(A) Immunostaining of E12.5 spinal cord shows Dystroglycan protein (magenta, left panel) expression in commissural axons (L1, green, middle panel). In the high magnification insets, arrows indicate the enriched expression of Dystroglycan in the basement membrane of the spinal cord proximal to the axons. (B) Commissural neurons from E12 dorsal spinal cord cultured for two days in vitro (2DIV) were stained with antibodies to Dystroglycan (magenta, left panel), TUJ1 (green, middle panel). Dystroglycan is present throughout the cell body, axon and growth cone (arrow). (C–F) DiI injections in open-book preparations of E12 spinal cords were used to examine the trajectory of commissural axons. In controls (C), axons extend through the floor plate, then execute an anterior turn (n=6 animals, 49 total injection sites). In Dag1F/-;Sox2Cre mice (D), axons stall within the floor plate and post-crossing axons exhibit anterior-posterior randomization (n=3 animals, 18 total injection sites). (E) Commissural axons in mice lacking the intracellular domain of Dystroglycan (Dag1βcyto/-) show normal crossing and anterior turning (n=3 animals, 34 total injection sites). Conditional deletion of Dystroglycan from commissural neurons in Dag1F/-;Wnt1Cre mice (F) did not affect floor plate crossing or anterior turning (n=8 animals, 59 total injection sites). Higher magnification insets for each image show the anterior (top) and posterior (bottom) trajectories of post-crossing commissural axons. (G) Quantification of open book preparations. On average, 97.62 3.39% of controls, 3.03 4.80% of Dag1F/-;Sox2Cre mutants, 89.52 4.80% of Dag1βcyto/- mutants, and 95.31 2.94% of Dag1F/-;Wnt1Cre mutants showed normal crossing and anterior turning. All of the Dag1F/-;Sox2Cre mutants with turning defects also showed stalling within the floor plate. *p< 0.001, one-way ANOVA, Tukey’s post hoc test. Scale bar = 100μm (A), 10μm (B) and 50μm (F–H).