca2+ homeostasis and signaling. The stim2a and stim2b double-knockout zebrafish have problems with vision, altered retinal pigment epithelium and thinner ganglion cell layer, and display decreased survival under low oxygen conditions. Also, genes related to light perception are downregulated in the (stim2a;stim2b)-/- zebrafish. In this study, we investigated the effect of lack of Stim2 on neuronal origin cells with single cell RNA sequencing. Utilizing a manifold learning-based method, we identified the subset of cells that were affected the most by the double-knockout. We found that genes involved in development of retina and trigeminal placode were downregulated in that subset of cells in the (stim2a;stim2b)-/- zebrafish. According to marker gene expression, (stim2a;stim2b)-/- zebrafish had decreased number of retinal ganglion cells expressing midkine b (mdkb), amacrine cells (ptmaa, ptmab), cells of the inner nuclear layer (calb2a), and trigeminal placode or sensory neurons (elavl4, slc17a6b) than (stim2a;stim2b)+/+ zebrafish. Furthermore, global downregulation of genes, especially ribosomal protein coding genes, linked with abnormality of retinal pigmented epithelium and brain development in general, and genes involved in oxidative phosphorylation and autophagy was observed in the subset of cells most affected by the double-knockout. Altogether, the results revealed potential mechanisms through which Stim2 may regulate vision/sensory circuitry, cellular response to hypoxia, and prevent neurodegeneration. Overall design: Neuronal origin cells from 5 dpf (stim2a;stim2b)+/+ and (stim2a;stim2b)-/- zebrafish were isolated based on fluorescence activated cell sorting according to the presence or absence of GCaMP5G fluorescence signal, and analyzed with scRNA-seq.