Journal: bioRxiv

Article Title: Dyrk1a gene dosage controls bipolar cell development and retinal connectivity

doi: 10.64898/2026.03.15.710015

Figure Lengend Snippet: (A – X) Representative immunohistochemical staining of coronal eye sections taken from Lhx2-Cre and Lhx2-Cre:Dyrk1a +/f adult mice (6 – 8 weeks) across four designated dorsoventral quadrants (D1, D2, V3 and V4) spanning the entire retina relative to the optic nerve head. (A – H) All quadrants in the INL of Lhx2-Cre (A – D) animals were comparably populated with bipolar cells (Chx10 + , BC, arrowheads). In contrast, the dorsoventral gradient of reduced INL thickness in Lhx2-Cre:Dyrk1a f/f animals (E – H) was accompanied by an apparent reduction in total bipolar cell number (Chx10 + , BC, arrowheads) which was most prominent in the D1 quadrant (E). (I – P) The INL and GCL of Lhx2-Cre animals (I – L) were densely populated with amacrine cells (INL, Pax6 + , AC, arrowheads) and RGCs (GCL, Pax6 + , RGC, arrowheads) across the four designated dorsoventral quadrants. In contrast, the dorsoventral gradient of reduced INL and GCL thickness in Lhx2-Cre:Dyrk1a f/f (M – P) animals led to a reduction in amacrine and RGC number (Pax6 + , RGC/AC, arrowheads) which was most prominent in the D1 quadrant (M). (Q – X) Horizontal cells (Calbindin + , HC, arrowheads) in the INL of Lhx2-Cre (Q – T) exhibited comparable numbers across the retina. In contrast, the dorsoventral gradient of reduced INL thickness in Lhx2-Cre:Dyrk1a f/f (U – X) animals led to a reduction in horizontal cell number (Calbindin + , HC, arrowheads) which was most prominent in the D1 quadrant (U). Scale bar: (A – X) 25 μm. Abbreviations: AC, amacrine cell; BC, bipolar cell; D, dorsal; GCL, ganglion cell layer; HC, horizontal cell, INL, inner nuclear layer; ONL, outer nuclear layer; RGC, retinal ganglion cell; V, ventral.

Article Snippet: The following primary antibodies and dilutions were used in this study: Calbindin (1:300, Sigma-Aldrich, #C9848); Calretinin (1:1000, Swant, #CG1); cleaved Caspase3 (Asp175) (1:500, Cell Signalling, #9661S); chAT (1:100, Millipore, #AB144P); Chx10 (1:50, SCBT, #SC365519); Dyrk1a (1:100, Novus, #NBP1-84032); Isl1/2 (recognises both Isl1 and Isl2 proteins) (1:500; DSHB, #39.4D5); M-opsin (1:500, Proteintech, #30975-1-AP); Pax6 (1:100, DSHB, #Pax6); Pikachurin (1:200, Proteintech, #14578-1-AP); Rhodopsin (1:500, Millipore, #MABN15); S-opsin (1:500, Proteintech, #24660-1-AP); TH (1:300, Millipore, #AB152); vGlut1 (1:2000, Millipore, #AB5905).

Techniques: Immunohistochemical staining, Staining

Journal: bioRxiv

Article Title: Dyrk1a gene dosage controls bipolar cell development and retinal connectivity

doi: 10.64898/2026.03.15.710015

Figure Lengend Snippet: (A – H) Representative flat mount retina images from Lhx2-Cre (A – D) and Lhx2-Cre:Dyrk1a +/f (E – H) adult mice (6 – 8 weeks) showing the mosaic distribution of bipolar cells across four defined dorsoventral quadrants (D1, D2, V3, and V4) spanning the entire central retina relative to the optic nerve head. Transgenic controls displayed an even distribution of bipolar cells across all quadrants. In contrast, heterozygous animals showed a reduced number of Chx10 + bipolar cells in the dorsal-most quadrant (E) while the remaining domains (F – H) were comparable to transgenic controls. (I – P) Representative Voronoi domain plots of bipolar cell distribution in Lhx2-Cre (I – L) and Lhx2-Cre:Dyrk1a +/f (M – P) adult mice (6 – 8 weeks). Transgenic controls showed evenly spaced bipolar cell mosaics with nearest neighbour (NNRI) and Voronoi domain regularity indices (VDRI) indicating robust spatial regularity. In contrast, the dorsal-most quadrant of heterozygous mice exhibited mosaic spacing variability reflected by irregular Voronoi domain areas and reduced regularity indices (M) while the remaining quadrants (N – P) retained regularity indices comparable to transgenic controls. (Q – T) Quantitative analysis of nearest neighbour distance in Lhx2-Cre ( n = 5) and Lhx2-Cre:Dyrk1a +/f ( n = 8) adult animals (6 – 8 weeks). Nearest neighbour distance was significantly increased in the dorsal-most quadrant of heterozygous animals owing to a reduced cell number (Q) while the other regions remained comparable to transgenic controls (R – T). (U – X) Quantitative analysis of Voronoi domain area in Lhx2-Cre ( n = 5) and Lhx2-Cre:Dyrk1a +/f ( n = 8) adult animals (6 – 8 weeks). A significant increase in Voronoi domain area was observed in the dorsal-most quadrant of heterozygous mice owing to decreased cell number (U) while the other quadrants showed no significant differences (V – X). All data represents the mean ± SEM. Statistical differences were calculated using Mann-Whitney tests. p-values are denoted as follows: **p≤ 0.01. Scale bar: (A – P) 50 μm. Abbreviations: CV, coefficient of variation; D, dorsal; N, nasal; NNRI, nearest neighbour regularity index; T, temporal; V, ventral; VDRI, Voronoi domain regularity index.

Article Snippet: The following primary antibodies and dilutions were used in this study: Calbindin (1:300, Sigma-Aldrich, #C9848); Calretinin (1:1000, Swant, #CG1); cleaved Caspase3 (Asp175) (1:500, Cell Signalling, #9661S); chAT (1:100, Millipore, #AB144P); Chx10 (1:50, SCBT, #SC365519); Dyrk1a (1:100, Novus, #NBP1-84032); Isl1/2 (recognises both Isl1 and Isl2 proteins) (1:500; DSHB, #39.4D5); M-opsin (1:500, Proteintech, #30975-1-AP); Pax6 (1:100, DSHB, #Pax6); Pikachurin (1:200, Proteintech, #14578-1-AP); Rhodopsin (1:500, Millipore, #MABN15); S-opsin (1:500, Proteintech, #24660-1-AP); TH (1:300, Millipore, #AB152); vGlut1 (1:2000, Millipore, #AB5905).

Techniques: Transgenic Assay, MANN-WHITNEY

Journal: Cell & Bioscience

Article Title: NOTCH2NLC GGC repeat expansions cause retinal neurodegeneration in neuronal intranuclear inclusion disease mouse model

doi: 10.1186/s13578-026-01542-x

Figure Lengend Snippet: Expression of NOTCH2NLC-polyG in the retina of NIID mice. A – F Co-immunostaining on retinas of NIID and control mice using anti-Flag (indicating NOTCH2NLC-polyG) with anti-VSX2 ( A ), anti-PAX6 ( B ), anti-RBPMS ( C ), anti-Rhodopsin ( D ), anti-Arrestin C ( E ), or anti-RPE65 ( F ). Red: VSX2 ( A ), PAX6 ( B ), RBPMS ( C ), Rhodopsin ( D ), Arrestin C ( E ) or RPE65 ( F ); Green: NOTCH2NLC-polyG; Blue: DAPI. Scale bar = 50 μm. The yellow dashed box indicates the region shown at higher magnification. G The percentage of cells co-expressing PolyG and VSX2, PAX6 or RBPMS. H The quantification of the number of VSX2-, PAX6-, and RBPMS-positive cells in NIID and control mice. Data are presented as mean ± SEM. N = 6 per group, ** P = 0.0012 (PAX6), ** P = 0.0021 (RBPMS), ns = no significance, two-tailed t-test. I Transmission electron microscopy images of intranuclear inclusions in the INL and RGC layer of the retinas of NIID mice. The yellow dashed box indicates the region shown at higher magnification. The yellow arrows indicate the round-shaped, filamentous, non-membranous intranuclear inclusions. Scale bar = 1 μm

Article Snippet: Primary antibodies used in this study include: RBPMS (Thermo Fisher Scientific, PA5-31231, 1/300), VSX2 (Proteintech, 25825-1-AP, 1/300), PAX6 (Abcam, ab195045, 1/300), Rhodopsin (Proteintech, 30438-1-AP, 1/200), Arrestin C (Proteintech, 85067-1-AP, 1/200), Flag (Sigma, F1804, 1/300), Flag (Thermo Fisher Scientific, PA5-31231, 1/300), Tuj1 (Sigma, MAB5564, 1/300), GFAP (Cell Signaling Technology, 3670S, 1/300), IBA1 (Wako, 019-19741, 1/300), GFP (Thermo Fisher Scientific, MA5-15256, 1/300).

Techniques: Expressing, Immunostaining, Control, Two Tailed Test, Transmission Assay, Electron Microscopy

Journal: Stem cell research & therapy

Article Title: Human embryonic stem cell-derived extracellular vesicles alleviate retinal degeneration by upregulating Oct4 to promote retinal Müller cell retrodifferentiation via HSP90.

doi: 10.1186/s13287-020-02034-6

Figure Lengend Snippet: Fig. 3 hESEVs promote the retrodifferentiation of Müller cells into retinal progenitor cells in vitro. Müller cells were treated with DiI labeled-hESEVs. Immunofluorescence was used to observe DiI labeled-hESEVs: green indicates DiI labeled-hESEVs and blue indicates DAPI-labeled nuclear DNA of Müller cells (bar = 25 μm) (a). The fluorescence intensities of Vimentin (b) and CHX10 (c) in retinal Müller cells at the 48th hours of treatment were assessed by immunofluorescence; hESEVs, human embryonic stem extracellular vesicles

Article Snippet: Following 5 min of PBS washing thrice, cells were subjected to the primary antibody against Vimentin (NB300-223, 1:5000) or CHX10 (NBP1-84476, 1:1000) (Novus, St. Louis, MO, USA) overnight at 4 °C for incubation.

Techniques: In Vitro, Labeling, Immunofluorescence, Fluorescence

Journal: Stem cell research & therapy

Article Title: Human embryonic stem cell-derived extracellular vesicles alleviate retinal degeneration by upregulating Oct4 to promote retinal Müller cell retrodifferentiation via HSP90.

doi: 10.1186/s13287-020-02034-6

Figure Lengend Snippet: Fig. 4 hESEVs boost retrodifferentiation of Müller cells through MVs in vitro. Müller cells were treated with DiI labeled-MVs or EXOs. Subsequently, TEM was utilized for measurement of morphologies of MVs and EXOs (a), NanoS90 for inspection of MV and EXO sizes (b), and western blot for determination of the expressions of MV and EXO specific proteins (c). DiI labeled-MVs or EXOs were observed by immunofluorescence: green indicates labeled-hESEVs and blue indicates DAPI labeled-nuclear DNA of Müller cells (bar = 25 μm) (d). The fluorescence intensities of Vimentin (e) and CHX10 (f) in retinal Müller cells were performed by immunofluorescence at the 48th hours of treatment; hESEVs, human embryonic stem extracellular vesicles; MVs, microvesicles; EXOs, exosomes; TEM, transmission microscope

Article Snippet: Following 5 min of PBS washing thrice, cells were subjected to the primary antibody against Vimentin (NB300-223, 1:5000) or CHX10 (NBP1-84476, 1:1000) (Novus, St. Louis, MO, USA) overnight at 4 °C for incubation.

Techniques: In Vitro, Labeling, Western Blot, Immunofluorescence, Fluorescence, Transmission Assay, Microscopy

Journal: Stem cell research & therapy

Article Title: Human embryonic stem cell-derived extracellular vesicles alleviate retinal degeneration by upregulating Oct4 to promote retinal Müller cell retrodifferentiation via HSP90.

doi: 10.1186/s13287-020-02034-6

Figure Lengend Snippet: Fig. 5 HSP90 in MVs enhances retrodifferentiation of Müller cells. Western blot analysis for HSP90 expression in MVs and EXOs (a); ***P < 0.001 vs. the EXOs group. qRT-PCR (b) and western blot (c) were employed for measurement of the mRNA and protein expressions of HSP90 in hESCs. After hESCs transfected with sh-NC, sh-HSP90, pcDNA3.1, or pcDNA3.1-HSP90, western blot was used to determine the protein expression of HSP90 in sh-NC-MVs, sh-HSP90-MVs, pcDNA3.1-MVs, or pcDNA3.1-HSP90-MVs groups (d); **P < 0.01, ***P < 0.001. Following 48 h of exposure to sh-NC-MVs,sh-HSP90-MVs, pcDNA3.1-MVs, or pcDNA3.1-HSP90-MVs, the fluorescence intensities of Vimentin (e) and CHX10 (f) in Müller cells were measured by immunofluorescence; MVs, microvesicles; EXOs, exosomes; hESCs, human embryonic stem cells; NC, negative control

Article Snippet: Following 5 min of PBS washing thrice, cells were subjected to the primary antibody against Vimentin (NB300-223, 1:5000) or CHX10 (NBP1-84476, 1:1000) (Novus, St. Louis, MO, USA) overnight at 4 °C for incubation.

Techniques: Western Blot, Expressing, Quantitative RT-PCR, Transfection, Fluorescence, Immunofluorescence, Negative Control