Superficial neuromast pattern and number is severely disrupted in the absence of innervation. (A–A′) Confocal images of the anterior LL anterodorsal (gAD) and anteroventral (gAV) ganglia labeled by Tg(Hgn39d:GFP) (yellow ovals) before (A) and immediately after (A′) laser ablation at 4 dpf. Scale = 50 µm. (B–D′) Images of a representative single 12 mm specimen that had undergone unilateral anterior LL ganglia ablation. (B) Dorsal view showing both control and ablated sides labeled with TMRE. The supraorbital superficial line (red bracket) is labeled parallel to the supraorbital canal line (yellow arrows) along with the anterior pit line (red asterisk). Scale = 1 mm. (C–C′) Lateral views showing control (C) and ablated (C′) sides. Yellow arrowheads indicate areas where superficial neuromast patterning was strongly reduced compared to the control side. The white boxes indicate the area magnified in (D–D′). Scale = 1 mm. (D–D′) Higher magnification confocal (10× objective) view of the opercular superficial neuromast line in the same specimen immunolabeled for Sox2, showing control (D) and ablated (D′) sides. Neuromasts are indicated with yellow arrowheads. Scale = 200 µm (E) Box plot showing change in canal neuromast number between the ablated side and internal unablated control side (n = 10). (F) Box plot showing change in superficial neuromast number between the ablated side and unablated control side (n = 10). Gray lines indicate paired values. A Shapiro–Wilk test was performed on all data to confirm normality before performing a paired t‐test. Not significant (ns) = p > 0.05; ****p < 0.0001. (G–G′) epifluorescent microscopy lateral views showing control (G) and ablated (G´) side nerves labeled by Tg(Hgn39d:GFP). Red boxes indicate the middle ganglion (gM) of the posterior LL system of each side, and blue boxes indicate the anteroventral ganglion (gAV). (G) Blue arrowheads indicate nerve tracts from gAV (blue box) innervating supraorbital and infraorbital superficial neuromasts (black dots), while gM is not visible (red box). (G′) Red arrowheads indicate nerve tracts from gM (red box) ectopically innervating supraorbital and infraorbital superficial neuromasts, while gAV has been ablated (blue box). Scale = 1 mm. e, eye, n, nostril, op, operculum.
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