Trans-Seq for mammalian retinotectal circuit mapping

The mouse visual system serves as an accessible model to understand mammalian circuit wiring. Despite rich knowledge in retinal circuits, the long-range connectivity map from distinct retinal ganglion cell (RGC) types to diverse brain neuron types remains unknown. Here we developed an integrated approach, named Trans-Seq, to map RGC to superior collicular (SC) circuits. Trans-Seq combines a fluorescent anterograde transsynaptic tracer, consisting of codon-optimized wheat germ agglutinin fused to mCherry, with single-cell RNA Sequencing. We used Trans-Seq to classify SC neuron types innervated by genetically-defined RGC types and predicted a neuronal pair from aRGCs to Nephronectin-positive wide-field neurons (NPWFs). We validated this connection using genetic labeling, electrophysiology, and retrograde tracing. We then utilized transcriptomic data from Trans-Seq to identify Nephronectin as a determinant for selective synaptic choice from aRGC to NPWFs via binding to Integrin-a8ß1. The Trans-Seq approach can be broadly applied for postsynaptic circuit discovery from genetically-defined presynaptic neurons. Overall design: Anterograde tracing with WGA-mCherry was combined with single-cell RNA sequencing to produce bioinformatic circuit maps from retinal ganglion cell subtypes into the superior colliculus. Intraocular injections of WGA-mCherry tracer were performed to transsynaptically label postsynaptic retinorecipient neurons in the superior colliculus. The superior colliculus was processed to obtain single-cell suspensions and mCherry+ connected cells were recovered by FACS. mWmC-positive SC neurons connected to all RGCs, alpha-RGCs (labelled through the reporter Kcng4-Cre), and ooDSGCs (labelled through Cart-Cre) were isolated and profiled in this study.