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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 inhibitors significantly suppresses cell proliferation. (A and B) Lateral line SCs are stained with Sox2, and the BrdU antibody shows dividing cells in the neuromasts of zebrafish. (C and D) Quantification of the ratio of BrdU+ SCs in control and TSA- or VPA-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 5: HDAC inhibitors significantly suppresses cell proliferation. (A and B) Lateral line SCs are stained with Sox2, and the BrdU antibody shows dividing cells in the neuromasts of zebrafish. (C and D) Quantification of the ratio of BrdU+ SCs in control and TSA- or VPA-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: Because the number of SCs in HDAC inhibitor-treated neuromasts is different from that of control neuromasts, we next examined the rates of SC division among different groups after neomycin damage at 24 h and 48 h by BrdU and Sox2 immunolabeling. Similar to the results described above, at 24 h after neomycin treatment we found fewer double-stained cells in the presence of TSA or VPA compared with the vehicle controls (Figure 5A). Furthermore, there was a significant difference between the ratio of BrdU+ SCs (Figures 5C,D). By 48 h after neomycin insult, the control larvae and the HDAC inhibitor-treated larvae showed a striking difference in the number of double-stained cells (Figure 5B). The ratio of BrdU+ SCs in the treated neuromasts were drastically reduced, and this most likely explains the reduction in the number of newly regenerated HCs in the inhibitor-treated embryos (Figures 5C,D). Taken together, these results show that HDAC inhibition has a significant negative impact on proliferation in the regenerating neuromast.


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 inhibitors significantly suppresses cell proliferation. (A and B) Lateral line SCs are stained with Sox2, and the BrdU antibody shows dividing cells in the neuromasts of zebrafish. (C and D) Quantification of the ratio of BrdU+ SCs in control and TSA- or VPA-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 5: HDAC inhibitors significantly suppresses cell proliferation. (A and B) Lateral line SCs are stained with Sox2, and the BrdU antibody shows dividing cells in the neuromasts of zebrafish. (C and D) Quantification of the ratio of BrdU+ SCs in control and TSA- or VPA-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: Because the number of SCs in HDAC inhibitor-treated neuromasts is different from that of control neuromasts, we next examined the rates of SC division among different groups after neomycin damage at 24 h and 48 h by BrdU and Sox2 immunolabeling. Similar to the results described above, at 24 h after neomycin treatment we found fewer double-stained cells in the presence of TSA or VPA compared with the vehicle controls (Figure 5A). Furthermore, there was a significant difference between the ratio of BrdU+ SCs (Figures 5C,D). By 48 h after neomycin insult, the control larvae and the HDAC inhibitor-treated larvae showed a striking difference in the number of double-stained cells (Figure 5B). The ratio of BrdU+ SCs in the treated neuromasts were drastically reduced, and this most likely explains the reduction in the number of newly regenerated HCs in the inhibitor-treated embryos (Figures 5C,D). Taken together, these results show that HDAC inhibition has a significant negative impact on proliferation in the regenerating neuromast.

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