E10.5 VM Neurosphere and Explant Culture

E10.5 VM neurospheres and E11.5 VM explants were generated using previously described methods^{7 (link), 21 (link)}. Heparin acrylic beads (Sigma), pre-incubated for 24 hrs in either PBS (control) or CHL1 (10μg/ml, R&D Systems), were attached to poly-D-lysine coated coverslips using rat tail collagen (2.1 mg/ml, Roche). The VM tissue (neurosphere or explant) was positioned adjacent to the beads, at a distance of approximately 300μm. Subsequently, both the VM tissue and beads were encapsulated in collagen gel and cultured in the presence of N2 media (consisting of a 1:1 mixture of F12 and MEM supplemented with 15 mM HEPES buffer, 1 mM glutamine, 6 mg/ml glucose, 1 mg/ml bovine serum albumin and N2 supplement) for 72 h, prior to fixation (4% paraformaldehyde, 20 min) and immunostaining.