Journal: bioRxiv

Article Title: Spontaneous differentiation across cell lineages of separate germ layer origin during progenitor cell-mediated regeneration of the central nervous system

doi: 10.1101/2025.11.24.690179

Figure Lengend Snippet: A. Schematic linear map of the AAV-SOX10-OLIG2-gRNA and AAV-mKate2 plasmid constructs. B. Schematic of the experimental overview. Animals received either AAV-SOX10-OLIG2-gRNA or AAV-mKate2 via tail vein injection, followed by doxycycline administration in drinking water from D12 to D16. At D14 post-injection, a focal lysolecithin-induced demyelinating lesion was created in the spinal cord. Remyelination was assessed at D40, three weeks after demyelination. C. Representative immunohistochemistry images of lesioned spinal cords at 21 dpl showing SOX10 + PRX + HA - cells (endogenously arising SCs) and SOX10 + PRX + HA + cells (OPC-derived SCs infected by the AAV) in both treatment groups. D. Quantification of HA+ and SOX10 + MPZ + HA - cells in the spinal cord lesion. E. Quantification of SOX10 + MPZ + HA + cells in the spinal cord lesion. F. Immunohistochemistry of the lesion area PRX, MPZ, and GFAP. G. Quantification of (E) shows a significant increase in the number of PRX + , MPZ + , and Sox10 + cells in animals treated with AAV-SOX10-OLIG2-gRNA compared to AAV-mKate2-treated animals. Values are presented as mean ± SD. (*p< 0.05, ns, not significant, by unpaired t-test). The above data was obtained from four biological replicates per group, with at least three fields of view quantified per replicate. H. Immuno-electron microscopy image showing the ultrastructure of an HA + SCs within the spinal cord. The SC body is pseudocoloured yellow, the associated axon in blue and the SC nucleus in red. The inset highlights immunogold particle labelling of HA in the SC nucleus.

Article Snippet: A second construct was generated using the same AAV backbone (AAV-CMVc-Cas9, Addgene #106431) and Sox10 U2 promoter, combined with rtTA, a T2A self-cleaving peptide, and WPRE elements (from pHR-EF1α-Tet-on 3G), together with mKate2 (from NFKBRp-mKate2-2xNLS-p2a-puroR, Addgene #82024) to enable fluorescent reporting of promoter activity.

Techniques: Plasmid Preparation, Construct, Injection, Immunohistochemistry, Derivative Assay, Infection, Immuno-Electron Microscopy

Journal: bioRxiv

Article Title: Spontaneous differentiation across cell lineages of separate germ layer origin during progenitor cell-mediated regeneration of the central nervous system

doi: 10.1101/2025.11.24.690179

Figure Lengend Snippet: A. Immunostaining of OLIG2, IBA1 and MBP in the lesion area. B. Quantification of the data presented in A. The bar graphs show the lesion size and the number of OLIG2 + , MBP + , and IBA1 + cells in animals receiving AAV-SOX10-OLIG2-gRNA or AAV-mKate2-treated animals. Values are expressed as mean ± SD. Statistical significance was determined by one-way ANOVA with Tukey’s multiple comparisons test. C. Quantification of the spatial relationship between PRX + SCs and GFAP + astrocytes in the lesion site.

Article Snippet: A second construct was generated using the same AAV backbone (AAV-CMVc-Cas9, Addgene #106431) and Sox10 U2 promoter, combined with rtTA, a T2A self-cleaving peptide, and WPRE elements (from pHR-EF1α-Tet-on 3G), together with mKate2 (from NFKBRp-mKate2-2xNLS-p2a-puroR, Addgene #82024) to enable fluorescent reporting of promoter activity.

Techniques: Immunostaining

Journal: Cell reports

Article Title: Cone photoreceptors transfer damaged mitochondria to Müller glia.

doi: 10.1016/j.celrep.2023.112115

Figure Lengend Snippet: Figure 2. Mislocalized mitochondria are abnormal and reside in and outside of cone photoreceptors (A) Transmission electron micrographs taken at the optic nerve. Cones with activated mKR have many mitochondria with disturbed, swollen morphology both in and outside of the ellipsoid clusters; an inset (black box) shows a cone ellipsoid containing multiple swollen mitochondria. Black arrowhead: ellipsoid cluster with typical mitochondria morphology, white arrowhead: ellipsoid cluster with swollen mitochondria. Scale bar: 5 mm. (B) Quantification of individual cone mitochondria from slices in (A). A greater fraction of mitochondria are swollen and mislocalized in mKR+ cones. All mislocalized mitochondria had swollen morphology. n = 2 kR fish (641 and 759 total mitochondria) and n = 3 kR+ fish (520, 613, and 566 total mitochondria). *p < 0.05 and ****p < 0.0001 using a two-way ANOVA. (C) Imaging of Tg(gnat2:mtBFP, gnat2:GFP) fish reveals that mislocalized cone mitochondria reside inside cones (filled arrowheads) and partially or completely outside of cones (unfilled arrowheads). Hi-C, high-contrast to enhance visibility of mito- chondria. Scale: 5 mm. (D) Quantification of mislocalized mitochondria by colocalization of cone GFP; cone includes mito- chondria that appear partially or completely out of cones (see C). mtKR increases cone mitochondria number both inside and outside cones. *p < 0.05 and ****p < 0.0001 using a two-way ANOVA. n = 13 fish each condition. (E) Pooled cone-mislocalized mitochondria totals across all fish binned by retinal region. IR, inner retina; PR, photoreceptor layer. (F) Immunohistochemistry images of mislocalized mitochondria (filled arrows) colocalized with MTCO1 in PR and inner retina IR. Fraction of mis- localized cone mitochondria colocalized with mitochondrial protein, MTCO1. mKillerRed: 74 mislocalized mitochondria from 4 eyes mt- KillerRed+: 104 mislocalized mitochondria from 5 eyes. Comparisons are ns with unpaired t test. Scale: 5 mm.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies MTCO1 Abcam, ab14705 RRID:AB_2084810 SDHB Abcam, ab14714 RRID:AB_301432 IRDye 680RD donkey anti-mouse IgG (H + L) LI-COR Biosciences, 925–32212 RRID: AB_2716622) FluoTag-X2 anti-TagFP Alexa Fluor 647 NanoTag Biotechnologies N0502-AF647-L Chemicals, peptides, and recombinant proteins Chloramphenicol Thermo Scientific Cat#AC227920250 dye CM-H2DCFDA Thermo Fischer Cat#C6827 RNase A New England Biolabs Cat#T3018-2 iTaqTM Universal SYBR Green Supermix Bio-Rad Cat#1725120 Critical commercial assays DNeasy Blood & Tissue kit Qiagen Cat#69504 Millipore ApopTag Fluorescein In Situ Apoptosis Detection Kit EMD Millipore Cat#S71100 Experimental models: Organisms/strains Tg(gnat2:mtSRAI)w268 This study N/A Tg(gnat2:mtBFP)w269 This study N/A Tg(gnat2:mtKillerRed)w270 This study N/A Tg(gnat2:mtmKate2)w271 This study N/A Tg(gnat2:LAMP1-GFP)w272 This study N/A Tg(GFAP:GFP-mCh-LC3)w273 This study N/A pde6cw59 Stearns et al.,33 http://zfin.org/ZDB-ALT-080206-1 Tg(gnat2:GFP)w206 Kennedy et al.,34 http://zfin.org/ZDB-ALT-181217-7 Tg(GFAP:TdTomato) Shin et al.,35 N/A Tg(gnat2:TdTomato) Sloan et al.,36 N/A Tg(GFAP:GFP) Bernardos e al.,37 http://zfin.org/action/feature/ view/ZDB-ALT-060623-4 Tg(mpeg1:GFP)gl22 Ellett et al.,16 http://zfin.org/ZDB-ALT-120117-1 Tg(TBP-GAL4;UAS:secA5-YFP) van Ham et al.,12 Blume et al.,38 N/A Recombinant DNA Su9-EGFP Addgene plasmid # 23214 RRID:Addgene_23214 mTagBFP Addgene plasmid # 75175 RRID:Addgene_75175 mKate2 Addgene plasmid # 48345 RRID:Addgene_48345 SRAI RIKEN DNA Bank #RDB18223 KillerRed Bulina et al.,11 N/A p5E-gfap Addgene plasmid # 82401 RRID:Addgene_82401 LAMP1 Drerup et al.,39 N/A GFP-mCh-LC3 George et al.,40 N/A Software and algorithms Imaris 9.9 Oxford Instruments RRID:SCR_007370 ImageJ Schindelin et al.,41 RRID:SCR_002285 Blender www.blender.org RRID:SCR_008606 10 Cell Reports 42, 112115, February 28, 2023

Techniques: Transmission Assay, Imaging, Hi-C, Immunohistochemistry

Journal: Cell reports

Article Title: The spatiotemporal distribution of LIN-5/NuMA regulates spindle orientation in the C. elegans germ line.

doi: 10.1016/j.celrep.2025.115296

Figure Lengend Snippet: Figure 3. Dynein and LIN-5/NuMA are required for germ cell spindle orientation and germline tissue organization (A) Confocal sections through the mid-plane of gonad arms from L4 larvae expressing DHC-1::AID::GFP (dynein, left) or LIN-5::mAID::mNG (NuMA, right), with a germline-specific TIR1 (sun-1p::TIR1), after a 40-min auxin treatment, as compared to untreated animals. The germ line is outlined in white. Scale bar, 10 mm (B) Quantification of the fluorescence intensity of each FP-tagged protein in the germ line. Measurements were normalized to the mean value for non-depleted germ lines. (C) Cumulative distribution of spindle angles at anaphase to the gonad surface normal vector, comparing control animals to those depleted of DHC-1/dynein or LIN-5/NuMA as in (A) and (B). Depletion of either DHC-1/dynein or LIN-5/NuMA reduces the spindle orientation bias to the gonad surface. The theoretical random distribution (yellow) is shown for reference. (D) Top: maximum-intensity projections of gonad arms from L4 larvae expressing mKate2::ANI-1, depleted (right) or not (left) of LIN-5/NuMA in the germ line by a 6-h auxin treatment. Bottom: reconstruction of the distal rachis surface using the mKate2::ANI-1 signal showing one rendered object in the non-depleted germ line (gray) and two objects in the depleted germ line (gray and yellow). Scale bars, 10 mm (E) Distal rachis volume in control and LIN-5/NuMA-depleted animals. Each dot represents the rachis for one gonad arm. Bars represent the mean ± standard deviation. (F) The number of rendered rachis objects in control and LIN-5/NuMA-depleted animals. For all panels, *p < 0.05, **p < 0.01, and ***p < 0.001. Summary statistics and statistical tests used are given in Table S3. See also Figure S3.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER C. elegans: Strain HS3911; genotype: lin-5(os205[lin-5::mAID::mNG] II This study HS3911 C. elegans: Strain PD1594; genotype: ccTi1594 [mex-5p::GFP::gpr-1::smu-1 30UTR + Cbr-unc-119(+), III: 680195] III CGC PD1594 Oligonucleotides Guide RNA sequence for generating LIN5:mAID:mNG: 50-GTCCAAGAAAAAGAA CCGTC-30 Heppert et al.86 N/A Primers for generating LIN-5:mAID:mNG, see Table S1 This study N/A Recombinant DNA L4440 (RNAi empty vector for feeding) Timmons and Fire90 RRID: Addgene_1654 gpr-1/2 feeding RNAi Kamath et al.79 sjj_C38C10.4 mAID-mClover-Hygro Natsume et al.91 RRID: Addgene_72828 mNG-mom-5 Heppert et al.86 mKate2̂ SEĈ 3xMyc Dickinson et al.92 RRID: Addgene_70685 lin-5:mAID:mNG This study pTN26 Software and algorithms Fiji 1.52v Schindelin et al.93 Fiji (RRID:SCR_002285) MATLAB 2020b MathWorks94 MATLAB (RRID:SCR_001622) Trackmate 6.0.0 Tinevez et al.95 https://imagej.net/plugins/trackmate/ Imaris 9.2.1 Oxford Instruments96 Imaris (RRID:SCR_007370) Julia 1.10.4 Bezanson et al.97 Julia Programming Language (RRID:SCR_021666) Python 3 Van Rossum and Drake98 Python Programming Language (RRID:SCR_008394) Custom scripts This study https://github.com/VincentPoupart/ Zellag2024 CentTracker scripts Zellag et al.37 https://github.com/yifnzhao/CentTracker Other Microfabricated silica wafer Zellag et al.37,38; Gerhold et al.99 N/A Glass slides with wells for gonad explant culture and imaging Fisher Scientific 30-2066A-BROWN 3 SQUARE 14mm with Bars Epoxy autoclavable Cell Reports 44, 115296, February 25, 2025 17

Techniques: Expressing, Plasmid Preparation, Control, Standard Deviation