Continuous innervation is required for regulating ribbons in hair cells. Ribbon number and size was assayed in 5dpf larvae with mature neuromast hair cells, whose lateral line fibers were transected on one side of the larva, after the nerves had formed synapses with hair cells. Tg(neuroD:GFP) transgenic animals were used to visualize lateral line afferent fibers in vivo before and after transection. (A) Lateral line afferents in the uncut side of the larva as visualized by acetylated tubulin staining. (B) Lateral line afferents transected (cut) at the level of L1 neuromast in the opposite side of the larva (blue arrow). Cut and uncut larvae were fixed at 24h post transfection and stained for hair cells (green, anti-parvalbumin antibody), ribbons (magenta, anti-RibeyeB antibody) and afferent neurons (cyan, anti-GFP antibody). Projections of confocal microscope images of the uncut (C-C′′) and cut (D,D′) side were acquired at the level of L3, L4 and L5 neuromasts (NM) in the posterior lateral line (A). The absence of afferent fibers in neuromasts of the cut side is seen in D′, which is an overlay of RibeyeB (magenta) and anti-GFP antibody staining (no cyan staining was present), whereas neuromasts in the uncut side continue to be innervated by afferent fibers (C′′). (E-F′) Lateral views of surface renderings of neuromasts in C-D′ generated by Huygens software. In the denervated hair cell, the number of ribbons/cell increased (G), whereas the size of ribbons decreased (H,I). Ribbon size and number were obtained from the surface renderings of the neuromasts (see Materials and Methods). **P=0.0036, ***P=0.0006. Statistical analysis of ribbons (unpaired two tailed t-test) was performed on averages of averages - we calculated the average of ribbons per cell in a given neuromast and then averaged this number across six total neuromasts (three neuromasts in two different larvae). Posterior lateral line neuromasts had an average of 10 hair cells per neuromast in cut larvae and an average of 13 hair cells/neuromast in uncut larvae. Results are mean±s.e.m.
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