EMBO/SourceData huggingface dataset:
Figure 1(A-B'') Nr2f1 (red) and Pax6 (green, retinal progenitors) immunofluorescences (IF) on sagittal sections of E10.5 and E12.5 mouse optic vesicles. Note the high-ventral to low-dorsal Nr2f1 gradient in the presumptive neural retina (pNR), and high Nr2f1 expression in both the presumptive dorsal and ventral optic stalk (pvOS and pdOS). Cr: crystal lens; Di, distal; Pr, proximal; presumptive retinal pigmented epithelium (pRPE). Scale bars: 50µm for mouse sections(C-D") IF on E13.5 mouse eye sagittal sections with Nr2f1 (red) and Sox2 (green, progenitors in C,C') or Brn3a (green, RGCs in D,D') showing Nr2f1 expression in NR progenitors and post-mitotic RGCs (insets in C'-D"). Scale bars: 50µm for mouse sections(E-F") Nr2f1 (red) and Brn3a (green) IF on E18.5 and P7 mouse eyes depicting Nr2f1 expression in virtually all RGCs in the ganglion cell layer (GCL). Scale bars: 50µm for mouse sections(G,G') Nr2f1 (red) and Sox2 (green) IF on P7 mouse retina illustrating low Nr2f1 expression in the inner nuclear layer (INL) and progenitors (NPs), and high levels in the GCL and ciliary marginal zone (CMZ). No expression in the outer nuclear layer (ONL). Scale bars: 50µm for mouse sections(H-K") IF on cross-sections of E13.5 optic stalks (OS), and E18.5 and P7 optic nerves (ONs) with Nr2f1 (red) and Glast (green, astrocytic progenitors in H-H''), NF1A (green, astrocytes in I-I'') or Sox10 (green, oligodendrocyte precursors in J-K'') showing Nr2f1 expression in both astrocytic and oligodendrocytic lineages. Arrowheads point to double labeled cells. Scale bars: 50µm for mouse sections(L-L") NR2F1 (red) and PAX6 (green; NR domain) IF on sagittal sections of human eye primordia at gestational week (GW) 11 illustrating high NR2F1 expression in both ON and NR cells. Scale bars: 100µm for human sections(M-N") NR2F1 (red) and S100β (green, astrocytes) IF on cross-sections of human GW11 and GW14 ONs. Higher magnifications (M'-N") show that most of S100β+ astrocytes co-express NR2F1. Scale bars: 100µm for human sections,(O-Q"') NR2F1 (red) and SOX2 (green in O-P') or BRN3a (green in Q-Q''') IF on sagittal sections of human GW14 eyes indicating high NR2F1 expression in virtually all NR progenitors (O''',P,P'), differentiating RGCs (Q-Q''') and in the majority of ON astrocytic progenitors (O'). Nuclei counterstaining (blue) was obtained with DAPI. Scale bars 100µm for human sections
Figure 3(A-F) Pax2 (magenta, astrocytic progenitors) and Tuj1 (green, RGC axons) IF on E13.5, E15.5 and E18.5 OS/ONs cross-sections showing an increase of Pax2-expressing cells within the optic nerve (ON) of KO embryos at all stages.Histograms quantifying the average number of Pax2+ (G,H) cells in WT, HET and KO at different stages and axial length of the ON, as indicated. Astrocytic progenitors are significantly increased along the nerve of mutants. data are represented as means ± SEM; N=3-5. Statistical significance was obtained by Student t-test (*P<0.05; **P<0.01; ***P<0.001). Nuclei (blue) were stained with DAPI. Scale bars: 50µmHistograms quantifying the average number of NF1A+ (I) cells in WT, HET and KO at different stages and axial length of the ON, as indicated. Astrocytic progenitors are significantly increased along the nerve of mutants. data are represented as means ± SEM; N=3-5. Statistical significance was obtained by Student t-test (*P<0.05; **P<0.01; ***P<0.001). Nuclei (blue) were stained with DAPI. Scale bars: 50µm(J,K) NF1A (red, astrocyte precursors) IF on P7 WT and KO ON cross-sections illustrating a high astrocytic density in KO ON (delineated by thin dashed lines). (L) Histogram confirming the increased percentage of NF1A+ astrocytes in HET and KO ONs compared to WT. data are represented as means ± SEM; N=3-5. Statistical significance was obtained by Student t-test (*P<0.05; **P<0.01; ***P<0.001). Nuclei (blue) were stained with DAPI. Scale bars: 50µm(M-O") Tuj1 (green, RGC axons) and S100β (red, astrocytes) IF on P7 WT, HET and KO ONs. Arrowheads point to S100β+ astrocyte groups (N',O') and absence of Tuj1+ fibers (N'',O'') in HET and KO ONs.(P.Q) TEM images of WT and KO P8 ONs showing thick astrocytic processes filled with intermediate filaments (red dotted lines and arrowheads) in KO nerves. Scale bars (500nm)(R-S') S100β (green, astrocytes) and Sox2 (red, reactive/inflamed astrocytes) IF on P28 WT and HET ONs illustrating strong Sox2 expression in reactive astrocytes.(T) Graph quantifying the average number of Sox2+ astrocytes per P28 ON section in KO and WT animals. data are represented as means ± SEM; N=3-5. Statistical significance was obtained by Student t-test (*P<0.05; **P<0.01; ***P<0.001). Nuclei (blue) were stained with DAPI. Scale bars: 50µm(U,V) EM sections of P28 WT and HET ONs illustrating the expanded astrocytic processes (surrounded by red dotted lines) among myelinated fibers of P28 ONs. Scale bars: (U,V) (4µm).
Figure 4(A-C) NF1A (red, astrocytes) and Sox10 (green, oligodendrocytes) IF on cross-sections of WT and KO P8 ONs showing altered ratio of astrocytes and oligodendrocytes in mutants, particularly in KOs, as quantified in (C). data are represented as means ± SEM. N=4-5 Statistical significance was obtained by Student t-test or by two-way ANOVA when comparing 2 or multiple conditions, respectively (*P<0.05; **P<0.01; ***P<0.001 Scale bars: 50µm(D-J) MBP (yellow, fully differentiated oligodendrocytes) IF on cross-sections of WT, HET and KO P7 optic nerves (ONs) at the chiasm (D-F) and between the chiasm and the eye (G-I). Note the dramatic decrease of MBP+ oligodendrocytes at the chiasm in HET and KO, quantified in (J). The ratio of MBP+ cells is calculated over the total DAPI+ cell number per chiasmal/ON section. data are represented as means ± SEM. N=4-5 Statistical significance was obtained by Student t-test or by two-way ANOVA when comparing 2 or multiple conditions, respectively (*P<0.05; **P<0.01; ***P<0.001). Nuclei (blue) were stained with DAPI. Scale bars: 50µm(K-N) EM thin sections of P8 WT, HET and KO ONs depicting myelin as a dense, dark staining around axonal fibers illustrate a significantly lower number of myelinated fibers in HET and KO, as quantified in (N). Higher magnification images (insets) showing different degrees of myelination in HET and KO ONs. data are represented as means ± SEM. N=3 Statistical significance was obtained by Student t-test or by two-way ANOVA when comparing 2 or multiple conditions, respectively (*P<0.05; **P<0.01; ***P<0.001). Nuclei (blue) were stained with DAPI. Scale bars: 50µm (4µm)TEM images of P28 WT and HET confirming persistent hypomyelination in mutants at P28 Scale bars: (4µm)P28 WT and HET confirming persistent hypomyelination in mutants at P28, as quantified in (Q). data are represented as means ± SEM. N=3 Statistical significance was obtained by Student t-test or by two-way ANOVA when comparing 2 or multiple conditions, respectively (*P<0.05; **P<0.01; ***P<0.001). Nuclei (blue) were stained with DAPI. Scale bars: 50µm(R,S) High magnification EM images of P28 WT and HET ONs showing the difference in myelin compaction between WT (arrowheads in R) and HET (arrowheads in S). Scale bars: (1µm).
Figure 5(A) Experimental design to treat HET pups from P2 to P8 with Miconazole. DMSO (controls) and Miconazole-treated pups were sacrificed at P8 or at P28 to evaluate the ON myelination. (B-E) Sox10 (red, oligodendrocyte precursors) and MBP (green, fully differentiated oligodendrocytes) IF on cross-sections of P8 HET ONs after 6 days of treatment with DMSO (D) or Miconazole (E). Miconazole increased the number of Sox10+ cells compared to HETs treated with DMSO (quantified in B) and rescued MBP staining pixel intensity of HET pups to level comparable to those of WT pups (quantified in C). Nuclei (blue) were stained with DAPI. , the error bars represent the SEM of the means; N=3 Statistical significance was obtained by ANOVA (*P<0.05; **P<0.01; ***P<0.001). Scale bars: 50µm(F-I) Sox10 (oligodendrocyte marker, red) and Phospho-ERK (dually phosphorylated forms of active ERK1 and ERK2, green) IF in WT and HET ONs, treated with DMSO (F) or Miconazole (G) between P2 and P8. As previously reported, Miconazole strongly activate ERK signaling pathway (G). Signal intensity quantification in whole ON area is shown in (H), while specific evaluation of pixel intensity around Sox10+ nuclei is shown in (I). Nuclei (blue) were stained with DAPI. the error bars represent the SEM of the means N=2 Statistical significance was obtained by ANOVA (*P<0.05; **P<0.01; ***P<0.001). Scale bars: 50µm(J-M) EM thin sections of P28 WT and HET ONs, treated with DMSO (J,K) or Miconazole (L) between P2 and P8. Different degrees of myelin compaction can be appreciated at high magnification, with HET ONs showing non compacted sheaths (white arrows in K) and vacuoles (yellow arrows). Miconazole treatment rescues almost normal myelin g-ratio, as quantified in M. Nuclei (blue) were stained with DAPI. the error bars represent the SEM of the means N=2 Statistical significance was obtained by ANOVA (*P<0.05; **P<0.01; ***P<0.001). Scale bars: 50µm, except (J-L) (500nm).(N-P') S100β (green, astrocytes) and Sox2 (red, reactive/proliferative astrocytes) IF in the ONs of WT (N,N') and HET (O-P') animals after 6 days of treatment with DMSO (N-O') or Miconazole (P,P'), then sacrificed at P28. Astrocytes undergo remodeling and express Sox2 in Nr2f1 HET animals (O',P'), even after Miconazole treatment.Histogram showing the average number of S100β+/Sox2+ astrocytes per P28 ON section in WT or HET animals treated with DMSO or Miconazole as indicated, with Sox2 high (magenta columns) and low levels (pink columns). Nuclei (blue) were stained with DAPI. the error bars represent the SEM of the means; ; N=2 Statistical significance was obtained by ANOVA (*P<0.05; **P<0.01; ***P<0.001). Scale bars: 50µm
Figure 6(B) Nissl-stained sections illustrating the location of the recording electrodes (arrows). dhc, dorsal hippocampal commissure; PAC, periaqueductal grey; Th, thalamus.(C) Examples of field potentials (averaged ≥ 20 times) evoked by flash stimulation in the recording sites for WT and HET mice. Dotted lines indicate the beginning of evoked field potentials.(D) Latency of evoked field potentials for WT (N=13) and HET (N=18) mice.(E) In a first series of experiments, animals [WT (N=13) and HET (N=18)] were trained to press a lever to obtain a pellet of food in an illuminated Skinner box with a fixed-ratio (1:1) schedule. After ten days of training, they were transferred to a light on/light off protocol where lever presses were only rewarded when a small light bulb located over the lever was switched on. Lever presses while the bulb was off were punished with a time penalty of ≤ 10 s during which the bulb would not turn on. (F,G) Mean days to criterion (F) and mean number of lever presses during the last two training sessions (G) for the fixed-ratio (1:1) paradigm. No significant differences were observed.(H) Performance of WT (N=13) and HET (N=18) mice in the light on/light off test. Criterion (dotted line) was that the animal had to press the lever more times during the small bulb light on period than during the light off one. WT mice performed better than HET animals and reached the criterion by the 6th session. The HET did not reached criterion for the duration of the test. While a linear fit to WT data increased significantly across training, the same linear fit to HET performance did not present any significant change.(I) The percentage of lever presses during the light on and light off periods for WT and HET mice was also different, because the WT group pressed the lever preferentially during the light period. Data were averaged from the 9th and 10th sessions. For statistical significance (*P<0.05; **P<0.01; ***P<0.001).
Figure 1Human first-trimester trophoblast cell line Sw.71 (left panel) and human primary culture (right panel) trophoblast cells were infected with ZIKV (MOI=2) for 1 h and refreshed with regular media over time. RNA was collected for measuring viral titers by qRT-PCR. Data represent as mean ± SEM; n=4 biological replicates. NT, no treatment group; HPC, human primary culture. (A) ZIKV titers in Sw.71 and HPC trophoblasts. *p < 0.05 by One-way ANOVA.Human first-trimester trophoblast cell line Sw.71 (left panel) and human primary culture (right panel) trophoblast cells were infected with ZIKV (MOI=2) for 1 h and refreshed with regular media over time. RNA was collected for measuring gene expression by qRT-PCR. Data represent as mean ± SEM; n=4 biological replicates. NT, no treatment group; HPC, human primary culture. (B) IFNα is not highly induced in response to ZIKV infection in trophoblast cells. IFNα mRNA expression was inhibited in the first 24 h.p.i. and maintained at low level during ZIKV infection. Difference not significant (n.s.) by One-way ANOVA.Human first-trimester trophoblast cell line Sw.71 (left panel) and human primary culture (right panel) trophoblast cells were infected with ZIKV (MOI=2) for 1 h and refreshed with regular media over time. RNA was collected for measuring gene expression by qRT-PCR. Data represent as mean ± SEM; n=4 biological replicates. NT, no treatment group; HPC, human primary culture. (C) ZIKV infection induced a time-dependent increase in IFNβ mRNA expression in Sw.71 and HPC. *p < 0.05, **p < 0.01 by One-way ANOVA.Human first-trimester trophoblast cell line Sw.71 (left panel) and human primary culture (right panel) trophoblast cells were infected with ZIKV (MOI=2) for 1 h and refreshed with regular media over time. (D) Expression levels of secreted IFNβ detected in trophoblast supernatants by ELLA assay. Supernatants were collected from ZIKV-infected and control trophoblast cultures at different times, and IFNβ protein secretion was quantified by ELLA assay. Note the increase of secreted IFNβ in the ZIKV-infected supernatant in a time-dependent manner. ****p < 0.0001 by Student's t-test against individual time point NT control.
Figure 2Sw.71 (left panel) and HPC cells (right panel) were treated with IFNβ to detect ISG20 mRNA expression. Data represent as mean ± SEM; n=3 biological replicates. NT, no treatment group; HPC, human primary culture. (A) Sw.71 and HPC cells were treated with different doses of IFNβ (3, 30, 300 ng/ml) for 8 h and RNA were collected for determining ISG20 mRNA expressions by qRT-PCR. Note the increase of ISG20 mRNA expression in a dose-dependent manner. *p < 0.05, **p < 0.01 by One-way ANOVA. (B) Sw.71 and HPC cells were treated with 300ng/ml IFNβ over time and RNA was collected for determining ISG20 mRNA expressions by qRT-PCR. Note the increase of ISG20 mRNA expression in a time-dependent manner. *p < 0.05 by One-way ANOVA. (C) Sw.71 (left panel) and HPC cells (right panel) were treated with IFNβ to detect ISG20 protein expression. (D) Sw.71 and HPC cells were treated with 300ng/ml IFNβ over time and protein was collected for determining ISG20 protein expressions by western blot. Note that there was no ISG20 protein expression in the no treatment group in both Sw.71 and HPC, only after IFNβ treatment, ISG20 protein expression exhibited a time-dependent manner. β-actin served as a loading control.
Figure 3(B) No ISG20 protein expression in ISG20-/- Sw.71 in ZIKV infection. Sw.71 and ISG20-/- Sw.71 cells were infected with ZIKV (MOI=2) for 1 h and refreshed with regular media for 48 h, and proteins were collected for western blot analysis. β-actin served as a loading control.(C) Higher ZIKA titer was shown in ISG20-/- Sw.71 cells. Sw.71 and ISG20-/- Sw.71 cells were infected with ZIKV (MOI=2) for 1 h and refreshed with regular media for 48 h, and RNA was collected for determining the viral titer and gene expression by qRT-PCR. Data represent as mean ± SEM; n=4 biological replicates; *p < 0.05 by Student's t-test.(D) More viral shedding in ZIKV-infected ISG20-/- Sw.71 cell culture supernatant. Supernatant from ZIKV-infected Sw.71 and ISG20-/- Sw.71 cells were collected and plaque assay was performed using Vero cells. A representative plaque assay picture is presented. Note that more plaques were formed in Vero cells by incubating with ZIKV-infected ISG20-/- Sw.71 cell culture supernatant. Scale bar=130μm. (E) Plaques were counted and plaque forming-unit (pfu) ratio was calculated for the comparison of the viral titers. Data represent as mean ± SEM; n=3 biological replicates. *p < 0.05 by Student's t-test.
Figure 4(B) ISGs mRNA expression stimulated by IFNβ in Sw.71 and ISG20-/- Sw.71 trophoblast cells. Sw.71 and ISG20-/- Sw.71 cells were treated with 30 ng/ml IFNβ for 24 h, and RNA was collected for determining the ISGs gene expression by qRT-PCR. Data represent as mean ± SEM; n=3 biological replicates; *p < 0.05, **p < 0.01 by Student's t-test against control.(D) IFNβ pre-treatment significantly prevented trophoblast cells from ZIKV infection, however, this protection was evidently attenuated due to lack of ISG20. Sw.71 and ISG20-/- Sw.71 cells were pre-treated with or without 30 ng/ml IFNβ for 24 h, followed by ZIKV infection (MOI=2) for 1 h and refreshed with regular media for 24 h, and RNA was collected to determine the viral titers by qRT-PCR. Data represent as mean ± SEM; n=4 biological replicates; *p < 0.05 by Student's t-test.
Figure 5(D) ISG20 secretion in the positive clone supernatant significantly decreased ZIKV infection in trophoblast cells. Supernatants from negative and positive clones were collected and added to ISG20-/- Sw.71 trophoblast cells together with ZIKA virus (MOI=2) for 1 h, followed by refreshing with new growth media for 48 h. RNA was then collected to determine viral titers by qRT-PCR. Data represent as mean ± SEM; n=3 biological replicates; **p < 0.01 by Student's t-test.(E) Recombinant ISG20-Fc degrades ZIKA viral RNA and HSV2 viral DNA. 50ng purified viral RNA (ZIKV) or DNA (HSV-2) were incubated with increasing concentrations of rISG20-Fc (5, 50, 500 ng) in the presence of RNase inhibitor for 90min at 37°C followed by quantification of viral titers by qRT-PCR, and agarose gel was used to evaluate the RNA degradation by electrophoresis. The representative picture of agarose gel is presented. rISG20-Fc was able to degrade both ZIKV RNA and HSV-2 DNA in a dose dependent manner; however, rISG20-Fc was more efficient in degrading viral RNA than DNA. Data represent as mean ± SEM; n=3 biological replicates; **p < 0.01. ***p < 0.001 and ****p < 0.0001 by One-way ANOVA.
Figure 6(B) Macroscopic evaluation of the pregnant uterus and fetus. Compare to the fetal death and reabsorptions in ZIKV-infected mice, rISG20-Fc treatment rescued this phenotype by decreasing pregnancy loss (resorption).
Figure 7(A) rISG20-Fc treatment alleviates maternal viral burden. Note that rISG20-Fc treatment significantly decreased the maternal serum viral titers. Data represent as mean ± SEM; n=3 mice for control PBS group, and n=4 mice for treatment groups; *p < 0.05 by Student's t-test. (B) rISG20-Fc treatment reduces ZIKV titers in the fetal brain. Although there was no difference of ZIKV titer in the placenta, at the fetal side, there was a significant decrease of viral titer in the fetal brain, suggesting rISG20-Fc can block viral transmission from placenta to fetus. Data represent as mean ± SEM; n=4 for each group, and 3-4 placentas/fetal brains from every mouse were analyzed; *p < 0.05 by Student's t-test. ( (C) rISG20-Fc treatment decreases Eotaxin significantly in maternal circulation. Data represent as mean ± SEM; n=2 for PBS control group, and n=3 for every treatment group; *p < 0.05 by One-way ANOVA; difference not significant for IL-9 and IFNγ by One-way ANOVA.
Figure 2(A) RT-qPCR of early passage RMS tumoroid models shows positivity for at least one gene used in standard-of-care pathology analysis (DES, MYOG, or MYOD1). Conventional RMS cell lines (RD and RH30) were used as positive controls, while two Synovial Sarcoma (SS000DAZ and SS077) tumoroid models were used as negative controls. Gene expression was normalized to the expression of a house-keeping gene and human reference RNA (HREF) via the ΔΔCq method. Each tumoroid line was measured once with four technical replicates with the error bars representing the standard deviation of said technical replicates.(C) Morphological (via H&E) and immunohistochemical (IHC) comparison of RMS tumors and derived RMS tumoroid models shows retained marker protein (Desmin, Myogenin and MYOD1) expression and cellular morphology. Scale bars equal 200 µm (RMS012, RMS102) or 100 µm (RMS000HQC).
Figure 3(A) Copy number frequency plots of RMS tumors (upper row) and derived RMS tumoroid models (lower row) divided by fusion-status (columns). Chromosomes are annotated on the x-axis from left to right while the y-axis shows the percentage of samples in this group carrying a gain (red) or loss (blue) in this genomic region.(B) Contribution of somatic mutational signatures per tumor and tumoroid model. SBS = single base substitution, TMZ = temozolomide, T = tumor, O = tumoroid.(C) Table depicting pathogenic single-nucleotide variants (SNVs) in RMS tumors (T) and tumoroid models (O). Circle color indicates SNV type while circle size indicates variant allele fraction (VAF). Vertical dotted lines separate samples derived from individual patients. Highlighted are genes previously reported for this RMS subtype.(D) Correlogram of bulk mRNA sequencing expression profiles of pediatric kidney tumors (controls) as well as RMS tumoroid models and RMS tumors. CCRCC = Clear Cell Renal Cell Carcinoma, CMN = Congenital Mesoblastic Nephroma, WT = Wilms Tumor, Cor = correlation.
Figure 5(A) Clustered heatmap of viability measurements per RMS tumoroid model (x axis) and drug (y axis), showing the Area Under the Curve (AUC) after treatment of the cells for 120 h with a dose-range of 0.1 nM to 10 µM. Low AUC (red) indicates high drug efficacy while high AUC (blue) indicates low drug efficacy. Annotated clusters of (1) MEK/ERK and γ-secretase inhibitors showing specific efficacy in RMS tumoroid models without (RMS007, RMS012, RMS444) or low (RMS000HQC) fusion transcript expression, and (2) drugs that show broad efficacy across RMS tumoroid models.
Figure 6(B) Western Blot analysis of TP53 wildtype (WT) and knockout (KO) RMS tumoroid line RMS012. Histone 3 (H3) served as loading control.(C) Dose-response curve of TP53 WT and KO cells treated with the Chk-1 inhibitor prexasertib. Thin lines with numbers indicate individual biological replicates (n = 3) while thick lines indicate fitted lines over all replicates. The statistical significance of the differences in fitted IC50 values between WT and KO were obtained using a two-sided t-test (p = 0.008).
Figure 1(C-J) All panels show Drosophila 3rd instar larval eye imaginal discs with eyeless-Flippase-induced clones of cells homozygous for EMC3Δ4 labelled by the absence of ubiGFP (green). The red channel shows ELAV, which labels the nuclei of photoreceptor cells. All UAS constructs were expressed under the control of GMR-GAL4. (C) Expression of UAS-Rh1 (4C5, in blue) and (D) Na+K+ATPase (A5-C, in blue) is strongly reduced in EMC3Δ4 homozygous mutant cells. (E) Expression of UAS-Xport-A-HA (anti-HA, in blue) and (F) UAS-fan-HA (anti-HA, in blue) is strongly reduced in EMC3Δ4 homozygous mutant cells. (G) Expression of UAS-rtv-HA (anti-HA, in blue) and (H) hid (anti-hid, in blue) is increased in EMC3Δ4 homozygous mutant cells. (I-J) Normal expression of two TA proteins in EMC3Δ4 homozygous mutant cells. (I) UAS-CG8814-HA (anti-HA, in blue) is a TA protein containing a TMD of low hydrophobicity (J) UAS-Syx13-HA is a TA protein with high hydrophobicity TMD. Scale bars represent 10 μm.