lmx1bb is required for V0v interneuron glutamatergic fates at later stages of development. Lateral view of zebrafish spinal cord at 48 h (a and b) and 7 dpf (c and d), anterior left, dorsal top. in situ hybridization slc17a6 (red) and EGFP immunohistochemistry (green) in WT (a) and lmx1bb mutant (b) Tg(evx1:EGFP) SU1 embryos. Single magnified confocal plane from white dashed box region (a′-a′′′ and b′-b′′′). Immunohistochemistry for EGFP (green) and DsRed (red) in WT (c) and lmx1bb mutant (d) Tg(slc17a6b(vglut2a):loxP-DsRed-loxP-GFP) nns14 ;Tg(evx1:EGFP) SU1 embryos. Single magnified confocal plane from white dashed box region (c′-c′′′ and d′-d′′′). * indicates co-labeled cell, x indicates single labeled EGFP-expressing (V0v) cell. (e and f) Mean number of cells (y-axis) expressing slc17a6 or DsRed (glutamatergic), EGFP (V0v) and slc17a6 or DsRed + EGFP (co-labeled) (x-axis) in WT (white) and lmx1bb homozygous mutants (grey). Error bars indicate standard error of the mean. Three independent experiments were conducted for (c and d). Cell count results were similar in each replicate. One experiment was conducted for (a and b). Data shown here (e and f) are average values of 5–12 embryos. Precise number of embryos counted and p values are provided in Table 7. The glutamatergic and V0v numbers include co-labeled cells. Statistically significant (p < 0.05) comparisons are indicated with square brackets and stars. Scale bar = 30 µm (a-d) and 25 µm (a′-d′′′)
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