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[Preprint]. 2024 Apr 11:2024.04.09.588777.
doi: 10.1101/2024.04.09.588777.

Transdifferentiation is uncoupled from progenitor pool expansion during hair cell regeneration in the zebrafish inner ear

Marielle O Beaulieu  1   2 , Eric D Thomas  3   4 , David W Raible  1   2   3   4
Affiliations

Transdifferentiation is uncoupled from progenitor pool expansion during hair cell regeneration in the zebrafish inner ear

Marielle O Beaulieu et al. bioRxiv. .

Update in

Abstract

Death of mechanosensory hair cells in the inner ear is a common cause of auditory and vestibular impairment in mammals, which have a limited ability to regrow these cells after damage. In contrast, non-mammalian vertebrates including zebrafish can robustly regenerate hair cells following severe organ damage. The zebrafish inner ear provides an understudied model system for understanding hair cell regeneration in organs that are highly conserved with their mammalian counterparts. Here we quantitatively examine hair cell addition during growth and regeneration of the larval zebrafish inner ear. We used a genetically encoded ablation method to induce hair cell death and observed gradual regeneration with correct spatial patterning over two weeks following ablation. Supporting cells, which surround and are a source of new hair cells, divide in response to hair cell ablation, expanding the possible progenitor pool. In parallel, nascent hair cells arise from direct transdifferentiation of progenitor pool cells uncoupled from progenitor division. These findings reveal a previously unrecognized mechanism of hair cell regeneration with implications for how hair cells may be encouraged to regenerate in the mammalian ear.

Keywords: hair cell; inner ear; regeneration; transdifferentiation; zebrafish.

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Conflict of interest statement

COMPETING INTERESTS No competing interests declared.

Figures

Figure 1.
Figure 1.. Inner ear organs of the larval zebrafish
A) Maximum intensity projection image of Tg(myo6b:GFP) 5dpf larval zebrafish ear. GFP-labeled hair cells are shown in cyan and DAPI-labeled nuclei are shown in grey. Dotted outlines delineate neuromast and inner ear organ boundaries. Scale bar = 50 μm. B) Diagram of 5dpf larval zebrafish ear. Color gradient indicates depth of organs where lighter colors indicate more superficial structures and darker colors indicate deeper structures. Dotted outlines delineate neuromast and inner ear organ boundaries, while color-filled areas indicate location of hair cells. ac = anterior crista, lc = lateral crista, nm = neuromast, o = otolith, pc = posterior crista, s = saccule, u = utricle.
Figure 2.
Figure 2.. Addition of hair cells during larval zebrafish growth
A) Maximum intensity projections of Tg(myo6b:NLS-Eos) anterior crista hair cells at standard lengths 4.0 mm, 6.0 mm, 7.5 mm, and 10.25 mm. Scale bar = 10μm. B) Maximum intensity projections of lateral crista hair cells at standard lengths 4.0 mm, 6.0 mm, 7.5 mm, and 10.0 mm. Scale bar = 10μm. C) Maximum intensity projections of utricle hair cells at standard lengths 4.25 mm, 6.0 mm, 7.5 mm, and 10.25 mm. Scale bar = 20μm. D) Quantification of hair cell number in the anterior and lateral cristae across the larval stage of development. Anterior crista data points are represented by black circles (n = 35), while the lateral crista is represented by grey triangles (n = 47). Each data point represents one ear from one fish. Anterior crista slope = 19.14 ± 1.33 HC per mm SL, R2 = 0.862. Lateral crista slope = 20.30 ± 1.26 HC per mm SL, R2 = 0.853. Simple linear regression indicates no significant difference between these two slopes (p = 0.529). E) Quantification of utricle hair cell number across the larval stage (n = 34). Linear regression of utricle hair cell number slope = 49.96 ± 4.28 HC per mm SL, R2 = 0.812.
Figure 3.
Figure 3.. Little hair cell turnover occurs in the larval zebrafish ear
A-B) Representative maximum intensity projection images of Tg(myo6b:NLS-Eos) anterior cristae A) immediately post-photoconversion at 8 days post-fertilization (dpf) or B) one week post-photoconversion at 15 dpf. HC that were photoconverted retain photoconverted (magenta) Eos signal while new HC have unconverted (cyan) Eos only. C) Quantification of anterior crista photoconverted hair cells at 8 and 15 dpf (n = 20 control, 20 ablated). D-F) Analogous results for the lateral crista (n = 20, 20) and G-I) for the utricle (n = 29, 25). Unpaired t tests indicate no significant difference between the number of photoconverted hair cells at these two timepoints (ant crista p = 0.125, lat crista p = 0.859, utricle p = 0.071). Scale bars = 10 μm. All data is presented as mean ± s.d.
Figure 4.
Figure 4.. Identification of inner ear hair cell subtypes during larval growth
A-C) Maximum intensity projection images of HCR-FISH probing for cabp1b expression in Tg(myo6b:NLS-Eos) anterior cristae at A) 2 days post-photoconversion (dpp) (10 dpf, n = 14); B) 7 days dpp (15 dpf, n = 12); and C) 14 dpp (22 dpf, n = 8). Old HC retain photoconverted (magenta) Eos signal while new HC have unconverted (cyan) Eos only. Peripheral-type hair cells are labeled by the cabp1b HCR probe (yellow). Dotted outline delineates central, cabp1b− region of the sensory patch. Scale bars = 10 μm. D) Increase in hair cell numbers over the course of the experiment. E) ratio of central (cabp1−) to peripheral (cabp1b+) hair cells over time. The increased ratio for old cells suggests phenotypic conversion from peripheral to central hair cell typeover time. All data is presented as mean ± s.d.
Figure 5.
Figure 5.. Trpv1-capsaicin hair cell ablation
A) Maximum intensity projection of a photoconverted 8dpf Tg(myo6b:NLS-Eos) larval inner ear one hour after capsaicin treatment. B) Maximum intensity projection of a sibling Tg(myo6b:NLS-Eos);Tg(myo6b:TrpV1-mClover) inner ear one hour after capsaicin treatment or C) four hours after capsaicin treatment. Images show photoconverted (magenta) and unconverted (cyan) Eos signal with and without DRAQ5-labeled nuclei. Dashed oval regions indicate anterior, lateral, and posterior cristae. Dashed yellow box indicates magnified anterior cristae region shown in A’-C’. D) Dose-response curve for hair cells following one hour of treatment with capsaicin at different concentrations. Control treatment represents DMSO alone. Each data point represents the number of hair cells in combined anterior, lateral, and posterior crista of one fish ear (n = 6–20). All data is presented as mean ± s.d.
Figure 6.
Figure 6.. Anterior crista hair cells regenerate during the two weeks following ablation
A) Tg(myo6b:NLS-Eos) sibling larvae with or without Tg(myo6b:TrpV1-mClover) were photoconverted and treated with capsaicin to ablate hair cells at 8dpf. Larvae were collected at five timepoints over the following two weeks: 1 (n = 22 control, 25 ablated), 2 (n = 13, 20), 4 (n = 19, 18), 7 (n = 16, 13), or 14 (n = 18, 15) days-post ablation. B) Representative maximum intensity projections of anterior crista in control and ablated fish at five timepoints following treatment. Nuclei of cells that survived capsaicin treatment contain photoconverted Eos (magenta). Hair cells newly added after capsaicin treatment have nuclei with only unconverted Eos (cyan). Scale bars = 10 μm. C) Quantification of new (cyan-only) hair cells in ablated and control anterior crista. Two-way ANOVA variation across condition p < 0.0001; Šídák’s multiple comparisons post-hoc test for 7 dpa adjusted p-value = 0.0021, 14 dpa adjusted p-value < 0.0001. D) Quantification of total hair cells in ablated and control anterior crista. Two-way ANOVA variation across condition p < 0.0001; Šídák’s multiple comparisons post-hoc test for 1 dpa adjusted p-value < 0.0001, 2 dpa adjusted p-value = 0.0006, 4 dpa adjusted p-value = 0.0015, 7 dpa adjusted p-value = 0.0342. All data is presented as mean ± s.d.
Figure 7.
Figure 7.. Hair cell central-peripheral patterning is restored following ablation
A) Representative maximum intensity projections of anterior crista in control and ablated fish at 2 dpa with cabp1b HCR-FISH. Photoconverted Eos (magenta) and cabp1b (yellow) channels are shown with and without unconverted Eos (cyan). Dotted outline delineates central, cabp1b− region of the sensory patch. B) Quantification of cabp1b+ new hair cells, shown as a percentage of all new (cyan-only) hair cells in control (n = 18) and ablated (n = 16) anterior cristae. Unpaired t test p < 0.0001. C-D) Analogous data to A-B for crista at 14 dpa (n = 18 control, 15 ablated). Unpaired t test p = 0.5226. Scale bars = 10 μm. All data is presented as mean ± s.d.
Figure 8.
Figure 8.. Support cells proliferate in response to hair cell ablation
A) Larvae were incubated in EdU for 24 hours immediately after hair cell ablation, at 3 dpa, or at 6 dpa and collected at the end of the 24-hour incubation. B) Quantification of EdU-labeled support cells in the anterior and lateral cristae combined in control and ablated fish incubated in EdU from 0–1 dpa (n = 13 control, 14 ablated), 3–4 dpa (n = 19, 12), or 6–7 dpa (n = 9, 7). Two-way ANOVA is significant across condition p = 0.0021, Šídák’s multiple comparisons post-hoc test 0–1 dpa adjusted p-value = 0.0004. All data is presented as mean ± s.d. C) Representative maximum intensity projections of anterior crista in control and ablated fish incubated with EdU from 0–1 dpa with Eos-labeled hair cells in cyan and EdU-labeled nuclei in yellow.
Figure 9.
Figure 9.. EdU-labeling of hair cells over the week following ablation
A) Larvae were incubated in EdU for 24 hours after photoconversion and hair cell ablation and collected either at the end of the incubation (1 dpa; n = 10 control, 7 ablated) or at 4 (n = 14, 8) or 7 (n = 9, 13) dpa. B) Representative maximum intensity projections of anterior crista in control and ablated fish at 7 dpa. White arrowheads indicate hair cells added since ablation with EdU signal (yellow), unconverted Eos (cyan), and without photoconverted eos (magenta). Scale bars = 10 μm. C) Quantification of EdU+ hair cells in the combined anterior and lateral cristae at each timepoint in control and ablated fish. Two-way ANOVA is significant across condition p = 0.0050, Šídák’s multiple comparisons post-hoc test 7 dpa adjusted p-value = 0.0034. D) Quantification of new (cyan-only) hair cells at each timepoint in control and ablated fish. Two-way ANOVA is significant across condition p < 0.0001, Šídák’s multiple comparisons post-hoc test 1 dpa adjusted p-value = 0.0123, 4 dpa adjusted p-value <0.0001, 7 dpa adjusted p-value = 0.0010. E) EdU+ hair cells as a percentage of new hair cells. Two-way ANOVA with Šídák’s multiple comparisons post-hoc test is not significant across condition at any timepoint. All data is presented as mean ± s.d.
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