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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 cells are derived from proliferating progenitors.(A) Wild type larvae were treated at 5 dpf with neomycin or copper for 1 h followed by recovery in 5 mM BrdU until 24 hpt followed by fresh embryo medium. Greater numbers of BrdU-positive hair cells were observed in all ototoxin-treated groups at 72 hpt. (B) One-way ANOVA followed by a Tukey post-hoc analysis of the proportion of BrdU-positive hair cells revealed significant increases in all ototoxin-treated groups compared to control (p<0.001). No significant difference was observed between 1 and 10 μM copper that would suggest a dose-dependent effect on proliferative regeneration. A small but significant difference was observed between neomycin and 1 μM copper (*, p<0.05). N = 8 fish per group. Error bars are + SD. (C) Example neuromast at 72 hpt from a fish treated with 1 μM copper. Arrows mark BrdU-positive hair cells.
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pone-0047257-g003: Hair cells are derived from proliferating progenitors.(A) Wild type larvae were treated at 5 dpf with neomycin or copper for 1 h followed by recovery in 5 mM BrdU until 24 hpt followed by fresh embryo medium. Greater numbers of BrdU-positive hair cells were observed in all ototoxin-treated groups at 72 hpt. (B) One-way ANOVA followed by a Tukey post-hoc analysis of the proportion of BrdU-positive hair cells revealed significant increases in all ototoxin-treated groups compared to control (p<0.001). No significant difference was observed between 1 and 10 μM copper that would suggest a dose-dependent effect on proliferative regeneration. A small but significant difference was observed between neomycin and 1 μM copper (*, p<0.05). N = 8 fish per group. Error bars are + SD. (C) Example neuromast at 72 hpt from a fish treated with 1 μM copper. Arrows mark BrdU-positive hair cells.

Mentions: To examine whether treatment with neomycin or copper might bias regeneration toward proliferative or non-proliferative mechanisms, we used a pulse-chase paradigm to label new hair cells arising after damage with BrdU, a thymine analog that incorporates into DNA during S phase (Figure 3). Larvae were treated with 200 μM neomycin, 1 μM copper, or 10 μM copper for 1 h and then incubated in 5 mM BrdU for the first 23 h of recovery (24 h from the beginning of treatment). The BrdU was replaced with fresh embryo medium, and larvae recovered for an additional 48 h at which time they were fixed and stained with antibodies recognizing BrdU and parvalbumin to reveal mature hair cells derived from dividing precursors. Hair cells without BrdU staining were presumably derived from non-dividing progenitor cells or from progenitors that proliferated after exposure to BrdU.


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

Mackenzie SM, Raible DW - PLoS ONE (2012)

Hair cells are derived from proliferating progenitors.(A) Wild type larvae were treated at 5 dpf with neomycin or copper for 1 h followed by recovery in 5 mM BrdU until 24 hpt followed by fresh embryo medium. Greater numbers of BrdU-positive hair cells were observed in all ototoxin-treated groups at 72 hpt. (B) One-way ANOVA followed by a Tukey post-hoc analysis of the proportion of BrdU-positive hair cells revealed significant increases in all ototoxin-treated groups compared to control (p<0.001). No significant difference was observed between 1 and 10 μM copper that would suggest a dose-dependent effect on proliferative regeneration. A small but significant difference was observed between neomycin and 1 μM copper (*, p<0.05). N = 8 fish per group. Error bars are + SD. (C) Example neuromast at 72 hpt from a fish treated with 1 μM copper. Arrows mark BrdU-positive hair cells.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3475690&req=5

pone-0047257-g003: Hair cells are derived from proliferating progenitors.(A) Wild type larvae were treated at 5 dpf with neomycin or copper for 1 h followed by recovery in 5 mM BrdU until 24 hpt followed by fresh embryo medium. Greater numbers of BrdU-positive hair cells were observed in all ototoxin-treated groups at 72 hpt. (B) One-way ANOVA followed by a Tukey post-hoc analysis of the proportion of BrdU-positive hair cells revealed significant increases in all ototoxin-treated groups compared to control (p<0.001). No significant difference was observed between 1 and 10 μM copper that would suggest a dose-dependent effect on proliferative regeneration. A small but significant difference was observed between neomycin and 1 μM copper (*, p<0.05). N = 8 fish per group. Error bars are + SD. (C) Example neuromast at 72 hpt from a fish treated with 1 μM copper. Arrows mark BrdU-positive hair cells.
Mentions: To examine whether treatment with neomycin or copper might bias regeneration toward proliferative or non-proliferative mechanisms, we used a pulse-chase paradigm to label new hair cells arising after damage with BrdU, a thymine analog that incorporates into DNA during S phase (Figure 3). Larvae were treated with 200 μM neomycin, 1 μM copper, or 10 μM copper for 1 h and then incubated in 5 mM BrdU for the first 23 h of recovery (24 h from the beginning of treatment). The BrdU was replaced with fresh embryo medium, and larvae recovered for an additional 48 h at which time they were fixed and stained with antibodies recognizing BrdU and parvalbumin to reveal mature hair cells derived from dividing precursors. Hair cells without BrdU staining were presumably derived from non-dividing progenitor cells or from progenitors that proliferated after exposure to BrdU.

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