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  Indian J Med Microbiol
 

Figure 3: Inhibition of ascl1a blocks TPMPA induced-proliferation. Tg(1016tuba1a:gfp) zebrafish were intravitreally injected with either 1 × PBS, 25 nmol of the gamma aminobutyric acid A receptor (GABAA-ρ receptor) inhibitor TPMPA, 0.75 nmol of control morpholino (Ctl-MO), 0.75 nmol ascl1a-MO targeting ascl1a, or combinations thereof. Fish were allowed to recover for 3 hours prior to electroporation, and an additional 45 hours before sectioning. (A) PBS/Ctl-MO co-injection. (B) PBS/ascl1-MO1 co-injection. (C) 25 nmol TPMPA/Ctl-MO co-injection. (D) 25 nmol TPMPA/ascl1-MO1 co-injection. Immunostaining was performed with antibodies against PCNA or GFP to monitor DNA replication or dedifferentiation of Müller glia, respectively. Scale bars: 50 μm. Green: tuba1a:GFP; red: PCNA; blue: TO-PRO-3 (A–D). GCL: Ganglion cell layer; INL: inner nuclear layer; ONL: outer nuclear layer. Number of optical slices: 41 (A), 46 (B), 46 (C), and 47 (D). (E, F) Quantification of PCNA+ cells, with either ascl1-MO1 (E), or ascl1-MO2 (F). n = 5 for PBS+Ctl-MO, n = 6 for PBS+ascl1-MO1, n = 9 for TPMPA+Ctl-MO, and n = 8 for TPMPA+ascl1-MO1 (E). n = 7 for PBS+Ctl-MO, n = 10 for PBS+ascl1-MO2, n = 8 for TPMPA+Ctl-MO, and n = 8 for TPMPA+ascl1-MO2 (F). Each data point is from a separate eye and is an average of two sections, counting all PCNA+ cells in the inner layer. One-way analysis of variance with Tukey's multiple comparison tests was used to test for significance. Error bars are the mean ± SEM. (E) ****P = 5.8 × 10–9 (PBS+Ctl-MO vs. TPMPA+Ctl-MO); ****P = 1.2 × 10–12 (PBS+ascl1-MO1 vs. TPMPA+Ctl-MO); ****P = 3.7 × 10–11 (TPMPA+Ctl-MO vs. TPMPA+ascl1-MO1). (F) ****P = 1.5 × 10–7 (PBS+Ctl-MO vs. TPMPA+Ctl-MO); ****P = 4 × 10–10 (PBS+ascl1-MO2 vs. TPMPA+Ctl-MO); ****P = 2.45 × 10–8 (TPMPA+Ctl-MO vs. TPMPA+ascl1-MO2). GFP: Green fluorescent protein; PCNA: proliferating cell nuclear antigen; TPMPA: (1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid.

Figure 3:  Inhibition of ascl1a blocks TPMPA induced-proliferation. 
Tg(<i>1016tuba1a:gfp</i>) zebrafish were intravitreally injected with either 1 × PBS, 25 nmol of the gamma aminobutyric acid A receptor (GABA<sub>A</sub>-ρ receptor) inhibitor TPMPA, 0.75 nmol of control morpholino (Ctl-MO), 0.75 nmol ascl1a-MO targeting ascl1a, or combinations thereof. Fish were allowed to recover for 3 hours prior to electroporation, and an additional 45 hours before sectioning. (A) PBS/Ctl-MO co-injection. (B) PBS/<i>ascl1</i>-MO1 co-injection. (C) 25 nmol TPMPA/Ctl-MO co-injection. (D) 25 nmol TPMPA/<i>ascl1</i>-MO1 co-injection. Immunostaining was performed with antibodies against PCNA or GFP to monitor DNA replication or dedifferentiation of Müller glia, respectively. Scale bars: 50 μm. Green: <i>tuba1a</i>:GFP; red: PCNA; blue: TO-PRO-3 (A–D). GCL: Ganglion cell layer; INL: inner nuclear layer; ONL: outer nuclear layer. Number of optical slices: 41 (A), 46 (B), 46 (C), and 47 (D). (E, F) Quantification of PCNA<sup>+</sup> cells, with either <i>ascl1</i>-MO1 (E), or <i>ascl1</i>-MO2 (F). <i>n</i> = 5 for PBS+Ctl-MO, <i>n</i> = 6 for PBS+<i>ascl1</i>-MO1, <i>n</i> = 9 for TPMPA+Ctl-MO, and <i>n</i> = 8 for TPMPA+<i>ascl1</i>-MO1 (E). <i>n</i> = 7 for PBS+Ctl-MO, <i>n</i> = 10 for PBS+<i>ascl1</i>-MO2, <i>n</i> = 8 for TPMPA+Ctl-MO, and <i>n</i> = 8 for TPMPA+<i>ascl1</i>-MO2 (F). Each data point is from a separate eye and is an average of two sections, counting all PCNA<sup>+</sup> cells in the inner layer. One-way analysis of variance with Tukey's multiple comparison tests was used to test for significance. Error bars are the mean ± SEM. (E) ****<i>P</i> = 5.8 × 10<sup>–9</sup> (PBS+Ctl-MO <i>vs</i>. TPMPA+Ctl-MO); ****<i>P</i> = 1.2 × 10<sup>–12</sup> (PBS+<i>ascl1</i>-MO1 <i>vs</i>. TPMPA+Ctl-MO); ****<i>P</i> = 3.7 × 10<sup>–11</sup> (TPMPA+Ctl-MO <i>vs</i>. TPMPA+<i>ascl1</i>-MO1). (F) ****<i>P</i> = 1.5 × 10<sup>–7</sup> (PBS+Ctl-MO <i>vs</i>. TPMPA+Ctl-MO); ****<i>P</i> = 4 × 10<sup>–10</sup> (PBS+<i>ascl1</i>-MO2 <i>vs</i>. TPMPA+Ctl-MO); ****<i>P</i> = 2.45 × 10<sup>–8</sup> (TPMPA+Ctl-MO <i>vs</i>. TPMPA+<i>ascl1</i>-MO2). GFP: Green fluorescent protein; PCNA: proliferating cell nuclear antigen; TPMPA: (1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid.