Fig. S5

In vivo bromodeoxyuridine labeling of 1- and 3-day postfertilization rtn4 morphant embryos. (A) Overview of the bromodeoxyuridine (BrdU) pulse chase experiment. To maximize the labeling of cells entering the S-phase, a 1-hour BrdU pulse was applied at 1 dpf. Half of the embryos were fixed immediately after the pulse (B), (D), (F), (J), (K) and (L), and the other half were fixed 2 days later (C), (E), (G), (M), (N) and (O). Confocal maximum projections of midbrain and hindbrain sections showed a considerable amount of BrdU-labeled cells at 1 dpf (green) (B), (D), (F), (J), (K) and (L). Nuclei were counterstained with 42,6-diamidino-2-phenylindole (DAPI) (red). At 1 dpf, the presumptive tectum of rtn4a morphants (D), especially rtn4b morphants (F), is reduced in size relative to control embryos (B). (C), (E), (G), (M), (N) and (O) BrdU retention in the tectum and hindbrain at 3 dpf. In addition to the reduced tectum in 1-dpf rtn4a and rtn4b morphants, cells in the 3-dpf rtn4b morphants show strong BrdU signaling 2 days after the BrdU chase (G) and (O). Only weak and diffused BrdU signaling was detected in rtn4a (E) and (N) and control (C) and (M) groups. (H) and (P) Quantification of total cells (red) and proliferating cells (green) in the tectum and hindbrain at 1 dpf in rtn4a and rtn4b morphants and the control group. (I) Quantification of total cells in the tectum at 3 dpf (red). (Q) Quantification of total (red) vs. BrdU-positive cells (yellow) in analyzed areas of the hindbrain at 3 dpf in rtn4a and rtn4b morphants and the control group.*Melanocytes. Control (1 dpf; n = 8), rtn4a (n = 11), rtn4b (n = 12), control 2 dpf (n = 6), rtn4a (n = 6) and rtn4b (n = 14). Scale bar = 50 μm.