Limits...
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 reduce the number of FM1-43FX+ cells. (A and B) We treated 5 dpf zebrafish with neomycin for 1 h and then treated them for 24 h and 48 h with TSA or VPA and subsequently imaged FM1-43FX+ cells (red). Nuclei are stained with DAPI and scale bars = 10 μm. (C and D) The average number of FM1-43FX+ cells per neuromast (NM) in larvae treated with or without TSA (C) and VPA (D) at 24 h and 48 h after neomycin damage. Bars are mean ± s.e.m. and n = total number of embryos. **p < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4230041&req=5

Figure 2: HDAC inhibitors reduce the number of FM1-43FX+ cells. (A and B) We treated 5 dpf zebrafish with neomycin for 1 h and then treated them for 24 h and 48 h with TSA or VPA and subsequently imaged FM1-43FX+ cells (red). Nuclei are stained with DAPI and scale bars = 10 μm. (C and D) The average number of FM1-43FX+ cells per neuromast (NM) in larvae treated with or without TSA (C) and VPA (D) at 24 h and 48 h after neomycin damage. Bars are mean ± s.e.m. and n = total number of embryos. **p < 0.001.

Mentions: To test the functionality of the regenerated HCs, larvae were stained with the vital dye FM1-43FX, which is a marker of functional mechanotransduction channels in HCs (Seiler and Nicolson, 1999). We imaged and quantified the FM1-43FX+ HCs in neuromasts in control and inhibitor-treated zebrafish larvae after neomycin exposure at two time points during the recovery period. Again, we found that 0.1 μM TSA or 100 μM VPA treatment induced a significant decrease in FM1-43FX+ cell numbers when compared to controls at 24 h and 48 h post-treatment (Figure 2). Therefore, we conclude that the regeneration process in larvae is severely impaired in the presence of HDAC inhibitors.


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 reduce the number of FM1-43FX+ cells. (A and B) We treated 5 dpf zebrafish with neomycin for 1 h and then treated them for 24 h and 48 h with TSA or VPA and subsequently imaged FM1-43FX+ cells (red). Nuclei are stained with DAPI and scale bars = 10 μm. (C and D) The average number of FM1-43FX+ cells per neuromast (NM) in larvae treated with or without TSA (C) and VPA (D) at 24 h and 48 h after neomycin damage. 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 2: HDAC inhibitors reduce the number of FM1-43FX+ cells. (A and B) We treated 5 dpf zebrafish with neomycin for 1 h and then treated them for 24 h and 48 h with TSA or VPA and subsequently imaged FM1-43FX+ cells (red). Nuclei are stained with DAPI and scale bars = 10 μm. (C and D) The average number of FM1-43FX+ cells per neuromast (NM) in larvae treated with or without TSA (C) and VPA (D) at 24 h and 48 h after neomycin damage. Bars are mean ± s.e.m. and n = total number of embryos. **p < 0.001.
Mentions: To test the functionality of the regenerated HCs, larvae were stained with the vital dye FM1-43FX, which is a marker of functional mechanotransduction channels in HCs (Seiler and Nicolson, 1999). We imaged and quantified the FM1-43FX+ HCs in neuromasts in control and inhibitor-treated zebrafish larvae after neomycin exposure at two time points during the recovery period. Again, we found that 0.1 μM TSA or 100 μM VPA treatment induced a significant decrease in FM1-43FX+ cell numbers when compared to controls at 24 h and 48 h post-treatment (Figure 2). Therefore, we conclude that the regeneration process in larvae is severely impaired in the presence of HDAC inhibitors.

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