Figure 4. Expression of skor1a, skor1b, skor2, ebf3a, uncx and uncx4.1 in Zebrafish evx1;evx2 Double Mutant and Wild-Type Embryos.

(A, B, D, E, G, H, J, K, M, N, P, Q) Lateral views of (A, D, G, J, M, P) wild-type and (B, E, H, K, N, Q) evx1^i232;i232;evx2^sa140;sa140 double mutant embryos (labeled evx1;evx2) at 30 h. Rostral, left. Dorsal, up. (C, F, I, L, O) Number of cells expressing (C) skor1a, (F) skor1b, (I) skor2, (L) ebf3a, and (O) uncx in a precisely-defined spinal cord region adjacent to somites 6–10 at 30 h. We could not reliable count the number of cells expressing uncx4.1, due to the wek, punctate nature of the expression. Data are depicted as individual value plots with the n-values shown below. For each plot, the wider red horizontal bar indicates the mean number of cells, and the red vertical bar depicts the S.E.M. (both values are also listed in Table 1). All counts are an average of at least three embryos. Statistically significant comparisons are indicated with brackets and asterisks. *** P <0.001. * P <0.05. White circles indicate wild-type data and black circles indicate evx1;evx2 double mutant data. All data were analyzed for normality using the Shapiro-Wilk test. Data in L is not normally distributed and so a Wilcoxon-Mann-Whitney test was performed. Data sets in C, F, I and O are normally distributed and so the F-test for equal variances was performed, followed by a type 2 Student’s t-test (for equal variances). P-values are provided in Table 1. (C, F, I, L, O) There is a statistically significant reduction in the number of spinal interneurons expressing skor1a, skor1b, skor2 and ebf3a, but not uncx, in evx1;evx2 double mutant embryos. (A, B) skor1a, (D, E) skor1b and (P, Q) uncx4.1 in situ hybridization experiments were performed with the molecular crowding reagent Dextran Sulfate. This was omitted for the (G, H) skor2, (J, K), ebf3a and (M, N) uncx in situ hybridization experiments. Scale bar: 50 μm.