, 2011). Taken together with our findings here on class 5 semaphorins and their role in regulating the establishment of neural connectivity within the retina, these

observations suggest that inner retinal lamination is initially orchestrated through a combination of spatially distinct transmembrane GSK-3 beta phosphorylation semaphorin repellent cues. Sema6A, which is expressed in inner retinal neuron subtypes, directs a small subset of select laminar stratification events within the IPL; in contrast, Sema5A and Sema5B guidance cues present in outer retinal neurons control inner retinal lamination by constraining a major portion of inner retinal neurites to the IPL. It seems likely that a combination of transmembrane semaphorin short-range

repulsive interactions and attractive interactions mediated by CAMs facilitates laminar stratification by regulating synapse formation among select neuronal subtypes that together participate in the formation of specific neural circuits in the retina. Outer retinal lamination events within the OPL are controlled by as yet unidentified repellents or attractants. Our observations showing how transmembrane R428 class 5 semaphorins and their PlexA1 and PlexA3 receptors function during retinal development provide an example of repulsive guidance cue regulation of mammalian IPL lamination and segregation of inner retinal neurites from the outer retina. Correct development of inner retinal lamination is critical for appropriate physiological retinal responses, demonstrating that establishment of retinal laminar organization and retinal circuit function are intimately related. It will be of interest to determine whether similar molecular mechanisms facilitate the elaboration of laminar organization in other regions of the CNS, including the spinal cord and the cerebral cortex, and to understand how laminar organization in these regions of the CNS is related to function. Defining this relationship will advance our understanding of lamination as an organizing however principle throughout the nervous system. The day of vaginal plug observation was designated

as E0.5 and the day of birth as P0. Genetically modified mouse lines and targeting strategies for the generation of Sema5A−/− and Sema5B−/− mice are described in Supplemental Experimental Procedures. Immunohistochemistry was performed as previously described (Matsuoka et al., 2011). The primary antibodies used in this study are listed in Supplemental Experimental Procedures. In situ hybridization was performed on either fresh-frozen or PFA-fixed retina sections (20 μm thickness) as described previously (Matsuoka et al., 2011). Digoxigenin-labeled antisense riboprobes specific for the coding sequences of Sema5A (3353–3860 bp), Sema5B (2808–3366 bp), PlexA1 (381–1314 bp), and PlexA3 (4977–5616 bp) were used for in situ hybridization.