Figure 6

Genetic ablation of Sema6A in BC cells leads to ectopic migration of motor neurons. (a) In situ hybridisation shows expression of Sema6A in BC cells at the MEP (black arrows) in transverse cryosections of E12 mouse embryos. Expression of egr2/krox-20 in an adjacent section (a') confirms the signal in (a) corresponds to BC cells. Bar = 100 μm. (b,c) Dual immunostaining of transverse cryosections of E13.5 Sema6A mouse embryos. Compared with wild-type littermates (b) where HB9 positive motor neuron somata (red) are exclusively confined to the ventral spinal cord, in Sema6A null embryos (c) many motor neurons can be seen in ectopic positions in the neurofilament-positive (green) presumptive white matter and ventral nerve roots. Bar = 100 μm. (d) A quantitative analysis of the distribution of HB9-positive ectopic motor neurons along the rostro-caudal axis of E13.5 mouse spinal cord shows a distinct peak at hindlimb level (yellow box) but not forelimb level (red box) in null (triangles) compared with heterozygous (squares) and wild-type (diamonds) embryo littermates. (e) A comparison of the cumulative counts of HB9 positive ectopic motor neurons in sections from posterior trunk (hindlimb containing) region of E13.5 Sema6A wild-type, heterozygous and null embryos. Consistent with the quantitative analysis shown in (d), there is a significant increase (p = 0.03; two-tail t-test) in ectopic motor neurons in the hindlimb region of Sema6A null mice (n = 4 each). (f,g) In situ hybridisation for egr2/krox-20 on transverse cryosections (30 μm) of E11.5 mouse embryos at hindlimb level. The results show no obvious difference in Egr2 expression at the DREZ or MEP in wild-type (f) or Sema6A null mice (g), indicating that formation of boundary caps is not perturbed by genetic ablation of Sema6A. Bar = 100 μm. *P ≤ 0.05; two-tailed t-test