Taken together, these defects confirm that B3gnt1 and ISPD function in the same genetic pathway to regulate dystroglycan glycosylation in vivo, and establish B3gnt1LacZ/M155T Z-VAD-FMK order and ISPDL79∗/L79∗ mice as mouse models of dystroglycanopathy. The defects observed in B3gnt1, ISPD, and dystroglycan mutants suggests a role for dystroglycan in mediating axon guidance in vivo. The axons of both the descending hindbrain projections and the dorsal funiculus extend along the basal surface of the hindbrain and spinal cord, respectively, suggesting

that dystroglycan may be required in the hindbrain and spinal cord for the proper development of these axonal tracts. In contrast to the well-characterized role of dystroglycan in the developing cortex, its function in the spinal cord is unclear. Similar to the developing cortex, levels of total dystroglycan protein in the spinal cord of B3gnt1LacZ/M155T and ISPDL79∗/L79∗ mutants are normal, while glycosylated alpha-dystroglycan and laminin binding activity are reduced to an undetectable amount ( Figures 3A and 3B). Examination of dystroglycan localization in the spinal cord by immunostaining shows that dystroglycan is enriched in the FK228 mw radial neuroepithelial endfeet, where it colocalizes with several extracellular matrix proteins including laminin, perlecan, and collagen IV to form a continuous

basement membrane surrounding the spinal cord ( Figures 3C and S5A). In B3gnt1LacZ/M155T, ISPDL79∗/L79∗ and Sox2cre; DGF/− embryos, the loss of functional dystroglycan results in the progressive fragmentation of the basement membrane beginning around E11.5 which is accompanied by detachment of radial neuroepithelial endfeet from the basal surface ( Figures S5A and S5B). This fragmentation first appears in the lateral portion of the spinal cord and progresses ventrally and dorsally as the spinal cord continues to develop.

Interestingly, in addition to its localization to the basement membrane surrounding the spinal cord, we found that dystroglycan is enriched in the floor plate, a specialized glial structure in the ventral neuraxis that spans Thalidomide the CNS anteroposterior axis from the midbrain to caudal spinal cord (Figures 3C and 3D). The spinal cord floor plate functions both as an organizer of ventral cell fates and as an intermediate target for commissural axons whose cell bodies reside within the dorsal spinal cord. The axons of commissural neurons are initially attracted ventrally to the floor plate by a number of floor plate derived cues, including Netrin, Shh, and VEGF (Charron et al., 2003; Ruiz de Almodovar et al., 2011; Serafini et al., 1996). Once commissural axons reach the floor plate, these attractive cues are silenced and repulsive floor plate-derived cues, including Slits (Long et al., 2004) and Sema3B (Zou et al.