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Proliferative regeneration of zebrafish lateral line hair cells after different ototoxic insults.

Mackenzie SM, Raible DW - PLoS ONE (2012)

Bottom Line: Certain treatments, including cisplatin and higher concentrations of dissolved copper, significantly delayed regeneration by one or more days.However, cisplatin did not block all regeneration as observed previously in the chick basilar papilla.The particular ototoxin did not appear to affect the mechanism of regeneration, as we observed evidence of recent proliferation in the majority of new hair cells in all cases.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Structure, University of Washington, Seattle, Washington, United States of America.

ABSTRACT
Sensory hair cells in the zebrafish lateral line regenerate rapidly and completely after damage. Previous studies have used a variety of ototoxins to kill lateral line hair cells to study different phenomena including mechanisms of hair cell death and regeneration. We sought to directly compare these ototoxins to determine if they differentially affected the rate and amount of hair cell replacement. In addition, previous studies have found evidence of proliferative hair cell regeneration in zebrafish, but both proliferation and non-mitotic direct transdifferentiation have been observed during hair cell regeneration in the sensory epithelia of birds and amphibians. We sought to test whether a similar combination of regenerative mechanisms exist in the fish. We analyzed the time course of regeneration after treatment with different ototoxic compounds and also labeled dividing hair cell progenitors. Certain treatments, including cisplatin and higher concentrations of dissolved copper, significantly delayed regeneration by one or more days. However, cisplatin did not block all regeneration as observed previously in the chick basilar papilla. The particular ototoxin did not appear to affect the mechanism of regeneration, as we observed evidence of recent proliferation in the majority of new hair cells in all cases. Inhibiting proliferation with flubendazole blocked the production of new hair cells and prevented the accumulation of additional precursors, indicating that proliferation has a dominant role during regeneration of lateral line hair cells.

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Hair cell regeneration is blocked by inhibition of mitosis.ET4:GFP larvae were treated at 5 dpf with neomycin or copper for 1 h and incubated in flubendazole for 48 h. (A) Those not treated with flubendazole continued to add mature hair cells. Two-way ANOVA found significantly more cells expressed GFP than parvalbumin (p<0.001), indicating the presence of hair cell precursors. Bonferroni post-hoc analysis confirmed a significant difference in control and ototoxin-treated groups between GFP- and parvalbumin-positive hair cell counts at 48 hpt (**, p<0.01; ***, p<0.001). (B) Those treated with flubendazole exhibited little or no increase in mature hair cells. There were at most 1 or 2 parvalbumin-negative precursors present per neuromast, indicating that undifferentiated precursors did not accumulate. N = 8 fish per group. Error bars are +/− SD.
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pone-0047257-g004: Hair cell regeneration is blocked by inhibition of mitosis.ET4:GFP larvae were treated at 5 dpf with neomycin or copper for 1 h and incubated in flubendazole for 48 h. (A) Those not treated with flubendazole continued to add mature hair cells. Two-way ANOVA found significantly more cells expressed GFP than parvalbumin (p<0.001), indicating the presence of hair cell precursors. Bonferroni post-hoc analysis confirmed a significant difference in control and ototoxin-treated groups between GFP- and parvalbumin-positive hair cell counts at 48 hpt (**, p<0.01; ***, p<0.001). (B) Those treated with flubendazole exhibited little or no increase in mature hair cells. There were at most 1 or 2 parvalbumin-negative precursors present per neuromast, indicating that undifferentiated precursors did not accumulate. N = 8 fish per group. Error bars are +/− SD.

Mentions: Given that proliferative regeneration occurred after treatment with neomycin and with both low and high concentrations of copper, we sought to determine if preventing proliferation would uncover evidence of direct transdifferentiation. We treated 5 dpf larvae with neomycin or copper for 1 h and then incubated them in 5 μM flubendazole throughout the recovery period. Flubendazole blocks tubulin polymerization, preventing assembly of a mitotic spindle required for chromosome segregation [47], and has been identified as a drug that blocks hair cell regeneration [49]. Importantly, flubendazole shows little toxicity to hair cells themselves, removing a potential confounding effect of other mitotic inhibitors such as genistein and colchicine [42]. Larvae were collected up to 48 hours post-treatment (hpt) to establish a time course of regeneration with and without flubendazole exposure. All ototoxic treatments resulted in near-complete loss of hair cells in 5 dpf larvae and substantial recovery by 48 hpt (Figure 4A). Treatment with flubendazole alone had little effect on hair cell number in control fish but significantly impaired regeneration (Figure 4B; p<0.001). These results support the idea that hair cells are derived from dividing precursors and do not support the idea that latent transdifferentiation can compensate in the absence of proliferation.


Proliferative regeneration of zebrafish lateral line hair cells after different ototoxic insults.

Mackenzie SM, Raible DW - PLoS ONE (2012)

Hair cell regeneration is blocked by inhibition of mitosis.ET4:GFP larvae were treated at 5 dpf with neomycin or copper for 1 h and incubated in flubendazole for 48 h. (A) Those not treated with flubendazole continued to add mature hair cells. Two-way ANOVA found significantly more cells expressed GFP than parvalbumin (p<0.001), indicating the presence of hair cell precursors. Bonferroni post-hoc analysis confirmed a significant difference in control and ototoxin-treated groups between GFP- and parvalbumin-positive hair cell counts at 48 hpt (**, p<0.01; ***, p<0.001). (B) Those treated with flubendazole exhibited little or no increase in mature hair cells. There were at most 1 or 2 parvalbumin-negative precursors present per neuromast, indicating that undifferentiated precursors did not accumulate. N = 8 fish per group. Error bars are +/− SD.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3475690&req=5

pone-0047257-g004: Hair cell regeneration is blocked by inhibition of mitosis.ET4:GFP larvae were treated at 5 dpf with neomycin or copper for 1 h and incubated in flubendazole for 48 h. (A) Those not treated with flubendazole continued to add mature hair cells. Two-way ANOVA found significantly more cells expressed GFP than parvalbumin (p<0.001), indicating the presence of hair cell precursors. Bonferroni post-hoc analysis confirmed a significant difference in control and ototoxin-treated groups between GFP- and parvalbumin-positive hair cell counts at 48 hpt (**, p<0.01; ***, p<0.001). (B) Those treated with flubendazole exhibited little or no increase in mature hair cells. There were at most 1 or 2 parvalbumin-negative precursors present per neuromast, indicating that undifferentiated precursors did not accumulate. N = 8 fish per group. Error bars are +/− SD.
Mentions: Given that proliferative regeneration occurred after treatment with neomycin and with both low and high concentrations of copper, we sought to determine if preventing proliferation would uncover evidence of direct transdifferentiation. We treated 5 dpf larvae with neomycin or copper for 1 h and then incubated them in 5 μM flubendazole throughout the recovery period. Flubendazole blocks tubulin polymerization, preventing assembly of a mitotic spindle required for chromosome segregation [47], and has been identified as a drug that blocks hair cell regeneration [49]. Importantly, flubendazole shows little toxicity to hair cells themselves, removing a potential confounding effect of other mitotic inhibitors such as genistein and colchicine [42]. Larvae were collected up to 48 hours post-treatment (hpt) to establish a time course of regeneration with and without flubendazole exposure. All ototoxic treatments resulted in near-complete loss of hair cells in 5 dpf larvae and substantial recovery by 48 hpt (Figure 4A). Treatment with flubendazole alone had little effect on hair cell number in control fish but significantly impaired regeneration (Figure 4B; p<0.001). These results support the idea that hair cells are derived from dividing precursors and do not support the idea that latent transdifferentiation can compensate in the absence of proliferation.

Bottom Line: Certain treatments, including cisplatin and higher concentrations of dissolved copper, significantly delayed regeneration by one or more days.However, cisplatin did not block all regeneration as observed previously in the chick basilar papilla.The particular ototoxin did not appear to affect the mechanism of regeneration, as we observed evidence of recent proliferation in the majority of new hair cells in all cases.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Structure, University of Washington, Seattle, Washington, United States of America.

ABSTRACT
Sensory hair cells in the zebrafish lateral line regenerate rapidly and completely after damage. Previous studies have used a variety of ototoxins to kill lateral line hair cells to study different phenomena including mechanisms of hair cell death and regeneration. We sought to directly compare these ototoxins to determine if they differentially affected the rate and amount of hair cell replacement. In addition, previous studies have found evidence of proliferative hair cell regeneration in zebrafish, but both proliferation and non-mitotic direct transdifferentiation have been observed during hair cell regeneration in the sensory epithelia of birds and amphibians. We sought to test whether a similar combination of regenerative mechanisms exist in the fish. We analyzed the time course of regeneration after treatment with different ototoxic compounds and also labeled dividing hair cell progenitors. Certain treatments, including cisplatin and higher concentrations of dissolved copper, significantly delayed regeneration by one or more days. However, cisplatin did not block all regeneration as observed previously in the chick basilar papilla. The particular ototoxin did not appear to affect the mechanism of regeneration, as we observed evidence of recent proliferation in the majority of new hair cells in all cases. Inhibiting proliferation with flubendazole blocked the production of new hair cells and prevented the accumulation of additional precursors, indicating that proliferation has a dominant role during regeneration of lateral line hair cells.

Show MeSH
Related in: MedlinePlus