Fig 2: FGF affects the proportion of symmetric proliferative divisions in RG culture. (A) Plot of the average value of pp-dd of the population of RG under SC (red) and SC+FGF (blue) conditions. Areas around the curves represent the 50% confidence interval. (B) Representative images showing Sox2 and EdU (stained in green and red, respectively) for ‘pulse’ and ‘chase’ time points. (C) Quantification of the number of Sox2- and EdU-positive cells for time-points for SC and SC+FGF conditions. Error bars correspond to s.e.m. between independent repeats of the experiment.

Fig 3: Afferent terminals within the regenerating neuromast colocalise with supporting cells A, Confocal images showing the afferent fibres (magenta: 1), hair cells (blue: 2) and supporting cells (cyan: antibody anti-Sox2: 3) within a neuromast from control and copper-treated zebrafish (2 hr incubation with 30 µM copper). The next three columns are merge images showing: afferent fibres and hair cells (4); afferent fibres and supporting cells (5); and afferent fibres, hair cells and supporting cells (6). The two images in the “expanded” column (7) highlight the presence of afferent terminals in close proximity to supporting cells. Experiments were done using 3 dpf Tg(NeuroD:EGFP; Tg(Myosin6b:R-GECO) zebrafish. Scale bar: 10 µm. B, Percentage of neuromast showing afferent terminals as a function of hpt. Control neuromasts were only analyzed at 10 and 24 hpt. C, Number of supporting cells per neuromast obtained in control (black) and copper-treated (red) zebrafish has a function of hpt. At 10 hpt, control vs copper: P = 0.1668; At 24 hpt, control vs copper: P = 0.9998, one-way ANOVA, Tukey’s post-test. D, Confocal images as described in panel A, for 5, 7 and 10 hpt neuromasts showing some additional examples of afferent terminals in the proximity of the supporting cells. Scale bar: 10 µm.

Fig 4: Afferent terminals and supporting cells are less sensitive to copper damage than hair cells A, Confocal images showing the afferent fibres and terminals (magenta: Tg(NeuroD:EGFP) zebrafish line), hair cells (blue: Tg(Myosin6b:R-GECO) zebrafish line) and supporting cells (cyan: anti-Sox2 antibody) within a neuromast from control and copper-treated zebrafish (2 hours incubation with 10, 30 or 50 µM copper sulphate). Experiments were done using 3 dpf zebrafish. Scale bar: 10 µm. B-D, Number of afferent terminals (B), hair cells (C) and supporting cells (D) per neuromast after the application of different concentrations of copper. The presence of afferent terminals was defined as an enlargement of the fibre within the neuromast region (presence of afferent terminals was scored as 100; absence with 0, which were then converted in % based on the number of neuromast investigated). For all three comparisons in panels B-D, P < 0.0001, one-way ANOVA.

Fig 5: Correlation of scATAC-seq peaks to conserved regions and to differentially expressed genes(A) Venn diagram of regeneration-responsive elements (RREs) identified in SCs, PCs, and HCs.(B) Schematic of the approach used to link scATAC-seq peaks in proximal and distal DNA elements to genes.(C) Schematic of predicted links between peaks near the gene, sox2. For each cell type, a track is shown, including peaks present in non-regenerating controls (SS, steady state), conserved non-coding elements (CNEs) from genomic four-way phastcons (Z, zebrafish; C, grass and common carp; GF, goldfish), and the deleted region in sox2hg138 mutants. Genome coordinates of peaks, CNEs, and deleted region are represented by black bars.(D) Bar graphs showing the number of RREs that are within 50 kb of differentially expressed genes (blue) and overlap with CNEs (red) in each cell type.(E) Intersections of differentially expressed (DE) genes with peaks and peaks with CNEs are statistically significant (p < 0.001).