Molecular signatures of retinal ganglion cells revealed through single cell profiling.

Retinal ganglion cells can be classified into more than 40 distinct subtypes, whether by functional classification or transcriptomics. The examination of these subtypes in relation to their physiology, projection patterns, and circuitry would be greatly facilitated through the identification of specific molecular identifiers for the generation of transgenic mice. Advances in single cell transcriptomic profiling have enabled the identification of molecular signatures for cellular subtypes that are only rarely found. Therefore, we used single cell profiling combined with hierarchical clustering and correlate analyses to identify genes expressed in distinct populations of Parvalbumin-expressing cells and functionally classified RGCs. RGCs were manually isolated based either upon fluorescence or physiological distinction through cell-attached recordings. Microarray hybridization and RNA-Sequencing were employed for the characterization of transcriptomes and in situ hybridization was utilized to further characterize gene candidate expression. Gene candidates were identified based upon cluster correlation, as well as expression specificity within physiologically distinct classes of RGCs. Further, we identified Prph, Ctxn3, and Prkcq as potential candidates for ipRGC classification in the murine retina. The use of these genes, or one of the other newly identified subset markers, for the generation of a transgenic mouse would enable future studies of RGC-subtype specific function, wiring, and projection.

Polo-Like Kinase 3 Appears Dispensable for Normal Retinal Development Despite Robust Embryonic Expression.

During retinogenesis seven different cell types are generated in distinct yet overlapping timepoints from a population of retinal progenitor cells. Previously, we performed single cell transcriptome analyses of retinal progenitor cells to identify candidate genes that may play roles in the generation of early-born retinal neurons. Based on its expression pattern in subsets of early retinal cells, polo-like kinase 3 (Plk3) was identified as one such candidate gene. Further characterization of Plk3 expression by in situ hybridization revealed that this gene is expressed as cells exit the cell cycle. We obtained a Plk3 deficient mouse and investigated changes in the retina's morphology and transcriptome through immunohistochemistry, in situ hybridization and gene expression profiling. These experiments have been performed initially on adult mice and subsequently extended throughout retinal development. Although morphological studies revealed no consistent changes in retinogenesis upon Plk3 loss, microarray profiling revealed potential candidate genes altered in Plk3-KO mice. Further studies will be necessary to understand the connection between these changes in gene expression and the loss of a protein kinase such as Plk3.