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Effect of histone deacetylase inhibitors trichostatin A and valproic acid on hair cell regeneration in zebrafish lateral line neuromasts.

He Y, Cai C, Tang D, Sun S, Li H - Front Cell Neurosci (2014)

Bottom Line: Additionally, BrdU immunostaining and western blot analysis showed that TSA or VPA treatment caused a significant decrease in the percentage of S-phase cells and induced p21(Cip1) and p27(Kip1) expression, both of which are likely to explain the decrease in the amount of newly regenerated hair cells in treated embryos.Finally, we showed that HDAC inhibitors induced no observable cell death in neuromasts as measured by cleaved caspase-3 immunohistochemistry and western blot analysis.Taken together, our results demonstrate that HDAC activity has an important role in the regeneration of hair cells in the lateral line.

View Article: PubMed Central - PubMed

Affiliation: Department of Otorhinolaryngology, Affiliated Eye and ENT Hospital of Fudan University Shanghai, China.

ABSTRACT
In humans, auditory hair cells are not replaced when injured. Thus, cochlear hair cell loss causes progressive and permanent hearing loss. Conversely, non-mammalian vertebrates are capable of regenerating lost sensory hair cells. The zebrafish lateral line has numerous qualities that make it well-suited for studying hair cell development and regeneration. Histone deacetylase (HDAC) activity has been shown to have an important role in regenerative processes in vertebrates, but its function in hair cell regeneration in vivo is not fully understood. Here, we have examined the role of HDAC activity in hair cell regeneration in the zebrafish lateral line. We eliminated lateral line hair cells of 5-day post-fertilization larvae using neomycin and then treated the larvae with HDAC inhibitors. To assess hair cell regeneration, we used 5-bromo-2-deoxyuridine (BrdU) incorporation in zebrafish larvae to label mitotic cells after hair cell loss. We found that pharmacological inhibition of HDACs using trichostatin A (TSA) or valproic acid (VPA) increased histone acetylation in the regenerated neuromasts following neomycin-induced damage. We also showed that treatment with TSA or VPA decreased the number of supporting cells and regenerated hair cells in response to hair cell damage. Additionally, BrdU immunostaining and western blot analysis showed that TSA or VPA treatment caused a significant decrease in the percentage of S-phase cells and induced p21(Cip1) and p27(Kip1) expression, both of which are likely to explain the decrease in the amount of newly regenerated hair cells in treated embryos. Finally, we showed that HDAC inhibitors induced no observable cell death in neuromasts as measured by cleaved caspase-3 immunohistochemistry and western blot analysis. Taken together, our results demonstrate that HDAC activity has an important role in the regeneration of hair cells in the lateral line.

No MeSH data available.


Related in: MedlinePlus

HDAC inhibition decreases the proportion of cells in S-phase. (A and B) Lateral line HCs are stained with Myosin VI, and the BrdU antibody shows dividing cells in the neuromasts of zebrafish. (C and D) BrdU+ cells were counted in control and inhibitors-treated larvae at 24 h and 48 h after neomycin damage. (E and F) Quantification of the ratio of BrdU+ HCs in control and inhibitors-treated larvae at 24 h and 48 h after neomycin incubation. Bars are mean ± s.e.m. and n = total number of embryos. **p < 0.001.
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Figure 4: HDAC inhibition decreases the proportion of cells in S-phase. (A and B) Lateral line HCs are stained with Myosin VI, and the BrdU antibody shows dividing cells in the neuromasts of zebrafish. (C and D) BrdU+ cells were counted in control and inhibitors-treated larvae at 24 h and 48 h after neomycin damage. (E and F) Quantification of the ratio of BrdU+ HCs in control and inhibitors-treated larvae at 24 h and 48 h after neomycin incubation. Bars are mean ± s.e.m. and n = total number of embryos. **p < 0.001.

Mentions: Previous studies have reported that most regenerated HCs arise from SC proliferation within 72 h after neomycin treatment (Harris et al., 2003; Ma et al., 2008), so we next determined whether inhibition of HDAC affects cell proliferation during this regeneration process. After neomycin injury, zebrafish larvae were continuously incubated in fresh egg water containing 10 mM BrdU and either TSA or VPA for 24 h and 48 h. Cellular proliferation was determined by counting the number of BrdU+ cells in the L1–L5 neuromasts. As Figure 4 illustrated, compared with control, 24 h groups treated with 0.1 μM TSA or 100 μM VPA showed significant decrease in the number of BrdU+ cells per neuromast (control larvae harbor 15.9 ± 0.5 BrdU+ cells, TSA treated-larvae harbor 6.9 ± 0.2, p < 0.001; control larvae harbor 15.5 ± 0.5 BrdU+ cells, VPA treated-larvae harbor 6.3 ± 0.3, p < 0.001). In 48 h groups, there was an intensive decrease of BrdU+ cells per neuromast after the exposure to TSA or VPA (control larvae harbor 24.8 ± 0.7, TSA treated-larvae harbor 9.6 ± 0.5, p < 0.001; control larvae harbor 25.3 ± 0.8, VPA treated-larvae harbor 10.1 ± 0.3, p < 0.001). These data suggest that TSA and VPA significantly inhibit the proliferation of neuromast cells.


Effect of histone deacetylase inhibitors trichostatin A and valproic acid on hair cell regeneration in zebrafish lateral line neuromasts.

He Y, Cai C, Tang D, Sun S, Li H - Front Cell Neurosci (2014)

HDAC inhibition decreases the proportion of cells in S-phase. (A and B) Lateral line HCs are stained with Myosin VI, and the BrdU antibody shows dividing cells in the neuromasts of zebrafish. (C and D) BrdU+ cells were counted in control and inhibitors-treated larvae at 24 h and 48 h after neomycin damage. (E and F) Quantification of the ratio of BrdU+ HCs in control and inhibitors-treated larvae at 24 h and 48 h after neomycin incubation. Bars are mean ± s.e.m. and n = total number of embryos. **p < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4230041&req=5

Figure 4: HDAC inhibition decreases the proportion of cells in S-phase. (A and B) Lateral line HCs are stained with Myosin VI, and the BrdU antibody shows dividing cells in the neuromasts of zebrafish. (C and D) BrdU+ cells were counted in control and inhibitors-treated larvae at 24 h and 48 h after neomycin damage. (E and F) Quantification of the ratio of BrdU+ HCs in control and inhibitors-treated larvae at 24 h and 48 h after neomycin incubation. Bars are mean ± s.e.m. and n = total number of embryos. **p < 0.001.
Mentions: Previous studies have reported that most regenerated HCs arise from SC proliferation within 72 h after neomycin treatment (Harris et al., 2003; Ma et al., 2008), so we next determined whether inhibition of HDAC affects cell proliferation during this regeneration process. After neomycin injury, zebrafish larvae were continuously incubated in fresh egg water containing 10 mM BrdU and either TSA or VPA for 24 h and 48 h. Cellular proliferation was determined by counting the number of BrdU+ cells in the L1–L5 neuromasts. As Figure 4 illustrated, compared with control, 24 h groups treated with 0.1 μM TSA or 100 μM VPA showed significant decrease in the number of BrdU+ cells per neuromast (control larvae harbor 15.9 ± 0.5 BrdU+ cells, TSA treated-larvae harbor 6.9 ± 0.2, p < 0.001; control larvae harbor 15.5 ± 0.5 BrdU+ cells, VPA treated-larvae harbor 6.3 ± 0.3, p < 0.001). In 48 h groups, there was an intensive decrease of BrdU+ cells per neuromast after the exposure to TSA or VPA (control larvae harbor 24.8 ± 0.7, TSA treated-larvae harbor 9.6 ± 0.5, p < 0.001; control larvae harbor 25.3 ± 0.8, VPA treated-larvae harbor 10.1 ± 0.3, p < 0.001). These data suggest that TSA and VPA significantly inhibit the proliferation of neuromast cells.

Bottom Line: Additionally, BrdU immunostaining and western blot analysis showed that TSA or VPA treatment caused a significant decrease in the percentage of S-phase cells and induced p21(Cip1) and p27(Kip1) expression, both of which are likely to explain the decrease in the amount of newly regenerated hair cells in treated embryos.Finally, we showed that HDAC inhibitors induced no observable cell death in neuromasts as measured by cleaved caspase-3 immunohistochemistry and western blot analysis.Taken together, our results demonstrate that HDAC activity has an important role in the regeneration of hair cells in the lateral line.

View Article: PubMed Central - PubMed

Affiliation: Department of Otorhinolaryngology, Affiliated Eye and ENT Hospital of Fudan University Shanghai, China.

ABSTRACT
In humans, auditory hair cells are not replaced when injured. Thus, cochlear hair cell loss causes progressive and permanent hearing loss. Conversely, non-mammalian vertebrates are capable of regenerating lost sensory hair cells. The zebrafish lateral line has numerous qualities that make it well-suited for studying hair cell development and regeneration. Histone deacetylase (HDAC) activity has been shown to have an important role in regenerative processes in vertebrates, but its function in hair cell regeneration in vivo is not fully understood. Here, we have examined the role of HDAC activity in hair cell regeneration in the zebrafish lateral line. We eliminated lateral line hair cells of 5-day post-fertilization larvae using neomycin and then treated the larvae with HDAC inhibitors. To assess hair cell regeneration, we used 5-bromo-2-deoxyuridine (BrdU) incorporation in zebrafish larvae to label mitotic cells after hair cell loss. We found that pharmacological inhibition of HDACs using trichostatin A (TSA) or valproic acid (VPA) increased histone acetylation in the regenerated neuromasts following neomycin-induced damage. We also showed that treatment with TSA or VPA decreased the number of supporting cells and regenerated hair cells in response to hair cell damage. Additionally, BrdU immunostaining and western blot analysis showed that TSA or VPA treatment caused a significant decrease in the percentage of S-phase cells and induced p21(Cip1) and p27(Kip1) expression, both of which are likely to explain the decrease in the amount of newly regenerated hair cells in treated embryos. Finally, we showed that HDAC inhibitors induced no observable cell death in neuromasts as measured by cleaved caspase-3 immunohistochemistry and western blot analysis. Taken together, our results demonstrate that HDAC activity has an important role in the regeneration of hair cells in the lateral line.

No MeSH data available.


Related in: MedlinePlus