Fig. 3

Antibody staining reveals a decrease in spinal interneurons. (A–H) Lateral views of whole-mount zebrafish spinal cord stained with anti-Hu antibody in wild-type fish at 1, 2, and 3 days in development (A–C), α2 mismatch AMO-injected (α2 MM) fish at 2 days in development (D), GlyR α2 knockdown (α2 k/d) fish at 1, 2, and 3 days in development (E–G), and α1 knockdown fish (α1 k/d) at 2 days in development (H). (I–K) Anti-GFP antibody labeling of neurog1-positive Rohon–Beard sensory neurons (labeled with arrowheads) of wild-type (I), GlyR α2 knockdown (J; α2 k/d), GlyR α2 mismatch AMO-injected (K; MM), and α1 knockdown (L; α1 k/d) zebrafish spinal cords. (M–P) Lateral view of Pax 2 (green) labeling of interneurons and HB9 (red) labeling of secondary motoneurons in 2-day zebrafish spinal cord of wild-type (M), GlyR α2 knockdown (N; α2 k/d); GlyR α2 mismatch AMO-injected (O; α2 MM), and α1 knockdown (P; α1 k/d) zebrafish spinal cords. Dotted lines demarcate dorsal (D) and ventral (V) limits of the spinal cord. (Scale bar: 30 μm.) (Q) Graph of average number of Hu-labeled cells in 100-μm segments of spinal cord between days 1 and 3 in development for wild-type (open circles, broken line) and GlyR α2 knockdown (α2 k/d; solid circles, solid line) fish. The number of Hu-labeled neurons in wild-type fish was significantly different from GlyR α2 knockdown fish at each developmental stage (P = 2 × 10^-3 at day 1; P = 4 × 10^-5 at day 2; P = 3 × 10^-5 at day 3). (R) Mean number of neurog1-labeled (gray bars), HB9-labeled (white bars), and Pax 2-labeled (black bars) cells in a 100-μm section of spinal cord for each experimental condition. n values from which means were taken are illustrated below (for wild type) and above (for GlyR α2 k/d) each circle in M and above each bar in N. _**, P = 10^-7.