Distinct progenitor populations mediate regeneration in the zebrafish lateral line
- University of Washington, United States
Figures
Figure 1
Hair cell progenitors are replenished via proliferation of other support cells.
(A, F) Timelines of single-ablation (A) and double-ablation (F) proliferation experiments. (B) Maximum projections of mock- (Mock) and neomycin-treated (Neo) neuromasts. EdU-positive cells are shown in magenta, anti-Parvalbumin-stained hair cells are shown in green, and DAPI-stained nuclei are shown in blue. Arrowheads indicate EdU-positive support cells. Scale bar = 10 μm. (C) Percentage of hair cells per neuromast labeled by EdU. Mock: 6.11 ± 8.69, n = 50 neuromasts (10 fish); Neo: 78.24 ± 20.69, n = 45 neuromasts (nine fish); mean ± SD; Mann Whitney U test, p<0.0001. (D) Total EdU-positive cells per neuromast. Mock: 2.78 ± 1.84, n = 50 neuromasts (10 fish); Neo: 8.18 ± 3.07, n = 45 neuromasts (nine fish); mean ± SD; Mann Whitney U test, p<0.0001. (E) Percentage of EdU-positive cells per neuromast that are either hair cells or support cells. Mock: 29.73% hair cells, 70.27% support cells, n = 50 neuromasts (10 fish); Neo: 72.02% hair cells, 27.98% support cells, n = 45 neuromasts (nine fish); mean ± SD. (G–N) Individual slices of a neuromast following two regenerations at two different planes: apical hair cell layer (G–J) and basal support cell layer (K–N). EdU (visualized by a Click-iT reaction) is labeled in magenta, BrdU (anti-BrdU) is labeled in cyan, and myo6b:GFP hair cells are labeled in green. Arrowheads indicate EdU/BrdU-positive hair cells, and asterisks indicate EdU-positive support cells. Scale bar = 10 μm.
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Figure 1—source data 1
Hair cell progenitors are replenished via proliferation of other support cells
- https://doi.org/10.7554/eLife.43736.004
Figure 2 with 3 supplements
Genetic labeling of distinct support cell populations.
(A, C, E) Maximum projections of neuromasts from sfrp1a:nlsEos (Peripheral, A), tnfsf10l3:nlsEos (AP, C), and sost:nlsEos (DV, E) fish. Converted nlsEos-positive cells are shown in magenta, and brn3c:GFP-positive hair cells are shown in green. Scale bar = 10 μm. (B, D, F) Percentage of hair cells per neuromast labeled by Peripheral (B), AP (D), and DV cells (F) at 5 and 8 dpf. (B) 5 dpf: 19.04 ± 19.86, n = 50 neuromasts (10 fish); 8 dpf: 19.46 ± 19.44, n = 50 neuromasts (10 fish); mean ± SD; Mann Whitney U test, p=0.7047. (D) 5 dpf: 5.71 ± 8.22, n = 50 neuromasts (10 fish); 8 dpf: 6.36 ± 9.57, n = 50 neuromasts (10 fish); mean ± SD; Mann Whitney U test, p=0.9668. (F) 5 dpf: 38.93 ± 13.46, n = 50 neuromasts (10 fish); 8 dpf: 55.78 ± 14.13, n = 50 neuromasts (10 fish); mean ± SD; Mann Whitney U test, p<0.0001.
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Figure 2—source data 1
Genetic labeling of distinct support cell populations
- https://doi.org/10.7554/eLife.43736.010
Figure 2—figure supplement 1
Asymmetry of support cell transgene expression in secondary neuromasts is orthogonal to primary neuromasts.
(A–F) Maximum projections of lateral views of trunks (taken at 20x zoom) (A,D) or of individual neuromasts (B–C, E–F) from tnfsf10l3:nlsEos (A–C), and sost:nlsEos fish (D–F). Unconverted nlsEos-positive cells are shown in green. In Low Mag images (A,D), primary neuromasts are labeled with 1°, and secondary neuromasts are labeled with 2°. Scale bar = 10 μm.
Figure 2—figure supplement 2
Support cell transgenes are not expressed in hair cells.
(A–C) Maximum projections of neuromasts from Tg[sfrp1a:GFP]w222 (Peripheral, A), Tg[tnfsf10l3:GFP]w223 (AP, B), and sost:NTR-GFP (DV, C) fish. GFP-positive cells are shown in green, and hair cells are shown in magenta via myo6:mKate2. In all three populations, there is no GFP expression in hair cells. Scale bar = 10 μm.
Figure 2—figure supplement 3
Expression of support cell transgenes does not alter hair cell development or regeneration.
(A) Total number of FM 1-43FX-labeled hair cells per neuromast at 5 dpf or following hair cell regeneration (72 hpt) in sfrp1a:nlsEos fish (nlsEos) and non-transgenic siblings (Sib). 5 dpf: 13.28 ± 1.32 (Sib) vs. 12.72 ± 1.93 (nlsEos), n = 25 neuromasts each (5 fish each); Regeneration: 6.76 ± 1.55 (Sib) vs. 7.52 ± 1.56 (nlsEos), n = 25 neuromasts each (5 fish each); mean ± SD; Mann Whitney U test, p=0.1414 (5 dpf), p=0.0658 (Regeneration). (B) Total number of FM 1-43FX-labeled hair cells per neuromast at 5 dpf or following hair cell regeneration (72 hpt) in tnfsf10l3:nlsEos fish (nlsEos) and non-transgenic siblings (Sib). 5 dpf: 12.88 ± 1.56 (Sib) vs. 12.64 ± 2.25 (nlsEos), n = 25 neuromasts each (five fish each); Regeneration: 9.04 ± 1.49 (Sib) vs. 8.72 ± 1.31 (nlsEos), n = 25 neuromasts each (five fish each); mean ± SD; Mann Whitney U test, p=0.9338 (5 dpf), p=0.2722 (Regeneration). (C) Total number of FM 1-43FX-labeled hair cells per neuromast at 5 dpf or following hair cell regeneration (72 hpt) in sost:nlsEos fish (nlsEos) and non-transgenic siblings (Sib). 5 dpf: 12.52 ± 1.76 (Sib) vs. 12.32 ± 1.46 (nlsEos), n = 25 neuromasts each (five fish each); Regeneration: 8.80 ± 1.41 (Sib) vs. 8.64 ± 1.35 (nlsEos), n = 25 neuromasts each (five fish each); mean ± SD; Mann Whitney U test, p=0.4527 (5 dpf), p=0.5396 (Regeneration).
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Figure 2—figure supplement 3—source data 1
Expression of support cell transgenes does not alter hair cell development or regeneration
- https://doi.org/10.7554/eLife.43736.009
Figure 3
Distinct support cell populations have different regenerative capacities.
(A) Timeline of nlsEos fate mapping experiment. Fish were photoconverted at 5 dpf, treated with neomycin, then fixed and imaged 72 hr post treatment (8 dpf). (B, C, D) Maximum projections of neuromasts from sfrp1a:nlsEos (Peripheral, B), tnfsf10l3:nlsEos (AP, C), and sost:nlsEos (DV, D) fish following photoconversion and hair cell regeneration. Converted nlsEos-positive cells are shown in magenta, and brn3c:GFP-positive hair cells are shown in green. Arrowheads indicate nlsEos-positive hair cells. Scale bar = 10 μm. (E) Percentage of hair cells per neuromast labeled by nlsEos following regeneration. Sfrp1a:nlsEos (Peripheral): 3.59 ± 8.87, n = 50 neuromasts (10 fish); tnfsf10l3:nlsEos (AP): 20.28 ± 20.58, n = 50 neuromasts (10 fish); sost:nlsEos (DV): 60.87 ± 12.37, n = 50 neuromasts (10 fish); mean ± SD; Kruskal-Wallis test, Dunn’s post-test, p=0.003 (Peripheral vs. AP), p<0.0001 (Peripheral vs. DV, AP vs. DV). (F) Total nlsEos-positive support cells per neuromast prior to hair cell ablation. Sfrp1a:nlsEos (Peripheral): 14.30 ± 4.17, n = 50 neuromasts (10 fish); tnfsf10l3:nlsEos (AP): 22.8 ± 4.40, n = 50 neuromasts (10 fish); sost:nlsEos (DV): 23.86 ± 4.45, n = 50 neuromasts (10 fish); mean ± SD; Kruskal-Wallis test, Dunn’s post-test, p<0.0001 (Peripheral vs. AP, Peripheral vs. DV), p>0.9999 (AP vs. DV).
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Figure 3—source data 1
Distinct support cell populations have different regenerative capacities
- https://doi.org/10.7554/eLife.43736.012
Figure 4
Notch signaling differentially regulates support cell populations.
(A, E, I) Maximum projections of neuromasts expressing sfrp1a:nlsEos (Peripheral, A), tnfsf10l3:nlsEos (AP, E), and sost:nlsEos (DV, I) following Notch-inhibited hair cell regeneration. Converted nlsEos-positive cells are shown in magenta, and brn3c:GFP-positive hair cells are shown in green. Scale bar = 10 μm. (B) Total number of hair cells per neuromast in sfrp1a:nlsEos fish following hair cell regeneration. Neo: 10.28 ± 1.88, n = 50 neuromasts (10 fish); Neo/LY: 19.07 ± 6.79, n = 46 neuromasts (10 fish); mean ± SD; Mann Whitney U test, p<0.0001. (C) Sfrp1a:nlsEos-positive hair cells per neuromast following hair cell regeneration. Neo: 0.62 ± 1.28, n = 50 neuromasts (10 fish); Neo/LY: 1.15 ± 2.16, n = 46 neuromasts (10 fish); mean ± SD; Mann Whitney U test, p=0.2481. (D) Percentage of sfrp1a:nlsEos-labeled hair cells per neuromast following hair cell regeneration. Neo: 6.31 ± 13.83, n = 50 neuromasts (10 fish); Neo/LY: 4.95 ± 8.82, n = 46 neuromasts (10 fish); mean ± SD; Mann Whitney U test, p=0.5148. (F) Total number of hair cells per neuromast in tnfsf10l3:nlsEos fish following hair cell regeneration. Neo: 8.84 ± 1.75, n = 50 neuromasts (10 fish); Neo/LY: 22.93 ± 5.45, n = 40 neuromasts (eight fish); mean ± SD; Mann Whitney U test, p<0.0001. (G) Tnfsf10l3:nlsEos-positive hair cells per neuromast following hair cell regeneration. Neo: 2.22 ± 1.94, n = 50 neuromasts (10 fish); Neo/LY: 11.38 ± 4.23, n = 40 neuromasts (eight fish); mean ± SD; Mann Whitney U test, p<0.0001. (H) Percentage of tnfsf10l3:nlsEos-labeled hair cells per neuromast following hair cell regeneration. Neo: 25.19 ± 21.72, n = 50 neuromasts (10 fish); Neo/LY: 50.68 ± 19.23, n = 40 neuromasts (eight fish); mean ± SD; Mann Whitney U test, p<0.0001. (J) Total number of hair cells per neuromast in sost:nlsEos fish following hair cell regeneration. Neo: 10.94 ± 2.23, n = 50 neuromasts (10 fish); Neo/LY: 27.06 ± 6.90, n = 48 neuromasts (10 fish); mean ± SD; Mann Whitney U test, p<0.0001. (K) Sost:nlsEos-positive hair cells per neuromast following hair cell regeneration. Neo: 7.40 ± 2.13, n = 50 neuromasts (10 fish); Neo/LY: 15.25 ± 6.36, n = 48 neuromasts (10 fish); mean ± SD; Mann Whitney U test, p<0.0001. (L) Percentage of sost:nlsEos-labeled hair cells per neuromast following hair cell regeneration. Neo: 67.86 ± 14.63, n = 50 neuromasts (10 fish); Neo/LY: 54.69 ± 14.01, n = 48 neuromasts (10 fish); mean ± SD; Mann Whitney U test, p<0.0001.
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Figure 4—source data 1
Notch signaling differentially regulates support cell populations
- https://doi.org/10.7554/eLife.43736.014
Figure 5
Differences in overlap between sost:NTR-GFP and sost:nlsEos populations.
(A–B) Maximum projections of neuromasts from sost:NTR-GFP; sost:nlsEos fish at 3 dpf (A) and 5 dpf (B). Sost:NTR-GFP cells are shown in green and sost:nlsEos cells are shown in magenta. Arrowheads indicate cells expressing sost:nlsEos but not sost:NTR-GFP. Scale bar = 10 μm. (C) Support cells per neuromast expressing either NTR-GFP and nlsEos (green) or nlsEos only (magenta) at 3 dpf and 5 dpf. NTR-GFP/nlsEos: 9.04 ± 2.39 (3 dpf) vs. 8.47 ± 2.27 (5 dpf), n = 49 neuromasts each (10 fish each); nlsEos only: 6.10 ± 2.27 (3 dpf) vs. 10.86 ± 2.72 (5 dpf), n = 49 neuromasts each (10 fish each); mean ± SD; Kruskal-Wallis test, Dunn’s post-test, p>0.9999 (NTR-GFP/nlsEos 3 dpf vs. 5 dpf), p<0.0001 (nlsEos only 3 dpf vs. 5 dpf). (D–F) Maximum projections of neuromasts from sost:NTR-GFP; sost:nlsEos fish following mock treatment (D; Mock), Mtz at 5 dpf (E; Mtz5), and Mtz at 3 dpf and 5 dpf (F; Mtz3/5). Sost:NTR-GFP cells are shown in green and sost:nlsEos cells are shown in magenta. Scale bar = 10 μm. (G) Support cells per neuromast solely expressing sost:nlsEos following Mtz treatment. Mock: 11.18 ± 2.04, n = 50 neuromasts (10 fish); Mtz5: 9.72 ± 2.03, n = 50 neuromasts (10 fish); Mtz3/5: 6.76 ± 2.12, n = 50 neuromasts (10 fish); mean ±SD; Kruskal-Wallis test, Dunn’s post-test, p=0.0288 (Mock vs. Mtz5), p<0.0001 (Mock vs. Mtz3/5, Mtz5 vs. Mtz3/5).
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Figure 5—source data 1
Differences in overlap between sost:NTR-GFP and sost:nlsEos populations
- https://doi.org/10.7554/eLife.43736.016
Figure 6 with 2 supplements
Ablation of DV cells decreases number of regenerated hair cells.
(A) Timeline of DV cell-ablation experiment. Larvae were treated with Mtz at 3 dpf, photoconverted, then treated with neomycin, then treated with Mtz again at 5 dpf, and fixed and immunostained at 72 hpt (8 dpf). (B–D) Maximum projections of neuromasts from sost:NTR-GFP; sost:nlsEos fish following neomycin (B; Neo), neomycin and Mtz (C; Neo/Mtz5), and Mtz, neomycin, and Mtz treatments (D; Mtz3/Neo/Mtz5). Sost:NTR-GFP cells are shown in green, sost:nlsEos cells are shown in magenta, and anti-Parvalbumin-stained hair cells are shown in cyan. Scale bar = 10 μm. (E) Total hair cells per neuromast following regeneration. Neo: 11.73 ± 2.10, n = 49 neuromasts (10 fish); Neo/Mtz5: 9.33 ± 1.88, n = 39 neuromasts (8 fish); Mtz3/Neo/Mtz5: 7.52 ± 1.74, n = 50 neuromasts (10 fish); mean ± SD; Kruskal-Wallis test, Dunn’s post-test, p=0.0001 (Neo vs. Neo/Mtz5), p<0.0001 (Neo vs. Mtz3/Neo/Mtz5), p=0.0016 (Neo/Mtz5 vs. Mtz3/Neo/Mtz5). (F) Sost:nlsEos-positive hair cells per neuromast following regeneration. Neo: 7.78 ± 2.36, n = 49 neuromasts (10 fish); Neo/Mtz5: 4.90 ± 2.02, n = 39 neuromasts (eight fish); Mtz3/Neo/Mtz5: 1.16 ± 1.46, n = 50 neuromasts (10 fish); mean ± SD; Kruskal-Wallis test, Dunn’s post-test, p=0.0003 (Neo vs. Neo/Mtz5), p<0.0001 (Neo vs. Mtz3/Neo/Mtz5, Neo/Mtz5 vs. Mtz3/Neo/Mtz5). (G) Percentage of hair cells per neuromast labeled by sost:nlsEos following regeneration. Neo: 65.81 ± 14.89, n = 49 neuromasts (10 fish); Neo/Mtz5: 51.40 ± 17.17, n = 39 neuromasts (8 fish); Mtz3/Neo/Mtz5: 14.29 ± 18.10, n = 50 neuromasts (10 fish); mean ± SD; Kruskal-Wallis test, Dunn’s post-test, p=0.0147 (Neo vs. Neo/Mtz5), p<0.0001 (Neo vs. Mtz3/Neo/Mtz5, Neo/Mtz5 vs. Mtz3/Neo/Mtz5).
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Figure 6—source data 1
Ablation of DV cells decreases number of regenerated hair cells
- https://doi.org/10.7554/eLife.43736.022
Figure 6—figure supplement 1
Mtz treatment does not inherently impact hair cell regeneration.
Total number of hair cells per neuromast following regular hair cell regeneration (Neo) or DV cell-ablated regeneration (Mtz3/Neo/Mtz5) in non-transgenic siblings of sost:NTR-GFP fish. Neo: 9.5 ± 1.50, n = 50 neuromasts (10 fish); Mtz3/Neo/Mtz5: 9.98 ± 1.51, n = 40 neuromasts (eight fish); mean ±SD; Mann Whitney U test, p=0.2317.
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Figure 6—figure supplement 1—source data 1
Mtz treatment does not inherently impact hair cell regeneration
- https://doi.org/10.7554/eLife.43736.019
Figure 6—figure supplement 2
Hair cell development and regeneration is unaffected in fish expressing both sost:NTR-GFP and sost:nlsEos.
Total number of FM 1-43FX-labeled hair cells per neuromast at 5 dpf or following hair cell regeneration (72 hpt) in sost:NTR-GFP fish (NTR-GFP), sost:nlsEos fish (nlsEos), sost:NTR-GFP; sost:nlsEos fish (NTR-GFP/nlsEos), and non-transgenic siblings (Sib). 5 dpf: 13.00 ± 1.41 (Sib) vs. 13.56 ± 2.26 (NTR-GFP) vs. 13.00 ± 1.26 (nlsEos) vs. 13.36 ± 1.98 (NTR-GFP/nlsEos), n = 25 neuromasts each (5 fish each); Regeneration: 8.72 ± 1.65 (Sib) vs. 8.32 ± 1.70 (NTR-GFP) vs. 8.00 ± 0.91 (nlsEos) vs. 8.52 ± 1.23 (NTR-GFP/nlsEos), n = 25 neuromasts each (5 fish each); mean ± SD; Kruskal-Wallis test, Dunn’s post-test; 5 dpf: p > 0.9999 (all comparisons); Regeneration: p=0.4448 (Sib vs. nlsEos), p>0.9999 (all other comparisons).
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Figure 6—figure supplement 2—source data 1
Hair cell development and regeneration is unaffected in fish expressing bothsost:NTR-GFP and sost:nlsEos
- https://doi.org/10.7554/eLife.43736.021
Figure 7
DV cell-ablation reduces the number of supernumerary hair cells formed during Notch-inhibited hair cell regeneration.
(A) Timeline of dual DV cell-ablation, Notch-inhibition experiment. Sost:NTR-GFP larvae were treated with Mtz at 3 dpf, treated with neomycin at 5dpf, then co-treated with Mtz and LY411575 for 8 hr, then washed out and treated with LY411575 for 16 additional hours (24 hr total LY). (B–E) Maximum projections of sost:NTR-GFP neuromasts following normal hair cell regeneration (B; Neo), Notch-inhibited hair cell regeneration (C; Neo/LY), DV cell-ablated hair cell regeneration (D; Mtz3/Neo/Mtz5), and DV cell-ablated and Notch-inhibited hair cell regeneration (E; Mtz3/Neo/Mtz5/LY). Sost:NTR-GFP cells are shown in green, and anti-Parvalbumin immunostained hair cells are shown in magenta. Scale bar = 10 μm. (F) Total number of hair cells per neuromast following hair cell regeneration. Neo: 9.42 ± 1.85, n = 50 neuromasts (10 fish); Neo/LY: 21.08 ± 4.42, n = 50 neuromasts (10 fish); Mtz3/Neo/Mtz5: 6.86 ± 1.76, n = 50 neuromasts (10 fish); Mtz3/Neo/Mtz5/LY: 15.06 ± 3.51, n = 50 neuromasts (10 fish); mean ± SD; Kruskal-Wallis test, Dunn’s post-test, p<0.0001 (Neo vs. Neo/LY; Mtz3/Neo/Mtz5 vs. Mtz3/Neo/Mtz5/LY), p=0.0058 (Neo vs. Mtz3/Neo/Mtz5), p=0.0029 (Neo/LY vs. Mtz3/Neo/Mtz5/LY).
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Figure 7—source data 1
DV cell-ablation reduces the number of supernumerary hair cells formed during Notch-inhibited hair cell regeneration
- https://doi.org/10.7554/eLife.43736.024
Figure 8
AP cells and DV cells define separate progenitor populations.
(A–C) Maximum projections of neuromasts from tnfsf10l3:nlsEos (AP, (A), sost:nlsEos (DV, (B), and tnfsf10l3:nlsEos/sost:nlsEos fish (AP + DV, (C) following photoconversion and regeneration. Converted nlsEos-positive cells are shown in magenta, and brn3c:GFP-positive hair cells are shown in green. Arrowheads indicate nlsEos-positive hair cells. Scale bar = 10 μm. (D) Number of nlsEos-positive hair cells per neuromast in each of the nlsEos lines following regeneration. Tnfsf10l3:nlsEos (AP): 2.4 ± 1.84, n = 50 neuromasts (10 fish); sost:nlsEos (DV): 6.34 ± 1.87, n = 50 neuromasts (10 fish); tnfsf10l3:nlsEos/sost:nlsEos (AP + DV): 8.24 ± 1.99, n = 50 neuromasts (10 fish); mean ±SD; Kruskal-Wallis test, Dunn’s post-test, p<0.0001 (AP vs. DV, AP vs. AP + DV), p=0.0031 (DV vs. AP + DV). (E) Percentage of hair cells per neuromast labeled by nlsEos lines following regeneration. AP: 27.59 ± 20.21, n = 50 neuromasts (10 fish); DV: 65.16 ± 13.89, n = 50 neuromasts (10 fish); AP + DV: 87.57 ± 13.02, n = 50 neuromasts (10 fish); mean ± SD; Kruskal-Wallis test, Dunn’s post-test, p<0.0001 (all comparisons).
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Figure 8—source data 1
AP cells and DV cells define separate progenitor populations
- https://doi.org/10.7554/eLife.43736.026
Figure 9
AP population doesn’t compensate for the loss of the DV population during hair cell regeneration.
(A–B) Maximum projections of tnfsf10l3:nlsEos; sost:NTR-GFP neuromasts following normal hair cell regeneration (A; Neo) or DV cell-ablated hair cell regeneration (B; Mtz3/Neo/Mtz5). Sost:NTR-GFP cells are shown in green, tnfsf10l3:nlsEos cells are shown in magenta, and anti-Parvalbumin-stained hair cells are shown in cyan. Arrowheads indicate nlsEos-positive hair cells. Scale bar = 10 μm. (C) Total number of hair cells per neuromast following hair cell regeneration. Neo: 10.36 ± 1.60, n = 50 neuromasts (10 fish); Mtz3/Neo/Mtz5: 7.98 ± 1.74, n = 50 neuromasts (10 fish); mean ± SD; Mann Whitney U test, p<0.0001. (D) Number of nlsEos-positive hair cells per neuromast following hair cell regeneration. Neo: 2.88 ± 1.83, n = 50 neuromasts (10 fish); Mtz3/Neo/Mtz5: 3.14 ± 1.43, n = 50 neuromasts (10 fish); mean ± SD; Mann Whitney U test, p=0.3855. (E) Percentage of hair cells per neuromast labeled by nlsEos following hair cell regeneration. Neo: 27.26 ± 16.00, n = 50 neuromasts (10 fish); Mtz3/Neo/Mtz5: 40.43 ± 19.44, n = 50 neuromasts (10 fish); mean ± SD; Mann Whitney U test, p=0.0002.
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Figure 9—source data 1
AP population doesn’t compensate for the loss of the DV population during hair cell regeneration
- https://doi.org/10.7554/eLife.43736.028
Figure 10
DV population regenerates via proliferation.
(A–B) Maximum projections of neuromasts from sost:NTR-GFP fish either untreated (A; Mock) or treated with 10 mM Mtz (B; Mtz). Sost:NTR-GFP cells are shown in green and EdU-positive cells are shown in magenta. Arrowheads indicate EdU-positive sost:NTR-GFP cells. Scale bar = 10 μm. (C) Total number of sost:NTR-GFP cells per neuromast following DV cell regeneration. Mock: 8.94 ± 1.62, n = 50 neuromasts (10 fish); Mtz: 5.34 ± 2.14, n = 50 neuromasts (10 fish); mean ± SD; Mann Whitney U test, p<0.0001. (D) Percentage of sost:NTR-GFP cells per neuromast labeled by EdU following DV cell regeneration. Mock: 14.47 ± 17.95, n = 50 neuromasts (10 fish); Mtz: 57.49 ± 32.34, n = 50 neuromasts (10 fish); mean ± SD; Mann Whitney U test, p<0.0001.
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Figure 10—source data 1
DV population regenerates via proliferation
- https://doi.org/10.7554/eLife.43736.030
Figure 11 with 2 supplements
DV cells are replenished by other support cell populations.
(A–B, D–E, G–H) Maximum projections of neuromasts expressing sost:NTR-GFP and sost:nlsEos (A–B), sfrp1a:nlsEos (D–E), and tnfsf10l3:nlsEos (G–H) in the absence of (A, D, G; Mock) or following Mtz-induced DV cell ablation (B, E, H; Mtz). Sost:NTR-GFP cells are shown in green and nlsEos-positive cells are shown in magenta. Arrowheads indicate nlsEos-positive sost:NTR-GFP cells. Scale bar = 10 μm. (C) Percentage of sost:NTR-GFP cells per neuromast labeled by sost:nlsEos following DV cell regeneration. Mock: 97.39 ± 7.14, n = 50 neuromasts (10 fish); Mtz: 56.09 ± 33.72, n = 50 neuromasts (10 fish); mean ± SD; Mann Whitney U test, p<0.0001. (F) Percentage of sost:NTR-GFP cells per neuromast labeled by sfrp1a:nlsEos following DV cell regeneration. Mock: 6.15 ± 11.14, n = 50 neuromasts (10 fish); Mtz: 31.27 ± 24.41, n = 50 neuromasts (10 fish); mean ± SD; Mann Whitney U test, p<0.0001. (I) Percentage of sost:NTR-GFP cells per neuromast labeled by tnfsf10l3:nlsEos following DV cell regeneration. Mock: 7.31 ± 9.55, n = 50 neuromasts (10 fish); Mtz: 21.11 ± 22.51, n = 50 neuromasts (10 fish); mean ± SD; Mann Whitney U test, p=0.0004.
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Figure 11—source data 1
DV cells are replenished by other support cell populations
- https://doi.org/10.7554/eLife.43736.036
Figure 11—figure supplement 1
Ablation of DV cells does not decrease the number of other support cell populations.
(A) Total number of sost:nlsEos-positive support cells per neuromast following DV cell regeneration. Mock: 21.42 ± 3.29, n = 50 neuromasts (10 fish); Mtz: 12.70 ± 3.35, n = 50 neuromasts (10 fish); mean ± SD; Mann Whitney U test, p<0.0001. (B) Total number of sfrp1a:nlsEos-positive support cells per neuromast following DV cell regeneration. Mock: 12.00 ± 2.54, n = 50 neuromasts (10 fish); Mtz: 11.32 ± 2.71, n = 50 neuromasts (10 fish); mean ± SD; Mann Whitney U test, p=0.2413. (C) Total number of tnfsf10l3:nlsEos-positive support cells per neuromast following DV cell regeneration. Mock: 18.40 ± 2.44, n = 50 neuromasts (10 fish); Mtz: 17.46 ± 2.49, n = 50 neuromasts (10 fish); mean ± SD; Mann Whitney U test, p=0.0699.
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Figure 11—figure supplement 1—source data 1
Ablation of DV cells does not decrease the number of other support cell populations
- https://doi.org/10.7554/eLife.43736.033
Figure 11—figure supplement 2
Expression of nlsEos transgenes does not alter number of sost:NTR-GFP cells.
Total number of sost:NTR-GFP cells per neuromast in sost:nlsEos (sost), sfrp1a:nlsEos (sfrp1a), and tnfsf10l3:nlsEos (tnfsf10l3) fish. Sost: 7.08 ± 1.59, n = 50 neuromasts (10 fish); Sfrp1a: 6.80 ± 1.92, n = 50 neuromasts (10 fish); Tnfsf10l3: 6.96 ± 1.70, n = 50 neuromasts (10 fish); mean ± SD; Kruskal-Wallis test, Dunn’s post-test, p>0.9999 (all comparisons).
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Figure 11—figure supplement 2—source data 1
Expression of nlsEos transgenes does not alter number of sost:NTR-GFP cells
- https://doi.org/10.7554/eLife.43736.035
Figure 12
Model of neuromast progenitor identity.
Sost:nlsEos-positive cells, located in the dorsoventral (DV) region of the neuromast, contain immature hair cell progenitors (shown in light pink) and mature hair cell progenitors (shown in magenta). Immature hair cell progenitors do not directly generate new hair cells (outlined in dark green) during regeneration, but do become mature hair cell progenitors, which comprise the majority of hair cell progenitors (see magenta-filled hair cells following regeneration). Tnfsf10l3:nlsEos-positive cells (shown in gold), located in the anteroposterior (AP) region of the neuromast, also serve as hair cell progenitors (see gold-filled hair cells following regeneration). Both of these populations are regulated by Notch signaling, and both can replenish immature hair cell progenitors. Finally, sfrp1a:nlsEos-positive cells (shown in light green), located in the periphery, do not serve as hair cell progenitors, nor are they regulated by Notch signaling. However, they are capable of replenishing immature hair cell progenitors, and can thus be classified as an upstream progenitor.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information | ||
|---|---|---|---|---|---|---|
| Strain, strain background (Danio rerio) | sfrp1a:nlsEos | This paper | Tg[sfrp1a:nlsEos]w217; CRISPR-mediated transgenesis | |||
| Strain, strain background (Danio rerio) | sfrp1a:GFP | This paper | Tg[sfrp1a:GFP]w222; CRISPR-mediated transgenesis | |||
| Strain, strain background (Danio rerio) | tnfsf10l3:nlsEos | This paper | Tg[tnfsf10l3:nlsEos]w218; CRISPR-mediated transgenesis | |||
| Strain, strain background (Danio rerio) | tnfsf10l3:GFP | This paper | Tg[tnfsf10l3:GFP]w223; CRISPR-mediated transgenesis | |||
| Strain, strain background (Danio rerio) | sost:nlsEos | This paper | Tg[sost:nlsEos]w215; CRISPR-mediated transgenesis | |||
| Strain, strain background (Danio rerio) | sost:NTR-GFP | This paper | Tg[sost:NTR-GFP]w216; CRISPR-mediated transgenesis | |||
| Strain, strain background (Danio rerio) | myo6:mKate2 | This paper | Tg[myosin6b:mKate2]w232; Gateway cloning and Tol2-mediated transgenesis | |||
| Strain, strain background (Danio rerio) | brn3c:GFP | PMID: 15930106 | ZFIN ID: ZDB-TGCONSTRCT-070117–142 | Tg[Brn3c:GAP43-GFP]s356t | ||
| Strain, strain background (Danio rerio) | myo6:GFP | PMID: 27991862 | ZFIN ID: ZDB-TGCONSTRCT-170321–2 | Tg[myosin6b:GFP]w186 | ||
| Antibody | anti-Parvalbumin (mouse monoclonal) | Millipore Sigma | MAB1572 | (1:500) | ||
| Antibody | anti-GFP (rabbit monoclonal) | Thermo Fisher Scientific | ABfinity G10362 | (1:500) | ||
| Antibody | anti-BrdU (mouse monoclonal) | Developmental Studies Hybridoma Bank | DHSB: G3G4; RRID: AB_2314035 | (1:100) | ||
| Recombinant DNA reagent | pBSK mbait-GFP | PMID: 25293390 | ||||
| Recombinant DNA reagent | mbait-nlsEos | This paper | pDEST vector expressing nuclear-localized Eos following the mbait-HSP sequence from pBSK mbait-GFP; see Materials and methods for more info. | |||
| Recombinant DNA reagent | pBSK mbait-NTR-GFP | This paper | Modified pBSK mbait-GFP vector expressing enhanced potency nitroreductase fused to GFP; generated via Gibson assembly | |||
| peptide, recombinant protein | Cas9 protein with NLS (injection ready) | PNA Bio | PNA Bio: CP02 | |||
| Commercial assay or kit | NucleoSpin Plasmid | Machery-Nagel | 740588.25 | |||
| Commercial assay or kit | NucleoSpin Gel and PCR Clean-up | Machery-Nagel | 740609.25 | |||
| Commercial assay or kit | Plasmid Maxi | Qiagen | 12163 | |||
| Commercial assay or kit | RNA Clean and Concentrator | Zymo Research | RCC-25 | |||
| Chemical compound, drug | Gateway BP Clonase II | Thermo Fisher Scientific | 11789020 | |||
| Chemical compound, drug | Gateway LR Clonase II | Thermo Fisher Scientific | 11791020 | |||
| Chemical compound, drug | 5x Isothermal Reaction Buffer | PMID: 19363495 | ||||
| Chemical compound, drug | Metronidazole | Sigma-Aldrich | Sigma-Aldrich: M1547 | |||
| Chemical compound, drug | LY411575 | Sigma-Aldrich | Sigma-Aldrich: SML0506 | |||
| Chemical compound, drug | Neomycin | Sigma-Aldrich | Sigma-Aldrich: N1142 | |||
| Chemical compound, drug | f-ara-EdU | Sigma-Aldrich | Sigma-Aldrich: T511293 | |||
| Chemical compound, drug | BrdU | Sigma-Aldrich | Sigma-Aldrich: B5002 | |||
| Chemical compound, drug | FM 1-43FX | Molecular Probes | Molecular Probes: F35355 | |||
| Software, algorithm | GraphPad Prism | GraphPad Software | www.graphpad.com | |||
| Software, algorithm | Slidebook | Intelligent Imaging Innovations (3i) | www.intellgent-imaging.com | |||
| Software, algorithm | Zen Black | Zeiss | www.zeiss.com | |||
| Software, algorithm | FIJI | PMID: 22743772 | ||||
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.43736.038
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Distinct progenitor populations mediate regeneration in the zebrafish lateral line
eLife 8:e43736.
https://doi.org/10.7554/eLife.43736
