Fig. 3

Spinal 5-HT antibody labeling transitions from pet1:EGFP- to pet1:EGFP+ cells during development. Tg(-3.2pet1:EGFP)^ne0214 larvae were labeled with antibodies to 5-HT every 4 h between 56 and 80 hpf. Confocal Z-stacks were collected from the midbody region of the spinal cord (centered on body segment 15; dashed lines in A represent the dorsal and ventral boundaries of the spinal cord, approximately the same location in all panels). White arrows indicate cells that expressed pet1:EGFP and were not labeled with 5-HT antibodies, white arrowheads indicate cells in which pet1:EGFP expression and 5-HT colocalized, and black arrows indicate cells that did not express pet1:EGFP but were faintly labeled with 5-HT antibodies. A-C: 5-HT (B) was detected in cells in the ventral spinal cord before expression of pet1:EGFP (A, merge in C). D-F: Pet1:EGFP expression (D) was initially observed at 60 hpf in cells that did not colabel with 5-HT (E, merge in F; white arrows). G-L: At 64 and 68 hpf, a subset of pet1:EGFP+ cells (G, J) were 5-HT+ (H, K, merge in I, L; white arrowheads). 5-HT antibodies continued to detect pet1:EGFP- cells in the ventral spinal cord at 64 and 68 hpf (red cells in merged panels I, L). M-R: Colabeling of cells with pet1:EGFP (M, P) and 5-HT antibodies (N, Q) continued at 72 and 76 hpf (merge in O, R). The number of pet1:EGFP-/5-HT+ cells detected, and the intensity of 5-HT antibody labeling in these cells, were both reduced (black arrows). S-U: 5-HT antibody labeling (T) of cell somas was restricted to pet1:EGFP+ cells (S) in the ventral spinal cord at 80 hpf (merge in U). Scale bar = 50 µm.