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HDAC3 But not HDAC2 Mediates Visual Experience-Dependent Radial Glia Proliferation in the Developing Xenopus Tectum

View Article: PubMed Central - PubMed

ABSTRACT

Radial glial cells (RGs) are one of the important progenitor cells that can differentiate into neurons or glia to form functional neural circuits in the developing central nervous system (CNS). Histone deacetylases (HDACs) has been associated with visual activity dependent changes in BrdU-positive progenitor cells in the developing brain. We previously have shown that HDAC1 is involved in the experience-dependent proliferation of RGs. However, it is less clear whether two other members of class I HDACs, HDAC2 and HDAC3, are involved in the regulation of radial glia proliferation. Here, we reported that HDAC2 and HDAC3 expression were developmentally regulated in tectal cells, especially in the ventricular layer of the BLBP-positive RGs. Pharmacological blockade using an inhibitor of class I HDACs, MS-275, decreased the number of BrdU-positive dividing progenitor cells. Specific knockdown of HDAC3 but not HDAC2 decreased the number of BrdU- and BLBP-labeled cells, suggesting that the proliferation of radial glia was selectively mediated by HDAC3. Visual deprivation induced selective augmentation of histone H4 acetylation at lysine 16 in BLBP-positive cells. Furthermore, the visual deprivation-induced increase in BrdU-positive cells was partially blocked by HDAC3 downregulation but not by HDAC2 knockdown at stage 49 tadpoles. These data revealed a specific role of HDAC3 in experience-dependent radial glia proliferation during the development of Xenopus tectum.

No MeSH data available.


HDAC2 or HDAC3 expression is decreased by HDAC2-MO or HDAC3-MO knockdown. (A) Western blot analysis of homogenates from Ctrl-MO and HDAC2-MO transfected brains using an anti-HDAC2 antibody. (B) Quantification revealed that HDAC2 expression was significantly decreased in the HDAC2-MO transfected tectum compared to controls. (C) Ctrl-MO and HDAC3-MO transfected brain homogenates were compared using an anti-HDAC3 antibody. (D) HDAC3 expression was significantly decreased in the HDAC3-MO tectum. Data is represented as an intensity ratio of HDAC2 or HDAC3 to GAPDH normalized to the control value. Two-tailed T-test, N = 3, **p < 0.01. (E) Representative immunofluorescent staining of HDAC2 at HDAC2-MO-transfected cells (E1–E4) and HDAC3 at HDAC3-MO-transfected cells (E5–E8) in stage 48 tadpoles. Arrows indicate HDAC2/3-MO-transfected RGs stained for HDAC2/3. Scale: 20 μm.
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Figure 4: HDAC2 or HDAC3 expression is decreased by HDAC2-MO or HDAC3-MO knockdown. (A) Western blot analysis of homogenates from Ctrl-MO and HDAC2-MO transfected brains using an anti-HDAC2 antibody. (B) Quantification revealed that HDAC2 expression was significantly decreased in the HDAC2-MO transfected tectum compared to controls. (C) Ctrl-MO and HDAC3-MO transfected brain homogenates were compared using an anti-HDAC3 antibody. (D) HDAC3 expression was significantly decreased in the HDAC3-MO tectum. Data is represented as an intensity ratio of HDAC2 or HDAC3 to GAPDH normalized to the control value. Two-tailed T-test, N = 3, **p < 0.01. (E) Representative immunofluorescent staining of HDAC2 at HDAC2-MO-transfected cells (E1–E4) and HDAC3 at HDAC3-MO-transfected cells (E5–E8) in stage 48 tadpoles. Arrows indicate HDAC2/3-MO-transfected RGs stained for HDAC2/3. Scale: 20 μm.

Mentions: To test whether HDAC2 or HDAC3 affected the proliferation of RGs, we used antisense morpholinos of HDAC2-MO or HDAC3-MO to downregulate each of the HDACs expression. Western blot analysis of brain homogenates demonstrated that HDAC2-MO injection in the tectum results in a 28.1% downregulation of endogenous HDAC2 (Figures 4A,B), whereas HDAC3-MO transfection results in a 19.7% knockdown of HDAC3 (Figures 4C,D). Immunofluorescent staining also indicated that HDAC2-MO or HDAC3-MO transfected cells showed less HDAC2 or HDAC3 expression (Figure 4E). Tadpoles were transfected with Ctrl-MO, HDAC2-MO or HDAC3-MO at stage 46 respectively and maintained in the Steinberg’s solution for 48 h (Figure 5A). Tadpoles at stage 48 were subjected to BrdU labeling and immunostained with the anti-BLBP and anti-BrdU antibodies for counting BrdU+ and BLBP+ cells (Figure 5A). Summary data showed that the numbers of BrdU+ cells were markedly reduced for the HDAC3-MO transfected tadpoles compared to untreated or Ctrl-MO controls (Figures 5A,B). The number of BLBP+ cells were also decreased in the HDAC3-MO brain tectum compared to control or Ctrl-MO brain (Figures 5A,C). However, the numbers of BrdU+ and BLBP+ cells in HDAC2-MO transfected tectum (Figure 5A) were not altered (Figures 5B,C). These data suggested that HDAC3 but not HDAC2 knockdown reduced the number of BrdU+ and BLBP+ RGs in the tectum.


HDAC3 But not HDAC2 Mediates Visual Experience-Dependent Radial Glia Proliferation in the Developing Xenopus Tectum
HDAC2 or HDAC3 expression is decreased by HDAC2-MO or HDAC3-MO knockdown. (A) Western blot analysis of homogenates from Ctrl-MO and HDAC2-MO transfected brains using an anti-HDAC2 antibody. (B) Quantification revealed that HDAC2 expression was significantly decreased in the HDAC2-MO transfected tectum compared to controls. (C) Ctrl-MO and HDAC3-MO transfected brain homogenates were compared using an anti-HDAC3 antibody. (D) HDAC3 expression was significantly decreased in the HDAC3-MO tectum. Data is represented as an intensity ratio of HDAC2 or HDAC3 to GAPDH normalized to the control value. Two-tailed T-test, N = 3, **p < 0.01. (E) Representative immunofluorescent staining of HDAC2 at HDAC2-MO-transfected cells (E1–E4) and HDAC3 at HDAC3-MO-transfected cells (E5–E8) in stage 48 tadpoles. Arrows indicate HDAC2/3-MO-transfected RGs stained for HDAC2/3. Scale: 20 μm.
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Figure 4: HDAC2 or HDAC3 expression is decreased by HDAC2-MO or HDAC3-MO knockdown. (A) Western blot analysis of homogenates from Ctrl-MO and HDAC2-MO transfected brains using an anti-HDAC2 antibody. (B) Quantification revealed that HDAC2 expression was significantly decreased in the HDAC2-MO transfected tectum compared to controls. (C) Ctrl-MO and HDAC3-MO transfected brain homogenates were compared using an anti-HDAC3 antibody. (D) HDAC3 expression was significantly decreased in the HDAC3-MO tectum. Data is represented as an intensity ratio of HDAC2 or HDAC3 to GAPDH normalized to the control value. Two-tailed T-test, N = 3, **p < 0.01. (E) Representative immunofluorescent staining of HDAC2 at HDAC2-MO-transfected cells (E1–E4) and HDAC3 at HDAC3-MO-transfected cells (E5–E8) in stage 48 tadpoles. Arrows indicate HDAC2/3-MO-transfected RGs stained for HDAC2/3. Scale: 20 μm.
Mentions: To test whether HDAC2 or HDAC3 affected the proliferation of RGs, we used antisense morpholinos of HDAC2-MO or HDAC3-MO to downregulate each of the HDACs expression. Western blot analysis of brain homogenates demonstrated that HDAC2-MO injection in the tectum results in a 28.1% downregulation of endogenous HDAC2 (Figures 4A,B), whereas HDAC3-MO transfection results in a 19.7% knockdown of HDAC3 (Figures 4C,D). Immunofluorescent staining also indicated that HDAC2-MO or HDAC3-MO transfected cells showed less HDAC2 or HDAC3 expression (Figure 4E). Tadpoles were transfected with Ctrl-MO, HDAC2-MO or HDAC3-MO at stage 46 respectively and maintained in the Steinberg’s solution for 48 h (Figure 5A). Tadpoles at stage 48 were subjected to BrdU labeling and immunostained with the anti-BLBP and anti-BrdU antibodies for counting BrdU+ and BLBP+ cells (Figure 5A). Summary data showed that the numbers of BrdU+ cells were markedly reduced for the HDAC3-MO transfected tadpoles compared to untreated or Ctrl-MO controls (Figures 5A,B). The number of BLBP+ cells were also decreased in the HDAC3-MO brain tectum compared to control or Ctrl-MO brain (Figures 5A,C). However, the numbers of BrdU+ and BLBP+ cells in HDAC2-MO transfected tectum (Figure 5A) were not altered (Figures 5B,C). These data suggested that HDAC3 but not HDAC2 knockdown reduced the number of BrdU+ and BLBP+ RGs in the tectum.

View Article: PubMed Central - PubMed

ABSTRACT

Radial glial cells (RGs) are one of the important progenitor cells that can differentiate into neurons or glia to form functional neural circuits in the developing central nervous system (CNS). Histone deacetylases (HDACs) has been associated with visual activity dependent changes in BrdU-positive progenitor cells in the developing brain. We previously have shown that HDAC1 is involved in the experience-dependent proliferation of RGs. However, it is less clear whether two other members of class I HDACs, HDAC2 and HDAC3, are involved in the regulation of radial glia proliferation. Here, we reported that HDAC2 and HDAC3 expression were developmentally regulated in tectal cells, especially in the ventricular layer of the BLBP-positive RGs. Pharmacological blockade using an inhibitor of class I HDACs, MS-275, decreased the number of BrdU-positive dividing progenitor cells. Specific knockdown of HDAC3 but not HDAC2 decreased the number of BrdU- and BLBP-labeled cells, suggesting that the proliferation of radial glia was selectively mediated by HDAC3. Visual deprivation induced selective augmentation of histone H4 acetylation at lysine 16 in BLBP-positive cells. Furthermore, the visual deprivation-induced increase in BrdU-positive cells was partially blocked by HDAC3 downregulation but not by HDAC2 knockdown at stage 49 tadpoles. These data revealed a specific role of HDAC3 in experience-dependent radial glia proliferation during the development of Xenopus tectum.

No MeSH data available.