Fig. 3

V0v candidate genes are co-expressed in subsets of V0v spinal interneurons. (A-D’’’) Lateral views of WT spinal cord at 27 h. Rostral, left. Dorsal, up. in situ hybridization for (A’) skor1b, (B’), skor2, (C’), uncx, and (D’) nefma genes is shown in red. (A’’, B’’, C’’, D’’) Immunohistochemistry for Tg(evx1:EGFP)^SU1, which exclusively labels V0v spinal interneurons, is shown in green. (A, A’’’, B, B’’’, C, C’’’, D, D’’’) Merged images. (A, B, C, D) Maximum intensity projection images. (A’-A’’’, B’-B’’’, C’-C’’’, D’-D’’’) High-magnification single confocal planes of the region indicated by white dotted boxes in A, B, C and D. Similar skor2 results were also reported in [14]. We are showing additional skor2 data here to demonstrate reproducibility of our co-expression experiments, and for ease of comparison with the skor1b, uncx and nefma data. White asterisks indicate double-labelled V0v interneurons. Cells that are green and not red could be V0v interneurons that do not express the gene in question, or V0v interneurons with low expression, not revealed in these experiments, of the gene detected in red. We often detect fewer cells expressing a particular gene in double-labelling experiments where the mRNA is detected with a red fluorophore, than in single in situ hybridization experiments where the mRNA is detected with NBT/BCIP (viewed as an opaque blue stain under visible light), suggesting that the weakest-expressing cells may not be detected in the former, probably due to the prolonged processing of samples necessitated by fluorescent double-labelling experiments, which can affect the stability of target mRNA molecules, and the lower sensitivity of the red label. Therefore, we cannot conclude for certain that single-labelled EGFP-positive cells, do not express the gene detected in red. Scale bar: (A, B, C, D) 50 µm, (A’-A’’’, B’-B’’’, C’-C’’’, D’-D’’’) 20 µm