Fig. 4

Effect of nicotinamide on the organization of the trunk musculature and the formation of the sarcomeres. At ~ 17 hpf, embryos had the terminal portion of the tail excised and then they were treated with (A) Danieau's solution alone or Danieau's solution containing nicotinamide at: (Bi) 1 mM, (Bii) 10 mM, or (Biii) 50 mM. All the embryos were fixed at ~ 24 hpf and dual-labelled with phalloidin and the F59 antibody, to visualize F-actin (in green) and myosin heavy chain (in red) in the trunk musculature, respectively. The panels show series of optical sections projected as single images at (A, Bi–Biii) low and (A*, Bi*–Biii*) higher magnification when the green and red channels are merged; overlapping regions are shown in yellow. The higher magnification images of the slow myofibers reveal the presence or absence of the sarcomeric banding pattern. The white arrows in panel (Biii) show the disorganized nature of some slow myofibers. Ant. and Pos. are anterior and posterior, respectively. Scale bars, 50 µm (in panels A, Bi–Biii), and 2 µm (in panels A*, Bi*–Biii*). (C–F) In order to determine the level of disruption on slow muscle cell development following nicotinamide treatment, various dimensions of the trunk musculature and slow myofibers were measured. These bar graphs show the mean ± SEM: (C) myotome width (all n = 15, from 5 embryos), (D) somite angle (n = 15, from 5 embryos), (E)myofiber length:somite length ratio (n = 60 from 5 embryos); and (F) myofiber width (n = 50, from 5 embryos). The black dashed line in panel (E) indicates a myofiber:somite length ratio of 1. (Gi) The NAADP cycling assay was employed to detect the endogenous level of NAADP in whole embryo extracts that were either untreated, or treated with nicotinamide or benzamide. This bar graph shows the normalized [NAADP] detected in sample extracts and the corresponding fold-change in [NAADP] (n = 3). (Gii) Chemical structure of nicotinamide and benzamide. In panels (C–G), the asterisks indicate statistically significant differences at p < 0.05 (*) and p < 0.001 (***), whereas NS indicates that no significant difference was observed.