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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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Flubendazole impairs division of hair cell progenitors.ET4:GFP larvae were treated at 5 dpf with neomycin or copper for 1 h and incubated in flubendazole for 24 h. Immunohistochemistry was performed for GFP and PHH3, which is upregulated during mitosis. (A) Two-way ANOVA followed by Bonferroni post-hoc analysis found significant increases in the proportion of PHH3-positive cells in neomycin- and copper-treated larvae when treated with flubendazole (*, p<0.05; ***, p<0.001), demonstrating that cell division was arrested in GFP-positive hair cell precursors. N = 8 fish per group. Error bars are + SD. (B) Example neuromast at 24 hpt from a fish treated with neomycin and flubendazole. Arrows mark PHH3-positive nuclei.
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pone-0047257-g005: Flubendazole impairs division of hair cell progenitors.ET4:GFP larvae were treated at 5 dpf with neomycin or copper for 1 h and incubated in flubendazole for 24 h. Immunohistochemistry was performed for GFP and PHH3, which is upregulated during mitosis. (A) Two-way ANOVA followed by Bonferroni post-hoc analysis found significant increases in the proportion of PHH3-positive cells in neomycin- and copper-treated larvae when treated with flubendazole (*, p<0.05; ***, p<0.001), demonstrating that cell division was arrested in GFP-positive hair cell precursors. N = 8 fish per group. Error bars are + SD. (B) Example neuromast at 24 hpt from a fish treated with neomycin and flubendazole. Arrows mark PHH3-positive nuclei.

Mentions: Impairment of microtubule assembly with flubendazole might impair differentiation of hair cells instead of blocking progenitor cell division. We therefore assayed the extent of regeneration during pharmacological inhibition of proliferation using transgenic ET4:GFP fish that express GFP in mature hair cells as well as pre-mitotic progenitors [37]. In the absence of flubendazole, the increase in GFP-positive cells significantly preceded the increase in parvalbumin-positive cells for control and both ototoxin-treated groups (Figure 4A; p<0.001). This is consistent with the acquisition of a progenitor cell fate before subsequent hair cell differentiation.However, there was no significant difference between GFP- and parvalbumin-positive cells in larvae treated with flubendazole (Figure 4B), indicating pre-mitotic progenitors did not accumulate. Analysis of larvae with antibodies against phosphohistone H3 (PHH3), which is expressed during mitosis, revealed incubation in flubendazole produced a significant increase in the proportion of PHH3-positive nuclei in GFP-positive cells during regeneration after neomycin (p<0.05; Figure 5) and copper (p<0.001). This result strongly suggests that flubendazole arrests hair cell progenitors during mitosis by interfering with spindle microtubules, not at a later stage of differentiation.


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

Mackenzie SM, Raible DW - PLoS ONE (2012)

Flubendazole impairs division of hair cell progenitors.ET4:GFP larvae were treated at 5 dpf with neomycin or copper for 1 h and incubated in flubendazole for 24 h. Immunohistochemistry was performed for GFP and PHH3, which is upregulated during mitosis. (A) Two-way ANOVA followed by Bonferroni post-hoc analysis found significant increases in the proportion of PHH3-positive cells in neomycin- and copper-treated larvae when treated with flubendazole (*, p<0.05; ***, p<0.001), demonstrating that cell division was arrested in GFP-positive hair cell precursors. N = 8 fish per group. Error bars are + SD. (B) Example neuromast at 24 hpt from a fish treated with neomycin and flubendazole. Arrows mark PHH3-positive nuclei.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3475690&req=5

pone-0047257-g005: Flubendazole impairs division of hair cell progenitors.ET4:GFP larvae were treated at 5 dpf with neomycin or copper for 1 h and incubated in flubendazole for 24 h. Immunohistochemistry was performed for GFP and PHH3, which is upregulated during mitosis. (A) Two-way ANOVA followed by Bonferroni post-hoc analysis found significant increases in the proportion of PHH3-positive cells in neomycin- and copper-treated larvae when treated with flubendazole (*, p<0.05; ***, p<0.001), demonstrating that cell division was arrested in GFP-positive hair cell precursors. N = 8 fish per group. Error bars are + SD. (B) Example neuromast at 24 hpt from a fish treated with neomycin and flubendazole. Arrows mark PHH3-positive nuclei.
Mentions: Impairment of microtubule assembly with flubendazole might impair differentiation of hair cells instead of blocking progenitor cell division. We therefore assayed the extent of regeneration during pharmacological inhibition of proliferation using transgenic ET4:GFP fish that express GFP in mature hair cells as well as pre-mitotic progenitors [37]. In the absence of flubendazole, the increase in GFP-positive cells significantly preceded the increase in parvalbumin-positive cells for control and both ototoxin-treated groups (Figure 4A; p<0.001). This is consistent with the acquisition of a progenitor cell fate before subsequent hair cell differentiation.However, there was no significant difference between GFP- and parvalbumin-positive cells in larvae treated with flubendazole (Figure 4B), indicating pre-mitotic progenitors did not accumulate. Analysis of larvae with antibodies against phosphohistone H3 (PHH3), which is expressed during mitosis, revealed incubation in flubendazole produced a significant increase in the proportion of PHH3-positive nuclei in GFP-positive cells during regeneration after neomycin (p<0.05; Figure 5) and copper (p<0.001). This result strongly suggests that flubendazole arrests hair cell progenitors during mitosis by interfering with spindle microtubules, not at a later stage of differentiation.

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