On and Off Retinal Circuit Assembly by Divergent Molecular Mechanisms

Introduction Direction-selective responses to visual cues depend upon precise connectivity between inhibitory starburst amacrine cells (SACs) and direction-selective ganglion cells (DSGCs). Motion is detected by SAC responses to illumination onset (On) or cessation (Off). On and Off SACs costratify in the inner plexiform layer of the vertebrate retina with distinct DSGC dendritic arborizations that mediate On or Off directional responses. Here, we study the molecular mechanisms that specify On versus Off SACs and the signaling pathways governing the functional assembly of murine retinal direction-selective circuitry. We show that signaling between the transmembrane guidance cue semaphorin 6A (Sema6A) and its receptor plexinA2 (PlexA2) regulates dendritic morphology of On but not Off SACs, thereby controlling direction-selective responses to visual stimuli. Development of direction-selective circuitry. On and Off mouse SACs normally stratify in discrete layers (top left) and exhibit radial dendrite morphology (top right). Sema6A and its PlexA2 receptor are expressed in On SACs, but only PlexA2 is expressed in Off SACs. In Sema6A mutants, SACs fail to stratify (bottom left) and On SACs are misshapen (bottom right), compromising responses to “light on” directional cues. Methods We analyzed SAC stratification in the inner plexiform layer, and dendritic morphology of individual On and Off SACs, throughout retinal development in Sema6A and PlexA2 mutant mice. We used dissociated retinal cultures to determine how neurites from genetically labeled SACs respond to exogenous Sema6A. We determined the light-evoked excitatory and inhibitory responses of Sema6A–/– On SACs. Finally, we analyzed direction-selective responses in isolated retinas by On-Off direction-selective ganglion cells. Results Sema6A is expressed in On, but not Off, SACs, whereas PlexA2 is expressed in all SACs. In vitro, exogenous Sema6A repels neurites only from PlexA2+, Sema6A– SACs, the in vivo expression profile of Off SACs. In PlexA2–/– or Sema6A–/– retinas, SAC dendritic stratifications fail to completely segregate from each other; therefore, in vitro and in vivo observations suggest that repulsive interactions between Sema6A and PlexA2 mediate SAC dendritic stratification. Analysis of dendritic morphology in individual SACs in the x-y plane reveals that On SACs in PlexA2–/– and Sema6A–/– mutants are missing extensive portions of their dendritic fields, have asymmetric dendritic arbors, and exhibit self-avoidance defects; Off SACs in these mutants have normal x-y plane dendritic arbors. Specific On-Off bistratified direction-selective ganglion cells in Sema6A–/– mutant retinas exhibit decreased tuning of On-directional motion responses, whereas Off responses in these same cells are unaffected, correlating the elaboration of symmetric SAC dendritic morphology and asymmetric responses to motion. Discussion Our findings show that, in addition to contributing to the separation between On and Off SAC dendritic stratifications into distinct inner plexiform layer laminae, Sema6A-PlexA2 signaling selectively regulates the elaboration of symmetric On SAC dendritic fields. Disruption of Sema6A-PlexA2 signaling ultimately results in compromised On, but not Off, directional tuning in a subclass of On-Off direction-selective ganglion cells. Our elucidation of molecular events critical for functional assembly of retinal direction-selective circuitry may have general implications for understanding the establishment of circuitry in which individual neurons participate in multiple distinct pathways. Wiring the Retina Starburst amacrine cells in the retina detect motion by responding to light going on or off. L. O. Sun et al. (10.1126/science.1241974) analyzed how the cellular circuits develop in the mouse retina to form the basis of motion detection. Expression patterns of semaphorin 6A and its receptor plexin A2 defined the shape and reactivity of the starburst amacrine cells. Semaphorin 6A expression was restricted to particular cells, generating two classes of starburst amacrine cells with distinct morphologies and opposing functions. Work in mice reveals how motion-detection circuitry is established during visual system development. Direction-selective responses to motion can be to the onset (On) or cessation (Off) of illumination. Here, we show that the transmembrane protein semaphorin 6A and its receptor plexin A2 are critical for achieving radially symmetric arborization of On starburst amacrine cell (SAC) dendrites and normal SAC stratification in the mouse retina. Plexin A2 is expressed in both On and Off SACs; however, semaphorin 6A is expressed in On SACs. Specific On-Off bistratified direction-selective ganglion cells in semaphorin 6A−/− mutants exhibit decreased tuning of On directional motion responses. These results correlate the elaboration of symmetric SAC dendritic morphology and asymmetric responses to motion, shedding light on the development of visual pathways that use the same cell types for divergent outputs.