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Figure 1 Expression of Semaphorin7A in the zebrafish’s lateral line. (A) A volumetric rendering of a PLL neuromast depicts the sensory axons (magenta) that branch from the lateral line nerve to arborize around the basolateral surface of the hair cell cluster (green). Additional cell types in the neuromast are not labeled. Larval age, 3 dpf. A=anterior, P=posterior, D=dorsal, V=ventral. (B) A schematic drawing of the two variants of the Sema7A protein molecule depicts the full-length GPI-anchored form and the smaller, potentially secreted form. Both the molecules include a signal sequence (green) and a conserved sema domain (orange). (C) Tg(myo6b:actb1-EGFP) larvae at 4 dpf were terminally anesthetized and their heads were removed with fine blades. The tails were then dissociated into the component cells, which were sorted by flow cytometry. Green cells represent GFP+ hair cells. A cDNA library was prepared from the isolated RNA. (D) Gel-based RT-PCR analysis indicates the presence of both the sema7a transcript variants and the expression of hair cell-specific genes, parvalbumin 8 (pvalb8) and S100 calcium binding protein T (s100t). (E) A surface micrograph of a neuromast at 3 dpf depicts two pairs of mature hair cells (green dashed lines) and a pair of immature hair cells (grey dashed lines). Inset: among the three pairs of hair cell apices, the immature pair is indicated by arrowheads. Immunolabeling reveals that the Sema7A protein (orange) occurs consistently at the subapical region (arrowheads) and at the basolateral surface (arrows) of a hair cell. In this and in each of the subsequent neuromast images, anterior is to the left and dorsal to the top. (F) A plot quantifies developmental changes in the average Sema7A intensity in both rostrally and caudally polarized hair cells of neuromasts from 1.5 dpf to 4 dpf. The data stem from 18, 36, 39, and 40 hair cells in neuromasts of respectively 1.5 dpf, 2 dpf, 3 dpf, and 4 dpf larvae. (G) A plot quantifies the distribution of average Sema7A intensity along the hair cell’s apicobasal axis. The results stem from 52, 57, and 54 hair cells of neuromasts from 2 dpf, 3 dpf, and 4 dpf larvae. (H) An immunofluorescence image at the nuclear level of a 4 dpf neuromast shows the contact of the sensory arbors (magenta) with the basolateral surface of the hair cells (green). Immunolabeling for Sema7A (orange) reveals enrichment of the protein at the hair cell bases and sensory-axon interfaces (arrowheads). (I) At the base of the hair cell, association of the td-Tomato+ sensory arbors positively correlated with the Sema7A intensity. The results stemmed from 48 hair cells of 13 neuromasts from 3 dpf larvae. HC, hair cell; scale bars, 5 µm; au, arbitrary unit; means ± SEMs; ρ, Spearman’s correlation coefficient; *** implies p<0.001 and ** implies p<0.01.