Figure 7

shRNA mediated knockdown of Sema6A in the neural crest, but not in the ventral spinal cord, induces ectopic motorneurons. (a,b) Confocal micrographs of vibratome sections of an HH stage 25 embryo, two days after electroporation in the neural crest with a Sema-6A shRNA-EGFP (green). Embryos were wholemount dual immunostained with antibodies against MNR2 and Islet2 (red) and counterstained with anti-neurofilament (NF, blue). MNR2 and Islet2-positive motor neurons (white arrowheads) can be seen in ectopic positions in the ventral roots but only when accompanied by a large number of Sema-6A shRNA expressing-EGFP-positive (green) neural crest derivatives. Note that EGFP-labelled cells are restricted to neural crest derivatives in the ventral root and that motor neurons, including those in ectopic positions are EGFP-negative. The small amount of EGFP labelling of structures in a ventro-medial position in the spinal cord corresponds to the axon projections of labelled commissural neurons in the dorsal spinal cord. Bar = 100 μm. (c) Quantitative analysis of the prevalence of ectopic motor neurons after electroporation with Sema-6A shRNA-EGFP targeted towards either the ventral neural tube (NT) or dorsal neural tube (neural crest, NC) shows a distinct effect only for neural crest-electroporated embryos. ***P < 0.001; two-tailed t-test. Conversely, knockdown of Npn-2 expression in the neural crest does not induce ectopic motor neurons, unlike knockdown of Npn-2 in the ventral neural tube, as shown earlier (Figure 2). (d) Confocal micrograph of a transverse cryosection (20 μm) immunolabelled with cad7(red) and neurofilament (blue) of an HH23 embryo 2 days after neural crest electroporation of Sema6A shRNA. GFP-positive neural crest cells targeted with Sema-6A shRNA populate the dorsal root ganglion (DRG), the DREZ and the MEP where they express high level of cad7 (red; white arrows), indicating that Sema6A shRNA does not interfere with boundary cap formation.