List of RT-qPCR primers used in this study.

tnfb is constitutively expressed in neuromasts during early development.

Tg(tnfb:eGFP-F);Tg(tnfb:mCherry-F) embryos were imaged during development using a Leica M205 stereo microscope. A) Earliest expression of transgenes was observed in neuromast (cyan arrows) at 32 hpf. The lateral line at this stage reaches to about halfway the length of the larvae. In some individuals, transgene-expressing cells were also observed in the extra-embryonic yolk sac. B) From 3 dpf onwards, transgene-expressing cells are visible in the neuromast along the lateral line, which at this stage extends to the tip of the tail. C) Representative maximum projection of a neuromast imaged with an Andor spinning disk confocal microscope at a 20x magnification. D) Representative images of zebrafish larvae acquired with a Zeiss Stemi 508 stereo microscope after wholemount in situ hybridisation of tnfb antisense (top panel), tnfb sense (negative control, middle panel) and mpx antisense probes (positive control, lower panel). Numbers following the probe name, indicate the number of larvae showing the depictured expression pattern and the total number of larvae analysed per condition. tnfb mRNA expression was detected in posterior neuromasts, matching the schematic drawing showing lateral neuromasts position in 3dpf zebrafish. tnfb sense probe showed no specific signal. mpx mRNA expression was mainly observed in the caudal hematopoietic tissue (CHT). E) Representative images of the trunk region from larvae processed in the same experiment as in D, reimaged using a Leica DMb6 microscope using a 20x magnification. Top panel: tnfb mRNA expression in neuromast, also visible in the inset focusing on a single neuromast (right). Middle panel: sense probe (negative control) showing no signal, confirming specificity of tnfb mRNA expression in neuromast. (*) indicates the position of hair cells in the centre of the neuromast. Lower panel: mpx mRNA expression in the CHT.

Mantle cells on the outer perimeter of neuromasts express tnfb:eGFP-F and tnfb:mCherry-F.

Neuromasts of either a Tg(tnfb:eGFP-F) (left panel) or of a Tg(tnfb:mCherry-F) (right panel) transgenic zebrafish larvae (3 dpf), were imaged with a Leica SP8-DIVE multiphoton microscope, at 20x magnification and 5x digital zoom. Top row: maximum projection of a neuromast in the trunk region (white arrowhead), showing tnfb:eGFP-F or tnfb:mCh-F expression localized on the outer perimeter of the neuromast, where transgene-expressing cells form a characteristic dome- or volcano-like shape, typical of mantle cells. Middle row (blue): orthogonal view, further detailing that transgene-expressing cells form a dome-like structure along the outer perimeter of neuromasts. Cells in the core and the top of the dome are tnfb:eGFP-F or tnfb:mCh-F negative. Bottom row (yellow): Single slice through the base of the neuromast, emphasizing a ring of tnfb:eGFP-F or tnfb:mCh-F positive cells around the neuromast, and the lack of transgene expression in the sensory and support cells at the centre of the dome-like structure.

Reporter lines Tg(tnfb:eGFP-F) and Tg(tnfb:mCherry-F) recapitulate transcriptional activation of tnfb in innate immune cells after wounding.

A-F) Larvae (2 dpf) were screened based on transgene expression in neuromasts. Caudal fins of 3 dpf larvae were amputated (amp) or left intact (control, ctrl). A) Confocal imaging of Tg(tnfb:eGFP-F;tnfb:mCherry-F). The top panel shows control caudal fin with few fluorescent cells. Grey dashed line indicates the outer perimeter of the caudal fin. White square delimits the wound site used for cell counting, defined by a width of 105 µm centred around the tip of notochord. White arrows indicate eGFP+mCherry- cells, blue arrows indicate eGFP+mCherry+ cells clearly increasing in number after wounding (lower panel). Asterisks (*), indicate neuromasts. B) Representative images of zebrafish larvae acquired with Zeiss Stemi 508 stereo microscope after wholemount in situ hybridisation of tnfb antisense (top panel), tnfb sense probes (negative control, lower panel). tnfb expression was detected in the amputated fin (black arrow). C) Kinetics of tnfb expression were determined at the wound site by manually counting transgene-expressing cells. Graphs show mean and SEM. Asterisks (*) indicates significant differences, at a given time point, between the same cell type in the amputated and intact group, as assessed by Two-Way ANOVA followed by Tukey’s post-hoc test. D) Individual date derived from the same tnfb expression data as C), at the indicated timepoints, number of cells per cell type are shown. Asterisks (*) indicates significant differences within treatment at the indicated timepoint, as assessed by Two-Way ANOVA followed by Tukey’s post-hoc test. E-F) Relative expression level of tnfb, tnfa, mpeg1.1, mfap4.2 and mpx in E) eGFP+- or F) mCherry+-sorted fractions 2-3 hpa from Tg(tnfb:eGFP-F) or Tg(tnfb:mCherry-F) larvae, using eef1a1l1 as a reference gene. Dots represent independent experiments; sorted fractions from the same experiment are connected. Asterisks (*) indicate significant differences as assessed by One-tailed Mann-Whitney test. G) Tg(tnfb:eGFP-F;tnfb:mCherry-F) larvae (3 dpf) were injected intramuscularly with 1 nl PVP or with 2x103 CFU E. coli crimson resuspended in PVP. Images were acquired 2 hpi with a Leica M205FA fluorescence stereo microscope with 61x zoom. The area around fluorescent reporter (dashed outline) was selected in FIJI for total fluorescent analysis. Asterisks (*), indicate neuromasts. H) Larvae were treated as in G. Corrected total cell fluorescence (CTCF) in PVP- or E. coli-injected individuals was measured within the dashed outlined area as described in G. Neuromasts were excluded from the total fluorescence quantification. Asterisks (*) indicate significant difference assessed by One-tailed Mann-Whitney test.

Macrophages and a subset of neutrophils express tnfb:mCh-F during wounding-induced inflammation.

Caudal fins of 3 dpf transgenic larvae were amputated (amp) or left intact (control, ctrl), and imaged with an Andor spinning disk confocal microscope using 20x magnification (n=5-11 larvae per group). To aid cell counting, Z-stacks of 21 µm were acquired at a 6-minute interval between 5.5-6.5 hpa. In each pair of panels (A-B through I-J), the upper panel shows a representative image either of a control or of an amputated caudal fin (Asterisks (*) indicate neuromast); the lower bar graph panel, shows the corresponding total cell count. A) Macrophages (mCherry+) and neutrophils (eGFP+) are recruited to the wound and increase in number compared to the control. C) The majority of macrophages (eGFP+) express tnfb (eGFP+mCherry+; white arrows), while few eGFP+mCherry-(mpeg1+tnfb-) cells can be seen within the yellow outline. The yellow inset shows the same field of view marked by the yellow outline, 12 minutes later, with improved visibility of eGFP+mCherry- cells (yellow arrow). E) Only a subpopulation of neutrophils (eGFP+) express tnfb (eGFP+mCherry+; blue arrow) in response to wounding. G) Macrophages (mCherry+) were the only cells expressing tnfa (mCherry+eGFP+) I). Macrophages (mCherry+) are the main producers of il1b (mCherry+eGFP+; white arrow), while few mCherry-eGFP+ (mpeg1- il1b+) cells can also be seen (blue arrow). B/D/F/H/J) Total cell count. Cells were counted manually within the grey dashed box. In B, asterisks (*) indicate significant differences between the control and amputated group within each cell type as assessed using Kluskal-Wallis test followed by a Dunn’s multiple comparison test; in D/F/H/J, significant differences were calculated within each cell type group, and between cell type groups within the same treatment as assessed using Kluskal-Wallis test followed by a Dunn’s multiple comparison test. Asterisks (*) indicates significant difference between the indicated groups.

Kinetics of tnfb, tnfa and il1b expression in innate immune cells after amputation.

Caudal fins of 3 dpf transgenic larvae were amputated (amp) or left intact (control, ctrl) and imaged during 9 hpa with an Andor spinning disk confocal microscope using 20x magnification (n=4-5 larvae per group). To aid manual counting of cells, Z-stacks of 21 µm were acquired at a 6-minute interval. A-B) Kinetics of expression of the indicated cytokines were determined at the wound site by manually counting transgene-expressing cells. Graphs show mean and SEM. Asterix (*) indicates significant differences between the same cell type in amputated and control fins at the same timepoint, as assessed by 2-WAY ANOVA with Geisser-Greenhouse correction for sphericity, followed by Tukey’s multiple comparisons test. C) Representative maximum projections of a control or amputated (grey outline) fin of Tg(tnfa:eGFP-F);Tg(tnfb:mCherry-F) larvae. Left panel shows a control fin; panels on the right show an amputated fin of the same individual at the indicated timepoints. Arrows indicate typical tnfa:eGFP-F+tnfb:mCh-F+ (cyan arrow) and tnfa:eGFP-F-tnfb:mCh-F+ (yellow arrow) cells. D) Tg(il1b:eGFP);Tg(tnfb:mCherry-F) larvae treated as in C. Arrows indicate typical il1b:eGFP+tnfb:mCh-F+ (cyan arrow), il1b:eGFP-tnfb:mCh-F+ (yellow arrows), and il1b:eGFP+tnfb:mCh-F- (white arrow) or immotile il1b:eGFP+tnfb:mCh-F - (grey arrow) cells.

Expression of tnfb, tnfa and il1b after E. coli injection.

Transgenic zebrafish (3 dpf) were injected intramuscularly with 1 nl PVP or with 2x103 CFU E. coli crimson resuspended in PVP, and images were acquired at 6 hpi with a Leica M205FA fluorescence stereo microscope using 58-61x digital zoom. In each pair of panels A-B) Tg(tnfa:eGFP-F);Tg(tnfb:mCherry-F) or C-D) Tg(il1b:eGFP);Tg(tnfb:mCherry-F), the left panel (A/C) shows representative images of PVP- or E. coli-injected larvae (Asterisks (*) indicate neuromast); the right panel (B/D) shows the corresponding total green or red fluorescence for the indicated cytokine-reporter measured within the dashed outlined area. Neuromasts were excluded from total fluorescence quantification. Asterisks (*) indicate significant difference assessed by one-tailed Mann-Whitney test.

Occurrence of innate immune cells expressing tnfb, tnfa and il1b after E. coli injection.

Transgenic zebrafish (3 dpf) were injected intramuscularly with 1 nl PVP or with 2x103 CFU E. coli crimson resuspended in PVP, and imaged at 2.5 hpi with an Andor spinning disk confocal microscope using 40x (A, C-E) or 20x (B) magnification. Representative maximum projections of PVP- and E. coli-injected larvae, using Z-stacks of 25-40 µm are shown. A) Macrophages (mCherry+) and neutrophils (eGFP+) are observed after PVP injection and in response to E. coli. B) Macrophages (eGFP+) express tnfb (eGFP+mCherry+; yellow arrow) and a second tnfb-expressing cell population is observed (eGFP-mCherry+; grey arrow) (Asterisks (*) indicate neuromast). C) Only a subpopulation of neutrophils (eGFP+) express tnfb (eGFP+mCherry+; yellow arrow) in response to E. coli. D) Macrophages (mCherry+) express tnfa (mCherry+eGFP+; yellow arrow), and E) Macrophages (mCherry+) express il1b (mCherry+eGFP+; yellow arrow), and mCherry-eGFP+ (mpeg1-il1b+; grey arrow) cells are observed.

Fluorescence-Activated Cell Sorting (FACS) gating strategy to identify tnfb-expressing cells.

Caudal fins of Tg(tnfb:eGFP-F) or Tg(tnfb:mCherry-F) larvae (3 dpf) were amputated, and 2-3 hpa FACS was used to sort eGFP+ and eGFP- fractions, or mCherry+ and mCherry- fractions, from whole larvae (n=300). Representative dot plots showing the gating strategy: A) Debris and dead cells were excluded based on the Forward (FSC-A) and Side Scatter (SSC-A) Area. B) Single cells were discriminated from doublets and clumps based on SSC-Width (SSC-W) vs SSC-Height (SSC-H) corresponding to Singlets 1 and C) by plotting FSC-Width (FSC-W) vs FSC-Height (FSC-H) corresponding to Singlets 2. D) Live cells were discriminated from dead cells based on Sytox Red viability staining, and Sytox Red–positive cells were excluded. E) Cells from wildtype larvae (Unlabelled cells), used to define gating boundaries for F) sorted eGFP+ and eGFP- fractions. G) Cells from wildtype larvae (Unlabelled cells), used to define gating boundaries for H) sorted mCherry+ and mCherry- fractions. To be more stringent on purity of sorted populations, gates have been designed to not overlap. Gene expression analysis for sorted populations is shown in main Figure 3E-F.

Wholemount in situ hybridization of mpx and tnfb mRNA in 3 dpf zebrafish.

A) Panel of tnfb mRNA expression. Larvae were scored positive when signal was visible in at least two neuromast of the posterior lateral line. Positions of lateral neuromasts in 3dpf zebrafish are depicted in the schematic drawing. 9 out of 10 larvae were positive, only A9 did not meet this criterium. B) tnfb sense probe did not show any specific signal in the neuromast. B1 did not show any coloration, different from the remaining larvae where some coloration in the head was observed. C) Panel of mpx mRNA expression. Larvae were scored positive when signal was visible in the caudal hematopoietic tissue (CHT). All larvae (8/8) met this criterium. Black outlined are the images of larvae also shown in main Figure 1D.

Mantle cells in homeostatic neuromasts or soon after hair cell ablation are enriched in tnfb.

Single-cell RNA-sequencing data of homeostatic neuromasts (A-C) was retrieved from Zebrafish Neuromast scRNAseq, and data from neuromasts at multiple timepoints after hair cell (HC) ablation (D-E) was retrieved from Neuromast regeneration scRNAseq. A) Heatmap of mantle cell’s lineage markers, including tnfb. Heat bar shows log2 fold change. Numbers 1-14 indicate cell clusters. Clusters under the same lineage represent a different developmental stage, for example clusters 3-5 are progenitor, young and mature hair cells, respectively. For the other lineages, this distinction is not as clear, including clusters 1-2, both marking the development of mantle cells. A/P: anterior-posterior, D/V: dorsal-ventral. B) t-distributed Stochastic Neighbour Embedding (t-SNE) plot of tnfb-expressing cells in blue, which are mostly located in the mantle cell’s lineage clusters 1 and 2. C) Violin plot of normalised tnfb expression per cell cluster. Mantle cell cluster 1 contains the highest number of tnfb-expressing cells and is most enriched with this gene. D) t-SNE plot of tnfb-expressing cells from homeostatic and from neuromast cells (0-10 hours) post HC ablation. tnfb-expressing cells are located within the cluster of mantle cells and in some cells from the central support cell cluster. Heat bar shows log2 fold change. E) Heatmap of tnfb expression within each cell cluster and at various timepoints after HC ablation. Homeostatic (ctrl) mantle cells are enriched in tnfb mRNA, and directly after HC ablation (0h), tnfb transcripts increase in mantle cells. At the same timepoint, tnfb transcripts also increase in central support cells. F) Violin plot of normalised tnfb expression within mantle cells in homeostatic (ctrl) cells and at several timepoints post-HC ablation. tnfb transcripts increase early and transiently following HC ablation.

Wholemount in situ hybridization of tnfb mRNA in zebrafish after wounding.

Wholemount in situ hybridization with tnfb antisense and sense probes in 3 dpf larvae, 2 hours after caudal fin fold amputation (black arrows). A) Panel of tnfb mRNA expression. Larvae were scored positive when signal was visible in the tail. B) tnfb sense probe did not show any specific signal, some coloration in the head was observed. Black outlined are the images of larvae also shown in main Figure 3B.

Innate immune cells of developing zebrafish are enriched in tnfb and il1b, and not tnfa transcripts.

Single-cell RNA-sequencing performed across multiple zebrafish developmental stages (3 – 120 hpf) was retrieved from Daniocell database and plots were generated using the web interface (A/C) or the companion application Daniocell Desktop (B). A) t-distributed Stochastic Neighbour Embedding (t-SNE) plots of cytokines tnfb, tnfa, and il1b in all cells, accompanied by a panel, plotted to the same scale, zoomed in on four innate immune cell clusters: 1. neutrophils, 2. macrophages, 3. microglia, 4. hematopoietic stem cell (myeloid). While tnfb and il1b are enriched at varying levels in macrophages, microglia, haematopoietic stem cells (myeloid) and in neutrophils, tnfa could only be detected at a low level in a few macrophages. B) Co-expression of tnfb/tnfa/il1b transcripts. Matrix indicates relative expression level per cell. Only a few macrophages are tnfa+tnfb+ or tnfa+il1b+. In contrast, tnfb+il1b+ cells are present in all four cell clusters, including in a subpopulation of neutrophils. C) Mean enrichment of cytokine transcripts across multiple developmental stages (3 – 120 hpf) in lateral line, periderm (non-immune tissue) and in immune cells. tnfb is initially detected in periderm, prior to the emergence of immune cells, and by 14-21 hpf onwards tnfb transcripts are enriched in lateral line and immune cells. Enrichment of il1b is also evident in immune cells. At the bottom, the timeline indicates the emergence of hematopoietic stem cells (myeloid), followed by differentiation and appearance of neutrophils and macrophages.