PROJECT SUMMARY The precise assembly of neural circuits ensures accurate neurological function and behavior. For example, to communicate specific aspects of the visual world to the brain, retinal ganglion cells (RGCs) find and form synaptic contacts with specific postsynaptic partners out of the heterogeneous neuronal population of retino-recipient areas in the brain. One such area is the superior colliculus (SC), which receives direct retinal inputs and sends commands for direct innate behaviors such as escape or prey capture. What are the molecular determinants for selective RGC to SC neuron wiring? How are parallel retinotectal circuits sorted onto different SC laminae and neuronal relays? How are distinct retinotectal circuits linked to defined visual evoked behaviors? This proposed study aims to answer these questions in the mouse visual system. To accomplish this goal, first, we will map out parallel retinotectal circuits. We have established an integrated anterograde-tracing and sequencing platform, Trans-Seq, that defines the outputome of a genetically- defined RGC subtype. We applied Trans-Seq to all RGC subtypes globally, α-RGCs, and On-Off direction- selective-ganglion-cells and reconstructed their differential outputomes onto superficial superior-collicular (sSC) neuron subtypes. We propose to apply Trans-Seq to other major RGC subtypes representing different visual features. The proposed studies will determine retinotectal circuit convergence and divergence at neuron subtype resolution. Second, we aim to understand cellular and molecular mechanisms regulating specific retinotectal circuit wiring. We have analyzed α-RGC specific outputomes and revealed a selective sSC neuron subtype, Nephronectin-positive-wide-field neurons (NPWFs). The α-RGC-to-NPWF circuit was genetically validated using imaging, electrophysiology, and retrograde tracing. We propose to study how Nephronectin mediates α-RGC selective axonal lamination onto the deep sSC layer and whether Nephronectin determines the subsequent synaptic specificity from α-RGCs to NPWFs. We will also investigate what molecular mechanisms mediate Nephronectin binding and lead to a selective mammalian retinotectal circuit assembly. Third, we will link specific retinotectal circuits to defined visual evoked behaviors. We propose to combine genetic and optogenetic tools established above to determine whether the α-RGC-to-NPWF circuit contributes to visual evoked innate behaviors, such as looming triggered defense responses. We will also examine whether molecular determinants for connectivity, such as Nephronectin, regulate this behavioral output via these retinotectal circuits. Our circuit mapping platform builds a precise connectivity map at neuronal subtype resolution. Further, this work will align the precise neuronal wiring diagram to innate visual evoked behaviors, informing future functional and behavioral analysis. The new knowledge gained here may include molecular principles unde...