INVESTIGATING THE MOLECULAR BASIS OF RETINAL DIRECTION-SELECTIVE CIRCUITRY

The mouse retina contains dozens of different cell types, each specialized for a particular aspect of visual function. One such cell type is the direction-selective ganglion cell (DSGC), which functions as a motion detector, only firing in response to movement in a particular direction. A DSGC’s directional preference arises from asymmetric inhibitory input from neighboring starburst amacrine cells (SACs). Some DSGCs respond to upward motion and others to downward motion, yet the molecular programs that determine a DSGC’s preferred direction are unknown. The first aim of my thesis was to identify genes that determine a DSGC’s directional preference, focusing primarily on DSGCs of the mouse accessory optic system (AOS) responsible for image stabilization. I performed single-cell RNA sequencing on several AOS DSGC types and found candidate genes that distinguish upward- and downward-preferring DSGCs. One of these genes, the homophilic cell adhesion molecule Ptprk, was necessary for upward motion detection and proper dendrite development in upward-preferring DSGCs. Deletion of another gene, the transcription factor Tbx5, abolished vertical motion detection and led to the selective loss of upward-preferring DSGCs. These results provide insight into the molecules regulating ganglion cell dendrite morphology and the fate specification of a particular ganglion cell subtype. The second aim of my thesis was to develop techniques to visualize SAC-DSGC connectivity using light microscopy. The first tool used rabies trans-synaptic tracing to visualize connected SACs in upward-preferring DSGCs. SACs were symmetrically distributed around DSGCs in the first postnatal week, but became asymmetrically distributed in the second postnatal week, consistent with prior work showing DS acquisition during the second week of life. The second tool used the Gphn.FingR intrabody, a genetically encoded inhibitory synapse sensor, to visualize inhibitory synapses in upward-preferring DSGCs. Inhibitory synapses were uniformly distributed throughout the DSGC dendritic arbor after normalizing for dendrite asymmetry, indicating that asymmetric SAC input does not arise from the spatial segregation of inhibitory synapses on one side of the DSGC arbor. Taken together, the current work provides the first description of genetic differences between AOS DSGCs and offers new tools to screen for genes that may regulate DS. The identification of Ptprk provides insight into the molecules DSGCs use to regulate dendrite morphology, which has consequences for motion detection. Likewise, the identification of Tbx5 improves our understanding of genes that regulate DSGC fate specification. The results of our RNAseq analysis, combined with the trans-synaptic tracing and inhibitory synapse visualization tools, provide a strong foundation for future studies investigating the molecular mechanisms of DS circuitry