Figure 8

Knockdown of MICAL3 by ventral targeted shRNA-EGFP induces large scale ectopic migration of spinal motor neurons. (a) In situ hybridisation on HH stage 23 chick embryo transverse cryosections shows distinct expression of MICAL3 in motor neurons. (b,c) Targeting of MICAL3 shRNA-EGFP in the ventral spinal cord induces large scale ectopic migration of dual labelled GFP-positive (green (b)) Islet2/MNR2 positive (red (b,c)) motor neuron somata into ventral root (white arrows in (b,c)). The section was counterstained with anti-neurofilament (NF; blue) to locate the dorsal and ventral roots and show the specific, ventral targeting of the electroporation. (d) The percentage of vibratome sections from HH stage 24 embryos containing dual labelled EGFP- and MNR2/Islet2 positive ectopic motor neurons out of the total of EGFP-positive sections, after ventral electroporations with two shRNA-EGFP constructs MICAL3-A and -B, is significantly higher than when EGFP alone was electroporated. ***P < 0.001; two-tailed t-test. (e,f) Cad-7 expressing BC cells at the MEP (red; white arrowheads) are unaffected by MICAL3 shRNA knock down in the ventral spinal cord (green in (e)) that induces large scale motor neuron migration (arrow in (e)). Bar = 100 μm.