EMBO/soda-nel huggingface dataset:
De novo DNA methylation controls neuronal maturation during adult hippocampal neurogenesis (2021)
Figure 4

Figure 4: Deletion of de novo DNA methyltransferases does not influence neurogenic activity of adult hippocampal NSPCs in vivo.

A, No difference in numbers of Ki67-positive, proliferating cells was observed between the subgranular zone (SGZ) of Dnmt3a/b-WT and Dnmt3a/b-KO mice one week after tamoxifen administration. Depicted are data points for every animal with genotype means ± SEM.

B, Bright field image of Ki67-positive cells stained using diaminobenzidine method. Scale bar: 100 µm.

C, KO of Dnmt3a/b did not affect numbers of new-born neurons in the dentate gyrus. Mice were administered with tamoxifen and analyzed 5 weeks after the first injection. BrdU was injected 3.5 weeks before analysis. Depicted are data points for every animal with genotype means ± SEM.

D, Fluorescence image of BrdU/NeuN-positive new-born neurons. Scale bar: 25 µm.

E, Experimental scheme for results depicted in Fig. 4F-I, Fig. EV3B-C and Fig. 5A-B. Recombined WT and KO cells were identified by Gfp expression in Nestin::Cre-ERT2 mice.

F, Gfp-positive cells were isolated by FACS from WT and KO mice and their transcriptional profile analyzed using single-cell RNA sequencing (n = 4 mice per genotype). t-distributed Stochastic Neighborhood Embedding (t-SNE) clustered cells into 4 populations which were labeled with T1-T4 and highlighted by color. Clusters T1b and T4b were identified as sub-clusters of T1 and T4, respectively.

G, Marker expression identified the cell clusters as distinct cell stages during adult hippocampal neurogenesis.

H, Distribution of WT (red) and KO cells (violet) into the different cell clusters.

I, Percentages of WT and KO cells in the different cell clusters relative to all sufficiently covered cells (in total n = 293 cells from 4 mice, WT; n = 344 cells from 4 mice, KO). Depicted are percentages of cells in cluster per animal (individual data points) with genotype means ± SEM.
De novo DNA methylation controls neuronal maturation during adult hippocampal neurogenesis (2021)
Figure 5

Figure 5: De novo DNA methyltransferases control maturation dynamics, dendritic outgrowth and spine formation of adult-born neurons in the hippocampus.

A, Dnmt3a/b-KO mice show reduced percentages of mature neurons (Calb1-positive, Dcx-negative) among Gfp-positive cells compared to WT mice (n = 3 mice, WT; n = 3 mice, KO). Immature neurons were identified as Dcx-positive and Calb1-negative. Experimental scheme as shown in Fig. 4E.

B, Representative fluorescence image of mature and immature neurons. Scale bar: 50 µm.

C, KO mice exhibit reduced total numbers of Calb2-positive cells relative to Dcx-positive cells in the dentate gyrus.

D, No difference in long-term survival of new-born cells (BrdU-positive) was observed between WT and KO mice. BrdU was injected three months before analysis (experimental scheme depicted in Fig. EV3D).

E, Long-term deficit in maturation of adult-born neurons in KO mice. Mature neurons were identified as BrdU-positive, Calb1-positive and Dcx-negative (n = 6 mice, WT; n = 8 mice, KO).

F, Representative fluorescence image of the immunostaining used for detection of mature neurons. Scale bar: 100 µm.

G, Sholl analysis of Gfp-positive cells revealed reduced dendritic outgrowth of adult-born neurons in KO mice (n = 20 neurons, WT; n = 23 neurons, KO). Depicted p-value reports genotype effect from two-way ANOVA (distance to soma: p < 0.001). Asterisks highlight data points with p < 0.05 after multiple testing correction (Holm method) of repeated t-tests. Data points are means ± SEM. Experimental scheme as depicted in Fig. 4E.

H, Representative neuronal traces of WT and KO mice.

I, Adult-born neurons of KO mice exhibit reduced numbers of dendritic spines in the outer molecular layer (n = 32 neurons from 3 mice, WT; n = 46 neurons from 4 mice, KO). Depicted are individual data points for every neuron with genotype means ± SEM (p-value from unpaired t-test).

J, Mean spine size per animal was reduced in KO mice (n = 50 spines per mouse).

K, Mean percentage of mushroom spines (diameter of spine head ≥ 0.45 µm) per animal was lower in KO mice compared to WT mice.

L, Representative fluorescence image of dendritic spines in WT and KO mice. Scale bar: 5 µm.

Data information: Depicted p-values in A, C-E, J-K are from unpaired t-test. Graphs show data points for every animal with genotype means ± SEM. Further statistical details are reported in Appendix Table S5.
De novo DNA methylation controls neuronal maturation during adult hippocampal neurogenesis (2021)
Figure 6

Figure 6: Deletion of de novo DNA methyltransferases does not abolish the pro-neurogenic effect of environmental enrichment but modifies hippocampal excitability.

A-B, Quantification of adult-born (BrdU-positive) cells in the dentage gyrus (DG) of Dnmt3a/b-KO and WT mice after five weeks or three months of environmental enrichment (ENR) or standard housing (STD). BrdU was injected 4.5 weeks before analysis. Depicted p-values correspond to genotype effects from two-way ANOVA. Influences of ENR were significant with p < 0.01. Depicted are data points for every animal with group means ± SEM.

C, Bright-field images of BrdU-positive cells from WT and KO mice housed in ENR for three months. Scale bar: 100 µm.

D, Fluorescence images of c-Fos-positive cells in the DG and CA3 of WT and KO mice. Scale bar: 200 µm.

E, Numbers of c-Fos-positive cells in the DG without (STD) and with acute stimulation in ENR for 1 h (n = 2, WT STD; n = 2, KO STD; n = 5, WT ENR; n = 5, KO ENR).

F, Numbers of c-Fos-positive cells in suprapyramidal and infrapyramidal blades of the DG after acute stimulation in ENR (n = 5 mice, WT; n = 5 mice, KO).

G, Percentage of new-born neurons (BrdU/NeuN-positive) that expressed c-Fos after ENR (n = 5 mice, WT; n = 5 mice, KO).

H, Representative fluorescent image of c-Fos expression in BrdU/NeuN-positive cells in the DG. Scale bar: 25 µm.

I, Percentage of c-Fos-positive cells that expressed Dcx in WT and KO mice (n = 5 mice, WT; n = 4 mice, KO).

J, Representative fluorescent image of c-Fos-positive cells and Dcx-positive cells in the DG. Scale bar: 100 µm.

K, Reduced numbers of c-Fos-positive cells in CA3 of KO mice (n = 4 mice, WT; n = 5 mice, KO).

L, Fluorescent image of c-Fos expression in Parvalbumin (Pv)-positive interneurons in CA3. Scale bar: 50 µm.

M, A higher percentage of Pv-positive interneurons in CA3 expressed c-Fos in KO mice (n = 4 mice, WT; n = 4 mice, KO).

Data information: Depicted p-values in panels E-M are from unpaired t-test. Graphs show data points for every animal with group means ± SEM. Further statistical details are presented in Appendix Table S5.
Autophagy enhances the presentation of endogenous viral antigens on MHC class I molecules during HSV-1 infection (2009)
figf1

(a) Activation of the gB-specific CD8+ T cell hybridoma (which expresses β-galactosidase as an indicator of T cell activation) by macrophages infected for various times (horizontal axis) with HSV-1, then incubated for 12 h at 37 °C with the hybridoma. A595, absorbance at 595 nm. (b) Activation of the hybridoma as described in a, with the addition of dimethyl sulfoxide (DMSO; negative control), bafilomycin A (Baf), brefeldin A (BFA) or MG-132 at 2 h after macrophage infection. (c) Activation of the hybridoma as described in a, with the addition of bafilomycin A at 2 h after macrophage infection. (d) Immunofluorescence microscopy of gB (blue) and LAMP-1 (red) in HSV-1-infected macrophages; pink indicates colocalization. Original magnification, × 40. (e) CD8+ T cell–stimulatory capacity (as described in a) of uninfected macrophages (Mock), of BMA (BMA-HSV) or J774 (J774-HSV) macrophages infected for 8 h with HSV-1, and of cocultures of J774 macrophages (H-2d) infected for 1 h with HSV-1, then mixed with uninfected BMA (H-2b) macrophages at a ratio of 1:1 and cultured together for 8 h (J774-HSV + BMA). Results in b,c,e are normalized to results obtained for CD8+ T cells stimulated with macrophages treated with DMSO (b,c) or infected BMA macrophages (e) and are presented in arbitrary units. Data are from one representative of three independent experiments (mean and s.d. of triplicate samples; a), are from three independent experiments (mean and s.e.m. of triplicate samples error bars; b,c,e) or are representative of three independent experiments (d).
Autophagy enhances the presentation of endogenous viral antigens on MHC class I molecules during HSV-1 infection (2009)
figf2

(a) Immunofluorescence microscopy of LC3 expression in macrophages infected for 2–8 h (left margin) with HSV-1. Original magnification, × 20. (b) Activation of the gB-specific CD8+ T cell hybridoma (described in Fig. 1a) by macrophages infected for various times (horizontal axis) with HSV-1, with (DMEM + 3-MA) or without (DMEM) the addition of 3-methyladenine 2 h after infection, then incubated for 12 h at 37 °C with the hybridoma. (c) Activation of the hybridoma (as described in b) by macrophages transfected for 60 h with control (Ctrl) siRNA or Atg5-specific siRNA, then infected for 8 h or 12 h with HSV-1. (d) Immunoblot analysis of Atg-5 in siRNA-treated macrophages. (e) Activation of the hybridoma (as described in b) by macrophages infected with HSV-1 and incubated at 37 °C (Basal), incubated for 12 h at 39 °C before being infected with HSV-1 (heat shock (HS)), or treated with rapamycin during HSV-1 infection (Rapa), with (+ Baf) or without the addition of bafilmycin A at 2 h after infection. (f) Activation of the hybridoma (as described in b) by macrophages transfected for 60 h with control siRNA or Atg5-specific siRNA, then infected for 8 h with HSV-1, with (+) or without (−) the addition of rapamycin at 2 h after infection. Results in b,c,e,f are normalized to results obtained for CD8+ T cells stimulated with macrophages infected for 8 h at 37 °C without further treatment (b,e) or infected macrophages treated with control siRNA (c,f) and are presented in arbitrary units. Data are representative of three independent experiments (a,d) or are from three independent experiments (mean and s.e.m. of triplicate samples; b,c,e,f).
Autophagy enhances the presentation of endogenous viral antigens on MHC class I molecules during HSV-1 infection (2009)
figf3

(a–c) Immunofluorescence microscopy of uninfected macrophages incubated at 37 °C (control; a), subjected to mild heat shock (b) or treated with rapamycin (c), then stained with anti-LC3. (d) Immunofluorescence microscopy of the expression of LC3, gB and GFP (VP26) by macrophages infected for various times (left margin) with HSV-1. White indicates colocalization. (e) Immunofluorescence microscopy of macrophages infected for 6 h or 8 h (left margin) with wild-type HSV-1 (WT) or HSV-1 lacking ICP34.5 (Δ34.5). Blue, staining of nuclei with DAPI (4,6-diamidino-2-phenylindole). Original magnification, × 100 (a–c) or × 63 (d,e). Results in a–e are representative of three independent experiments. (f) Activation of the gB-specific CD8+ T cell hybridoma (as described in Fig. 1a) by macrophages infected for various times (horizontal axis) with wild-type HSV-1 strain 17+ (WT 17+) or Δ34.5 HSV-1. Data are from three independent experiments (mean and s.e.m. of triplicate samples). (g,h) Immunoblot analysis (IB; g) and flow cytometry (h) of the expression of HSV-1 proteins (g) and gB (h) in macrophages infected for various times (above lanes (g) or plots (h)) with wild-type or Δ34.5 HSV-1. Ctrl, control (uninfected BMA macrophages; h). Data are representative of two (g) or three (h) independent experiments. (i) Activation of the gB-specific CD8+ T cell hybridoma (as described in Fig. 1a) by macrophages infected for various times (below graph) with wild-type or Δ34.5 HSV-1, with (+) or without (–) the addition of bafilomycin A at 2 h after infection. Results in f,i are normalized to results obtained for CD8+ T cells stimulated with macrophages infected for 6 h with wild-type virus (f) or with infected macrophages incubated without bafilomycin (i) and are presented in arbitrary units. Data are from three independent experiments (mean and s.e.m. of triplicate samples).
Autophagy enhances the presentation of endogenous viral antigens on MHC class I molecules during HSV-1 infection (2009)
figf5

Immunoelectron microscopy of macrophages 10 h after infection with HSV-1. (a–c) Accumulation of LC3 (a,b) and gB (c). (d) Fusion of four-layered membrane structures and lysosomes preloaded with bovine serum albumin–gold (BSA-gold; black dots). Original magnification, × 54,800 (a,b), × 69,000 (c) or × 38,000 (d). Images are representative of three (a–c) or two (d) independent experiments.
Autophagy enhances the presentation of endogenous viral antigens on MHC class I molecules during HSV-1 infection (2009)
figf6

(a) Activation of the gB-specific CD8+ T cell hybridoma (as described in Fig. 1a) by macrophages exposed to DMSO (negative control), mild heat shock, IL-1β or IFN-γ. (b) Dansylcadaverin staining of untreated macrophages (Basal) or macrophages exposed to mild heat shock, IFN-γ or IL-1β and infected for 8 h with wild-type HSV-1, normalized to the signal obtained in basal conditions and presented in arbitrary units. (c–f) Activation of the gB-specific CD8+ T cell hybridoma (as described in Fig. 1a) by macrophages incubated at 37 °C (c) or exposed to mild heat shock (d), IL-1β (e) or IFN-γ (f) and infected for 8 h with wild-type HSV-1 with the addition of DMSO (negative control), 3-methyladenine, bafilomycin, brefeldin A or MG-132 at 2 h after infection. Results in a,c–f are normalized to results obtained for CD8+ T cells stimulated with macrophages incubated with DMSO in each condition and are presented in arbitrary units. Data are from three independent experiments (mean and s.e.m. of triplicate samples).
APC/C-dependent degradation of Spd2 regulates centrosome asymmetry in Drosophila neural stem cells (2023)
Figure 1

Figure 1. The cellular levels of the conserved PCM protein Spd2 are regulated by APC/C-dependent proteolysis in vivo

(A) A schematic diagram showing the genetic compositions in the Drosophila lines that express different levels of Spd2 proteins in NBs used in this study. pSpd2 and pUAS indicate the native spd2 promoter and UAS sequences, respectively. Gal4 is expressed specifically in NBs by the control of a NB-specific worniu gene promoter.

(B) The Western blot analysis of GFP, Spd2 and αTub (loading control) in the brain extracts from wild type (Oregon R, OR), Spd2WT-OE and Spd2DK-OE third instar larva. Representative immune blot images are shown on the left. The signal intensities of Spd2 bands were quantified and normalised against αTub values. The values relative to control of each biological replicate (n = 2) and their means are shown as dots and bars, respectively, in a scatted dot plot on the right. The numbers above the bars indicate means ± Range. GFP-Spd2DK proteins in Spd2DK-OE larval brain extracts showed approximately 2.04-fold higher accumulation than GFP-Spd2WT proteins in Spd2WT-OE brain extracts.

(C) The Western blot analysis of Spd2, CycB and αTub in OR, fzrRNAi, Spd2WT-RES and Spd2DK-RES brain extracts. The signal intensities of Spd2 bands were quantified and normalised against αTub values. The values relative to control and their means are presented in a scatted dot plot on the right (biological replicate n = 2). Means ± Range are shown above the bars. RFP-Spd2DK proteins in Spd2DK-RES larval brain extracts showed approximately 2.08-fold higher accumulation than RFP-Spd2WT proteins in Spd2WT-RES brain extracts.

(D) Larval brains of OR, Spd2WT-RES, fzrRNAi and Spd2WT-OE flies were stained by an anti-Spd2 antibody. Representative images of NBs are shown on the top. Scale bar: 10 µm. The local signal intensities of Spd2 immunofluorescence at the centrosomes and in the cytoplasm of individual NBs in each brain were measured, and the normalised Spd2 signal intensities in individual NBs were presented as scattered dot plots (see Materials and Methods for detail). The lines indicate means ± SD. The numbers of NBs (n) and the numbers of brains analysed (N) are control: n= 29 (N = 6), Spd2WT-RES: n = 30 (N = 4), fzrRNA: n = 30 (N = 6), and Spd2WT-OE: n = 27 (N = 10). P values were calculated using non-parametric two-tailed Mann-Whitney tests. Based on these results, the relative centrosomal Spd2 protein levels in NBs can be estimated as wildtype (or control) : Spd2WT-RES: fzrRNAi: Spd2DK-RES: Spd2WT-OE: Spd2DK-OE ­= 1: 1.00~1.18: 1.06~1.29: 2.08~2.45 (= 1.00~1.18 x 2.08): 1.93~3.62: 3.94 ~ 7.38 (= 1.93~3.62 x 2.04).
APC/C-dependent degradation of Spd2 regulates centrosome asymmetry in Drosophila neural stem cells (2023)
Figure 3

Figure 3. Altered Spd2 levels partially randomises the NB division axis

(A) A diagram summarising the division axis maintenance in wild type larval NBs and NBs with altered levels of Spd2. Wild type NBs maintain division axes over consecutive divisions while NBs with increased Spd2 levels partially randomise division axes.

(B) Selected images from time-lapse microscopy of NBs in the whole-mount larval brains in the control (pUbq-sas6-GFP) and Spd2WT-OE flies (see also Movies EV7, EV8). Centrosomes were visualised by Sas6-GFP or GFP-Spd2 signals and microtubules by mCh-αTub signals. Multi-channel images showing GFP signals in green and mCh signals in red are shown in the upper panels. Single-channel images showing mCh-αTub signals in grey are shown in the lower panels, in which dotted white lines outline NBs and asterisks indicate forming GMCs. The pale blue and pink arrows indicate the NB division axis upon the first and second mitosis, respectively. Deviations between the division axes (𝜃) were determined. Control NBs mostly maintain the division axes over consecutive divisions (top panels) whereas Spd2DK-OE NBs often divide at significantly different angles, over 90° (lower panels). Scale bars: 10 µm.

(C) In larval NBs in the indicated fly brains, division angles were determined in the 3D context, and deviations between two consecutive divisions (𝜃) were quantified (see Loyer and Januschke, 2018 and Materials and Methods for detail)(Loyer & Januschke, 2018). The number of NB divisions (n) analysed from at least four brains and the average deviations (mean ± SD) were Control: 28.24° ± 20.43 (n = 36), Spd2WT-RES: 35.57° ± 17.10 (n =21), fzrRNAi: 39.62° ± 27.24 (n = 66), Spd2DK-RES: 28.13° ± 47.7 (n = 81), Spd2DK-OE: 60.14° ± 38.79 (n = 16), Spd2WT-OE combined with fzrRNAi (Spd2WT-OE+fzrRNAi): 46.00° ± 31.05 (n = 74). Spd2WT-OE brains were not analysed due to the low sample number (n = 4). p-values were calculated using Mann-Whitney U tests comparing with control and are shown above the plots. fzrRNAi, Spd2DK-OE and Spd2WT-OE+fzrRNAi showed statistically significant differences in division angle deviations.

(D) Division axis deviations were separately analysed in NBs with apically attached interphase centrosomes ("attached") and those with centrosomes being detached from the cortex ("detached") for each line, using the same dataset as in Fig. 3C. The same control data as in Fig. 3C is shown for reference. fzrRNA detached (mean ± SD: 34.62° ± 23.39), Spd2DK-OE detached (64.01° ± 33.93), and Spd2WT-OE+fzrRNAi attached (39.55° ± 27.08) and detached (59.08° ± 34.88) brains showed statistically significant differences from control, while NBs with cortically attached centrosomes in any of the lines did not show significant differences. n: the number of NB divisions analysed from at least four brains for each line. p-values were calculated using Mann-Whitney U tests comparing with control and are shown above the dots.
APC/C-dependent degradation of Spd2 regulates centrosome asymmetry in Drosophila neural stem cells (2023)
Figure 4

Figure 4 Spd2 accumulation compromises the segregation pattern of the daughter centrosome

(A) A schematic representation of the centrosome segregation patterns of NBs with or without the detachment of the daughter centrosome from the apical cortex. The daughter and mother centrosomes are represented by blue and yellow circles, respectively; the centrosomes become inactive and detached from the cortex are represented by red circles. In wild type NBs, the daughter centrosome matures earlier than the daughter centrosome upon mitotic entry and is segregated into the daughter NB (top). In rare cases, NBs mis-segregated the daughter centrosome into the GMC, either by polarity reversal or spindle rotation. In most of the NBs in which both centrosomes were inactive during interphase, one centrosome (possibly, the daughter centrosome) still matured earlier than the other (see Fig. EV4), like in control NBs, which can be segregated into either the daughter NB or the GMC.

(B) Selected images from time-lapse microscopy of NBs in control (wor>mCh-Tub, pUbq-GFP-Fzr, top panels) and Spd2DK-OE (lower panels) larval brains (Movies EV1, EV10). In the control NB, the daughter centrosomes (blue arrowheads) first matured (-5.0 min) and was segregated into the daughter NB (12.0 min), while the mother centrosome (yellow arrowheads) matured a little later (0.0 min) and segregated into the GMC. In contrast, in the Spd2DK-OE NB, both centrosomes lost microtubule nucleation activity in interphase (red arrowheads). The centrosome that matured earlier (blue arrowheads, -6.0 min) was incorrectly segregated into the GMC (12.0 min) while the centrosome that matured later (yellow arrowheads, 0.0 min) was segregated into the NB (12.0 min). Dotted white lines outline the dividing NBs and the newly formed GMC, which are also marked by asterisks. Scale bars: 5 µm.

(C, D) The NB centrosome segregation patterns were quantified in each line, based on the segregation patterns in A. The segregation patterns of the NBs with the apically attached centrosomes were analysed in C and those of the NBs with both centrosomes being detached in D. n: the total numbers of NB divisions analysed in at least three brains in each line. Except for the Spd2WT-OE brains, NBs with apically attached centrosomes could correctly segregate the daughter centrosome into the daughter NBs. In fzrRNAi and Spd2DK-RES lines, most NBs with centrosome detachment could segregate the first matured centrosomes (i.e., the daughter centrosomes). However, in the brains with even higher Spd2 levels, NBs frequently mis-segregated the centrosomes.