Journal: Cell reports

Article Title: Critical examination of Ptbp1-mediated glia-to-neuron conversion in the mouse retina

doi: 10.1016/j.celrep.2022.110960

Figure Lengend Snippet: (A) Experimental design for testing MG-to-RGC conversion. Subretinal injection of PHP.eB-AAVs was performed in Rosa-CAG-LSL-Sun1-GFP reporter mice at 5 weeks of age, followed by immunohistochemistry analysis using the pan-RGC marker RBPMS at 4 weeks after AAV injection. IHC, immunohistochemistry. (B) Schematic illustration showing that AAV-GFAP-Cre subretinal injection results in Sun1-GFP expression after Cre-dependent recombination in Sun1-GFP reporter mice. (C) Quantification of Sun1-GFP + Rbpms + cells in the ganglion cell layer (GCL) at 4 weeks after AAV injection. n = 4 retinas per group. Data are presented as the mean ± SEM. No significant difference was found between any two groups, p > 0.5, unpaired t test. (D–G) Confocal images showing expression of Sun1-GFP and RBPMS immunohistochemistry after Ptbp1 knockdown in retinas receiving GFAP-Cre and GFAP-CasRx- Ptbp1 . White arrowheads: Sun1-GFP-labeled cells were RBPMS positive in the GCL. (H–K) Confocal images showing expression of Sun1-GFP and RBPMS immunohistochemistry in retinas receiving GFAP-Cre and GFAP-CasRx. White arrowheads: Sun1-GFP-labeled cells were RBPMS positive in the GCL. (L–O) Confocal images showing expression of Sun1-GFP and RBPMS immunohistochemistry in retinas receiving GFAP-Cre only. White arrowheads: Sun1-GFP-labeled cells were RBPMS positive in the GCL. (D–O) Scale bars, 100 μm. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. See also .

Article Snippet: AAV-GFAP-CasRx (Addgene #15400) and AAV-GFAP-CasRx- Ptbp1 (Addgene #154001) were deposited from Dr. Hui Yang’s laboratory to Addgene.

Techniques: Injection, Immunohistochemistry, Marker, Expressing, Knockdown, Labeling

Journal: Cell reports

Article Title: Critical examination of Ptbp1-mediated glia-to-neuron conversion in the mouse retina

doi: 10.1016/j.celrep.2022.110960

Figure Lengend Snippet: (A) Experimental design for testing MG-to-RGC conversion in Sun1-GFP fate-mapping mice. Subretinal injection of PHP.eB-AAVs was performed in Sun1-GFP fate-mapping mice receiving tamoxifen-induced labeling of MG at 5 weeks of age. Ptbp1 mRNA and protein levels were measured from purified MG using fluorescence-activated cell sorting (FACS) and immunofluorescence (IF), respectively, at 2 weeks after AAV injection. Immunohistochemistry analysis using the pan-RGC marker RBPMS was performed at 4 weeks after AAV injection. IHC, immunohistochemistry. (B) Experimental design for FACS purification of Sun1-GFP labeled MG with or without AAV infection. (C) Real-time qPCR analysis of AAV-transduced (tdTomato + ) Sun1-GFP labeled MG confirmed the expression of AAV-GFAP-CasRx or AAV-GFAP-CasRx- Ptbp1 . The relative expression level of CasRx in Sun1-GFP-labeled MG without AAV infection was set as 1. Data are presented as the mean ± SEM, n = 3. (D) Confocal images of the indicated markers showing low-efficiency knockdown of Ptbp1 by the CRISPR-CasRx system 2 weeks after AAV infection. Scale bars, 100 μm. (E) Real-time qPCR analysis of Ptbp1 mRNA levels in AAV-transduced (tdTomato + ) Sun1-GFP-labeled MG showing a mild knockdown effect. The relative expression level of Ptbp1 in AAV-GFAP-CasRx (control)- and AAV-GFAP-tdTomato (infection marker)-transduced MG was set as 1. Data are presented as the mean ± SEM, n = 3, **p < 0.01, unpaired t test. (F) Quantification of PTBP1 immunofluorescence intensity in AAV-transduced (tdTomato + ) Sun1-GFP-labeled MG showing low-efficiency knockdown of Ptbp1. n = 4 retinas per group. Data are presented as the mean ± SEM, **p < 0.01, unpaired t test. (G) Total numbers of Sun1-GFP-labeled MG examined in retinas at 4 weeks after receiving GFAP-tdTomato (an infection marker), together with GFAP-CasRx (control) or GFAP-CasRx- Ptbp1 (Ptbp1 downregulation). Data are presented as mean ± SEM, n = 7–9 retinas per group. (H) Total numbers of Sun1-GFP-labeled MG that were also immunoreactive for the pan-RGC marker RBPMS per retina after Ptbp1 downregulation. (I–M) Confocal images showing expression of Sun1-GFP and RBPMS immunohistochemistry in retinas receiving GFAP-CasRx and GFAP-tdTomato. (N–R) Confocal images showing expression of Sun1-GFP and RBPMS immunohistochemistry in retinas receiving GFAP-CasRx- Ptbp1 and GFAP-tdTomato. (I–R) Scale bars, 100 μm. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. See also and .

Article Snippet: AAV-GFAP-CasRx (Addgene #15400) and AAV-GFAP-CasRx- Ptbp1 (Addgene #154001) were deposited from Dr. Hui Yang’s laboratory to Addgene.

Techniques: Injection, Labeling, Purification, Fluorescence, FACS, Immunofluorescence, Immunohistochemistry, Marker, Infection, Expressing, Knockdown, CRISPR, Control

Journal: Cell reports

Article Title: Critical examination of Ptbp1-mediated glia-to-neuron conversion in the mouse retina

doi: 10.1016/j.celrep.2022.110960

Figure Lengend Snippet: (A) Experimental design for testing MG-to-RGC conversion in Sun1-GFP fate-mapping mice. Intravitreal injection of ShH10-CMV-LSL-AAVs was performed inSun1-GFP fate-mapping mice at 5 weeks of age, followed by tamoxifen administration 2 days later to induce Sun1-GFP expression in MG and Ptbp1 depletion. Fluorescence-activated cell sorting (FACS) and immunofluorescence (IF) were used, respectively, to measure Ptbp1 mRNA and protein levels at 2 weeks after tamoxifen injection. Immunohistochemistry analysis was performed using the pan-RGC marker RBPMS at 4 weeks after tamoxifen injection. IHC, immunohistochemistry. (B) Experimental design for FACS purification of Sun1-GFP-labeled MG receiving ShH10-CMV-LSL-RFP (control) or CMV-LSL-RFP-shPtbp1 ( Ptbp1 depletion). (C) Real-time qPCR analysis in AAV-transduced (RFP + ) Sun1-GFP-labeled MG showing high-efficiency knockdown of Ptbp1. The relative expression level of Ptbp1 in AAV-CMV-LSL-RFP (control) transduced MG was set as 1. Data are presented as the mean ± SEM, n = 3, ****p < 0.0001, unpaired t test. (D–H) Confocal images showing Ptbp1 expression in Sun1-GFP fate-mapping mice receiving intravitreal injection of ShH10-CMV-LSL-RFP (control). Scale bar, 100 μm. The boxed areas are enlarged in (D′–H′). Pink arrowheads indicate that Ptbp1 levels were maintained in RFP + Sun1-GFP-labeled MG. (I–M) Confocal images showing Ptbp1 expression in Sun1-GFP fate-mapping mice receiving intravitreal injection of ShH10-CMV-LSL-RFP-shPtbp1 ( Ptbp1 depletion). Scale bar, 100 μm. The boxed areas are enlarged in (I′–M′). Blue arrowheads indicate that Ptbp1 levels were depleted in RFP + Sun1-GFP-labeled MG. Yellow arrow-heads indicate that Ptbp1 levels were maintained in RFP − Sun1-GFP-labeled MG (non-transduced MG, serving as an internal control for Ptbp1 depletion in transduced MG). (N) Quantification of PTBP1 immunofluorescence intensity in AAV-transduced (RFP + ) Sun1-GFP-labeled MG showing high-efficiency knockdown of Ptbp1. n = 4–5 retinas per group. Data are presented as the mean ± SEM, ****p < 0.0001, unpaired t test. (O–S) Confocal images showing expression of Sun1-GFP and RBPMS immunohistochemistry in retinas receiving CMV-LSL-RFP. (T–X) Confocal images showing expression of Sun1-GFP and RBPMS immunohistochemistry in retinas receiving CMV-LSL-RFP-shPtbp1. (O–X) Scale bar, 100 μm. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. (Y) Total numbers of Sun1-GFP-labeled MG examined in retinas (4 weeks after tamoxifen injection) receiving CMV-LSL-RFP (control) or CMV-LSL-RFP-shPtbp1 ( Ptbp1 depletion). Data are presented as mean ± SEM, n = 6–8 retinas per group. (Z) Total numbers of Sun1-GFP-labeled MG that were also immunoreactive for the pan-RGC marker RBPMS per retina after Pbtp1 depletion. See also and .

Article Snippet: AAV-GFAP-CasRx (Addgene #15400) and AAV-GFAP-CasRx- Ptbp1 (Addgene #154001) were deposited from Dr. Hui Yang’s laboratory to Addgene.

Techniques: Injection, Expressing, Fluorescence, FACS, Immunofluorescence, Immunohistochemistry, Marker, Purification, Labeling, Control, Knockdown

Journal: Cell reports

Article Title: Critical examination of Ptbp1-mediated glia-to-neuron conversion in the mouse retina

doi: 10.1016/j.celrep.2022.110960

Figure Lengend Snippet: (A) Experimental design for testing MG-to-RGC conversion in NMDA-injured retinas of Sun1-GFP fate-mapping mice. Intravitreal injection of NMDA (1.5 μL, 200 mM) was performed in Sun1-GFP fate-mapping mice receiving tamoxifen-induced labeling of MG at 5 weeks of age, followed by subretinal injection of PHP.eB-AAVs at 2 weeks after injury. Immunohistochemistry analysis using the pan-RGC marker RBPMS was performed at 4 weeks after AAV injection. IHC, immunohistochemistry. (B) Total numbers of Sun1-GFP-labeled MG examined in NMDA-injured retinas at 4 weeks after receiving GFAP-tdTomato (an infection marker), together with GFAP-CasRx (control) or GFAP-CasRx- Ptbp1 (Ptbp1 downregulation). Data are presented as mean ± SEM, n = 3–4 retinas per group. (C) Total numbers of Sun1-GFP-labeled MG that were also immunoreactive for the pan-RGC marker RBPMS per retina after Ptbp1 downregulation. (D–H) Confocal images showing expression of Sun1-GFP and RBPMS immunohistochemistry in NMDA-injured retinas receiving GFAP-CasRx and GFAP-tdTomato. (I–M) Confocal images showing expression of Sun1-GFP and RBPMS immunohistochemistry in NMDA-injured retinas receiving GFAP-CasRx- Ptbp1 and GFAP-tdTomato. (D–M) Scale bars, 100 μm. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. (N) Experimental design for testing MG-to-RGC conversion in NMDA-injured retinas of Sun1-GFP fate-mapping mice. Intravitreal injection of NMDA (1.5 μL, 200 mM) was performed in Sun1-GFP fate-mapping mice at 5 weeks of age, followed by intravitreal injection of ShH10-CMV-LSL-AAVs 2 weeks later. Tamoxifen was administrated 2 days later to induce Sun1-GFP expression in MG and Ptbp1 depletion. Immunohistochemistry analysis was performed using the pan-RGC marker RBPMS at 4 weeks after tamoxifen injection. IHC, immunohistochemistry. (O) Total numbers of Sun1-GFP-labeled MG examined in NMDA-injured retinas at 4 weeks receiving CMV-LSL-RFP (control) or CMV-LSL-RFP-shPtbp1 ( Ptbp1 depletion). Data are presented as mean ± SEM, n = 4–5 retinas per group. (P) Total numbers of Sun1-GFP-labeled MG that were also immunoreactive for the pan-RGC marker RBPMS per retina after Pbtp1 depletion. (Q–U) Confocal images showing expression of Sun1-GFP and RBPMS immunohistochemistry in NMDA-injured retinas receiving CMV-LSL-RFP. (V–Z) Confocal images showing expression of Sun1-GFP and RBPMS immunohistochemistry in NMDA-injured retinas receiving CMV-LSL-RFP-shPtbp1. (Q–Z) Scale bars, 100 μm.

Article Snippet: AAV-GFAP-CasRx (Addgene #15400) and AAV-GFAP-CasRx- Ptbp1 (Addgene #154001) were deposited from Dr. Hui Yang’s laboratory to Addgene.

Techniques: Injection, Labeling, Immunohistochemistry, Marker, Infection, Control, Expressing

Journal: Cell reports

Article Title: Critical examination of Ptbp1-mediated glia-to-neuron conversion in the mouse retina

doi: 10.1016/j.celrep.2022.110960

Figure Lengend Snippet:

Article Snippet: AAV-GFAP-CasRx (Addgene #15400) and AAV-GFAP-CasRx- Ptbp1 (Addgene #154001) were deposited from Dr. Hui Yang’s laboratory to Addgene.

Techniques: Recombinant, Software