Retinal ganglion cell-derived semaphorin 6A segregates starburst amacrine cell dendritic scaffolds to organize the mouse inner retina

To form functional circuits, neurons must settle in their appropriate cellular locations, and then project and elaborate neurites to contact their target synaptic neuropils. Laminar organization within the vertebrate retinal inner plexiform layer (IPL) facilitates pre- and postsynaptic neurite targeting, yet the precise mechanisms underlying establishment of functional IPL subdomains are not well understood. Here, we explore mechanisms defining the compartmentalization of OFF and ON neurites generally, and OFF and ON direction-selective neurites specifically, within the developing mouse IPL. We show that semaphorin 6A (Sema6A), a repulsive axon guidance cue, is required for delineation of OFF versus ON circuits within the IPL: in the Sema6a null IPL, the boundary between OFF and ON domains is blurred. Furthermore, Sema6A expressed by retinal ganglion cells (RGCs) directs laminar segregation of OFF and ON starburst amacrine cell dendritic scaffolds, which themselves serve as a substrate upon which other retinal neurites elaborate. These results demonstrate that RGCs, the first type of neuron born within the retina, play an active role in functional specialization of the IPL. mRNA from each sample corresponds to an isolated single TdTomato-labeled starburst amacrine cell isolated by FACS across early postnatal development (P1, P4, P8, P11, and P16). scRNA-seq libraries were generated using a modified SmartSeq2 protocol (Chevee et al., 2018, doi: 10.1016/j.celrep.2017.12.046) and profiled by NextSeq mid output 2x75 paired-end sequencing.