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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 inhibitor treatment decreases regeneration of HCs in lateral line neuromasts. (A and B) We treated 5 dpf Tg(pou4f3:gap43-GFP) zebrafish with 400 μM neomycin for 1 h and then treated them for 24 h or 48 h with TSA or VPA and subsequently imaged GFP+ HCs (green). Nuclei are stained with DAPI and scale bars = 10 μm. (C and D) Lateral line HCs are stained with Myosin VI. (E and F) The average number of Myosin VI+ cells per neuromast (NM) in larvae treated with or without HDAC inhibitor for 24 h or 48 h after neomycin damage. Bars are mean ± s.e.m. and n = total number of embryos. *p < 0.05.
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Figure 1: HDAC inhibitor treatment decreases regeneration of HCs in lateral line neuromasts. (A and B) We treated 5 dpf Tg(pou4f3:gap43-GFP) zebrafish with 400 μM neomycin for 1 h and then treated them for 24 h or 48 h with TSA or VPA and subsequently imaged GFP+ HCs (green). Nuclei are stained with DAPI and scale bars = 10 μm. (C and D) Lateral line HCs are stained with Myosin VI. (E and F) The average number of Myosin VI+ cells per neuromast (NM) in larvae treated with or without HDAC inhibitor for 24 h or 48 h after neomycin damage. Bars are mean ± s.e.m. and n = total number of embryos. *p < 0.05.

Mentions: To induce regeneration, we treated 5 dpf zebrafish larvae with 400 μM neomycin for 1 h to kill mature lateral line HCs. To facilitate visualization of regeneration, we used Tg(brn3c:mGFP) zebrafish that expressed GFP in differentiated HCs under the control of the pou4f3 promoter. After neomycin treatment, most of the HCs were damaged and lost but regeneration occurred rapidly in the fish over the following 2 days (Figures 1A1,B1; Supplemental Figure 1). To investigate the role of HDAC inhibition in HC regeneration, neomycin-treated larvae were placed in 6-well plates and exposed to TSA for 24 h and 48 h recovery periods. We found that larvae treated with 0.1 μM TSA for both 24 h and 48 h (Figures 1A2,B2) had fewer regenerated HCs relative to DMSO vehicle controls (Figures 1A1,B1). To quantify changes in the numbers of regenerated HCs after neomycin-induced damage, specific labeling of regeneration of HCs was observed and quantified using Myosin VI immunostaining. The numbers of HCs in neuromasts L1–L5 were counted in 6–13 fish at each time period. In the 24 h group, we found an average of five Myosin VI+ HCs in neuromasts of the vehicle control (Figure 1C1), but at the most only three Myosin VI+ HCs were seen in the TSA-treated neuromasts (Figure 1C2). At 48 h post-treatment, we found an average of 10 Myosin VI+ HCs in the DMSO controls (Figure 1D1) compared to at most five in the TSA-treated neuromasts (Figure 1D2).


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 inhibitor treatment decreases regeneration of HCs in lateral line neuromasts. (A and B) We treated 5 dpf Tg(pou4f3:gap43-GFP) zebrafish with 400 μM neomycin for 1 h and then treated them for 24 h or 48 h with TSA or VPA and subsequently imaged GFP+ HCs (green). Nuclei are stained with DAPI and scale bars = 10 μm. (C and D) Lateral line HCs are stained with Myosin VI. (E and F) The average number of Myosin VI+ cells per neuromast (NM) in larvae treated with or without HDAC inhibitor for 24 h or 48 h after neomycin damage. Bars are mean ± s.e.m. and n = total number of embryos. *p < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: HDAC inhibitor treatment decreases regeneration of HCs in lateral line neuromasts. (A and B) We treated 5 dpf Tg(pou4f3:gap43-GFP) zebrafish with 400 μM neomycin for 1 h and then treated them for 24 h or 48 h with TSA or VPA and subsequently imaged GFP+ HCs (green). Nuclei are stained with DAPI and scale bars = 10 μm. (C and D) Lateral line HCs are stained with Myosin VI. (E and F) The average number of Myosin VI+ cells per neuromast (NM) in larvae treated with or without HDAC inhibitor for 24 h or 48 h after neomycin damage. Bars are mean ± s.e.m. and n = total number of embryos. *p < 0.05.
Mentions: To induce regeneration, we treated 5 dpf zebrafish larvae with 400 μM neomycin for 1 h to kill mature lateral line HCs. To facilitate visualization of regeneration, we used Tg(brn3c:mGFP) zebrafish that expressed GFP in differentiated HCs under the control of the pou4f3 promoter. After neomycin treatment, most of the HCs were damaged and lost but regeneration occurred rapidly in the fish over the following 2 days (Figures 1A1,B1; Supplemental Figure 1). To investigate the role of HDAC inhibition in HC regeneration, neomycin-treated larvae were placed in 6-well plates and exposed to TSA for 24 h and 48 h recovery periods. We found that larvae treated with 0.1 μM TSA for both 24 h and 48 h (Figures 1A2,B2) had fewer regenerated HCs relative to DMSO vehicle controls (Figures 1A1,B1). To quantify changes in the numbers of regenerated HCs after neomycin-induced damage, specific labeling of regeneration of HCs was observed and quantified using Myosin VI immunostaining. The numbers of HCs in neuromasts L1–L5 were counted in 6–13 fish at each time period. In the 24 h group, we found an average of five Myosin VI+ HCs in neuromasts of the vehicle control (Figure 1C1), but at the most only three Myosin VI+ HCs were seen in the TSA-treated neuromasts (Figure 1C2). At 48 h post-treatment, we found an average of 10 Myosin VI+ HCs in the DMSO controls (Figure 1D1) compared to at most five in the TSA-treated neuromasts (Figure 1D2).

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