D , E The mRNA levels of PD - 1 , NFATc1 , or the mRNA and protein levels of granzyme B in lung infiltrating CD8 + T cells were examined at day 26 ( mean of triplicates ± SD , and the mRNA samples were prepared from at least 3 mice per group ) .

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D, E The mRNA levels of PD-1, NFATc1, or the mRNA and protein levels of granzyme B in lung infiltrating CD8+ T cells were examined at day 26 (mean of triplicates ± SD, and the mRNA samples were prepared from at least 3 mice per group).


F CTLA - 4 levels in the lung - infiltrated CD44hiCD8 + T cells or the percentages of CD4 + Foxp3 + T cells in lungs , MLN , and spleen were measured at day 26 ( mean ± SD , n ≥ 5 mice ) .

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F CTLA-4 levels in the lung-infiltrated CD44hiCD8+ T cells or the percentages of CD4+Foxp3+ T cells in lungs, MLN, and spleen were measured at day 26 (mean ± SD, n ≥ 5 mice).


A The same procedures as in Fig4A were performed with WT and ADAP − / − C57BL / 6 mice . HE staining of lung tissue was examined on day 26 ( representative graph of 5 mice ) .

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A The same procedures as in Fig4A were performed with WT and ADAP−/− C57BL/6 mice. HE staining of lung tissue was examined on day 26 (representative graph of 5 mice).


B , C PD - 1 expression in CD44hiCD8 + or CD44hiCD4 + T cells ( mean ± SD , n ≥ 5 mice per group ) or granzyme B expression in CD44hiCD8 + T cells were examined from the lungs at day 26 ( representative data of 3 mice per group ) .

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B, C PD-1 expression in CD44hiCD8+ or CD44hiCD4+ T cells (mean ± SD, n ≥ 5 mice per group) or granzyme B expression in CD44hiCD8+ T cells were examined from the lungs at day 26 (representative data of 3 mice per group).


D , E CTLA - 4 levels in lung infiltrating CD44hiCD8 + T cells and the percentages of CD4 + Foxp3 + T cells in the lungs or MLN were measured at day 26 ( mean ± SD , n ≥ 5 mice per group ) .

D, E CTLA-4 levels in lung infiltrating CD44hiCD8+ T cells and the percentages of CD4+Foxp3+ T cells in the lungs or MLN were measured at day 26 (mean ± SD, n ≥ 5 mice per group).


A , B WT and SKAP55 − / − mice were s . c . injected with MO5 melanoma cells followed by two injection at day 7 and day 14 of OVA257 - 264 - pulsed DCs or the PBS control respectively ( n ≥ 5 ) . The growth of tumors under skin was measured every three days according to tumor diameter ( mean ± SEM , n ≥ 5 mice per group ) ( A ) . Surface expression levels of PD - 1 were checked on OVA257 - 264 - specific Vα2high / Vβ5 + CD8 + effector cells at day 30 ( mean ± SD , n ≥ 5 mice ) ( B ) .

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A, B WT and SKAP55−/− mice were s.c. injected with MO5 melanoma cells followed by two injection at day 7 and day 14 of OVA257-264-pulsed DCs or the PBS control respectively (n ≥ 5). The growth of tumors under skin was measured every three days according to tumor diameter (mean ± SEM, n ≥ 5 mice per group) (A). Surface expression levels of PD-1 were checked on OVA257-264-specific Vα2high/Vβ5+ CD8+ effector cells at day 30 (mean ± SD, n ≥ 5 mice) (B).


C WT and SKAP55 − / − mice were s . c . injected with MO5 melanoma cells followed by s . c . two injection of OVA257 - 264 - pulsed DCs at day 7 and day 14 . anti - PD - 1 monoantibody or IgG controls were i . v . injected three times at day 7 , day 10 , and day 14 ( 50 μg / injection ) . The diameter and volume of tumors were measured every three days ( mean ± SEM , n ≥ 5 mice ) .

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C WT and SKAP55−/− mice were s.c. injected with MO5 melanoma cells followed by s.c. two injection of OVA257-264-pulsed DCs at day 7 and day 14. anti-PD-1 monoantibody or IgG controls were i.v. injected three times at day 7, day 10, and day 14 (50 μg/injection). The diameter and volume of tumors were measured every three days (mean ± SEM, n ≥ 5 mice).


D The WT recipient mice were s . c . injected with MO5 melanoma cells followed by injection of 10 nM OVA257 - 264 - stimulated WT or SKAP55 − / − CTLs at day 8 ( n ≥ 8 ) . The diameter and volume of tumors were measured every three days ( mean ± SEM , n ≥ 8 mice ) .

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D The WT recipient mice were s.c. injected with MO5 melanoma cells followed by injection of 10 nM OVA257-264-stimulated WT or SKAP55−/− CTLs at day 8 (n ≥ 8). The diameter and volume of tumors were measured every three days (mean ± SEM, n ≥ 8 mice).


A , B During generation of OVA257 - 264 peptide - specific CTLs , naïve OT - I splenocytes were stimulated with 10 nM OVA257 - 264 with or without CsA ( 10 nM ) for 3 days , then washed , and further cultured in the RPMI growth medium until day 6 . The CsA - treated or untreated OVA257 - 264 peptide - specific CTLs were incubated with OVA257 - 264 - pulsed EL - 4 cells for 4 h to assess the in vitro cytotoxicity ( A , mean of triplicates ± SD ) or i . v . injected into WT mice to measure in vivo cytotoxicity ( B , mean ± SD , n = 4 mice ) .

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A, B During generation of OVA257-264 peptide-specific CTLs, naïve OT-I splenocytes were stimulated with 10 nM OVA257-264 with or without CsA (10 nM) for 3 days, then washed, and further cultured in the RPMI growth medium until day 6. The CsA-treated or untreated OVA257-264 peptide-specific CTLs were incubated with OVA257-264-pulsed EL-4 cells for 4 h to assess the in vitro cytotoxicity (A, mean of triplicates ± SD) or i.v. injected into WT mice to measure in vivo cytotoxicity (B, mean ± SD, n = 4 mice).


C WT mice were s . c . injected with B16 - MO5 melanoma cells followed by injection of the CsA - treated or untreated OVA257 - 264 peptide - specific CTLs at day 9 ( n ≥ 12 ) . The diameter and volume of tumors were measured every 3 days ( mean ± SEM , n ≥ 12 mice ) .

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C WT mice were s.c. injected with B16-MO5 melanoma cells followed by injection of the CsA-treated or untreated OVA257-264 peptide-specific CTLs at day 9 (n ≥ 12). The diameter and volume of tumors were measured every 3 days (mean ± SEM, n ≥ 12 mice).


C : Time course of PIKfyve downregulation in brains of prion - infected mice ( 3 mice / time point ) . PIKfyve ( but not FIG4 , calnexin , NeuN and synaptophysin ) was suppressed between 60 and 120 dpi . T : terminal disease stage .

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C: Time course of PIKfyve downregulation in brains of prion-infected mice (3 mice/time point). PIKfyve (but not FIG4, calnexin, NeuN and synaptophysin) was suppressed between 60 and 120 dpi. T: terminal disease stage.


E : Prion - infected Gt1 cells ( 75 dpi ) stained with Alexa - Fluor647 - succinimidyl ester which stains vacuoles negatively ( arrows ) .

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E: Prion-infected Gt1 cells (75 dpi) stained with Alexa-Fluor647-succinimidyl ester which stains vacuoles negatively (arrows).


G : tga20 COCS were treated with pooled IgG or POM1 ( 72 hours ) . POM1 suppressed PIKfyve but not FIG4 and VAC14 . Right : Quantification of immunoblots . Each dot represents an independent experiment . * * : p < 0 . 01 ; * * * : p < 0 . 001 ; unpaired t - test Error bars represent s . e . m .

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G: tga20 COCS were treated with pooled IgG or POM1 (72 hours). POM1 suppressed PIKfyve but not FIG4 and VAC14. Right: Quantification of immunoblots. Each dot represents an independent experiment. **: p<0.01; ***: p<0.001; unpaired t-test Error bars represent s.e.m.


H : Immunoblots of human CJD brains ( red : sampled region ) . Gt1Sc : prion - infected Gt1 cells . Loading controls : actin and calnexin ( Cnx )

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H: Immunoblots of human CJD brains (red: sampled region). Gt1Sc: prion-infected Gt1 cells. Loading controls: actin and calnexin (Cnx)


C : Treatment of COCS with POM1 reduces the levels of PIKfyve starting 48h post treatment . Each dot represents an individual experiment . Statistics : Unpaired t - test . * * : p < 0 . 01 .

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C: Treatment of COCS with POM1 reduces the levels of PIKfyve starting 48h post treatment. Each dot represents an individual experiment. Statistics: Unpaired t-test. **: p<0.01.


G : Left : partially restored PIKfyve levels in prion - infected tga20 mice treated with LIN5044 ( LCP ) or vehicle . Right : quantification of the western blot . Each dot represents an individual biological replicate . * : p0 . 05 ; unpaired t - test Error bars represent s . e . m .

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G: Left: partially restored PIKfyve levels in prion-infected tga20 mice treated with LIN5044 (LCP) or vehicle. Right: quantification of the western blot. Each dot represents an individual biological replicate. *: p0.05; unpaired t-test Error bars represent s.e.m.


H : Left , tga20 COCS were inoculated with RML or NBH , optionally treated with GSK2606414 starting at 21 dpi , and lysed at 35 dpi . Depletion of PIKfyve in prion - infected samples was largely rescued by GSK2606414 ( GSK ) . Right , quantification of western blot . Each dot represents an individual biological replicate . Statistics : Unpaired t - test . * * : p < 0 . 01 ; unpaired t - test ; Error bars represent s . e . m .

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H: Left, tga20 COCS were inoculated with RML or NBH, optionally treated with GSK2606414 starting at 21 dpi, and lysed at 35 dpi. Depletion of PIKfyve in prion-infected samples was largely rescued by GSK2606414 (GSK). Right, quantification of western blot. Each dot represents an individual biological replicate. Statistics: Unpaired t-test. **: p<0.01; unpaired t-test; Error bars represent s.e.m.


E , F : Acyl - rac of Gt1 cells subjected to siRNA against zDHHC9 , zDHHC21 or both ( 72 hours post transfection ) , showing synergistic decrease of PIKfyve acylation by suppression of zDHHC9 and 21 ( quantified in F ) . Each dot represents a separate experiment ( unpaired t - test ) . * * : p < 0 . 01 . Error bars represent s . e . m .

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E, F: Acyl-rac of Gt1 cells subjected to siRNA against zDHHC9, zDHHC21 or both (72 hours post transfection), showing synergistic decrease of PIKfyve acylation by suppression of zDHHC9 and 21 (quantified in F). Each dot represents a separate experiment (unpaired t-test). **: p<0.01. Error bars represent s.e.m.


H - I : Flag - tagged zDHHC9 or zDHHC21 were expressed in Gt1 cells were transfected with plasmids encoding . After 72 hours , zDHHC9 / 21 ( magenta ) were localized to the Golgi apparatus ( GM130 + , cyan ) in NBH - exposed cells but not in prion - infected cells . n = 3 individual biological replicates .

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H-I: Flag-tagged zDHHC9 or zDHHC21 were expressed in Gt1 cells were transfected with plasmids encoding. After 72 hours, zDHHC9/21 (magenta) were localized to the Golgi apparatus (GM130+, cyan) in NBH-exposed cells but not in prion-infected cells. n=3 individual biological replicates.


A : Upper row : prion - infected Gt1 cells ( 75 dpi ) immunostained for LAMP1 showing prominent vacuoles . Control : NBH - inoculated cells . Middle row : Gt1 cells transfected with shRNA targeting PIKfyve or luciferase ( control ) for 5 days and immunostained for LAMP1 . Depletion of PIKfyve resulted in LAMP1 + vacuoles ( yellow ) . DAPI : nuclear stain . Lower row : prion - infected and NBH treated cells ( 75 dpi ) contained LAMP2 - vacuoles . n = 3 individual biological replicates .

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A: Upper row: prion-infected Gt1 cells (75 dpi) immunostained for LAMP1 showing prominent vacuoles. Control: NBH-inoculated cells. Middle row: Gt1 cells transfected with shRNA targeting PIKfyve or luciferase (control) for 5 days and immunostained for LAMP1. Depletion of PIKfyve resulted in LAMP1+ vacuoles (yellow). DAPI: nuclear stain. Lower row: prion-infected and NBH treated cells (75 dpi) contained LAMP2- vacuoles. n=3 individual biological replicates.


D : tga20 COCS treated with POM1 or control IgG ( 10 days ) and immunostained with NeuN and LAMP1 . Nuclei : DAPI . POM1 - treated COCS showed increased LAMP1 expression ( quantification : Appendix Fig . S4B ) . NeuN + cells exhibited vacuoles ( arrowheads ) .

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D: tga20 COCS treated with POM1 or control IgG (10 days) and immunostained with NeuN and LAMP1. Nuclei: DAPI. POM1-treated COCS showed increased LAMP1 expression (quantification: Appendix Fig. S4B). NeuN+ cells exhibited vacuoles (arrowheads).


G : Lysosomal enzymes , but not β - actin , were elevated in brains of terminally scrapie - sick mice . Panels show independent triplicates . Statistics : ANOVA . . * * : p < 0 . 01 . Error bars represent s . e . m .

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G: Lysosomal enzymes, but not β-actin, were elevated in brains of terminally scrapie-sick mice. Panels show independent triplicates. Statistics: ANOVA.. **: p<0.01. Error bars represent s.e.m.


A : Time course of TFEB phosphorylation in prion - infected C57BL / 6 mice . Total TFEB was unchanged but reduced TFEB phosphorylation was evident at ≥ 90 days . Quantification : Each dot represents an individual biological replicate . Statistics : Unpaired t - test . * : p0 . 05 ; * * : p < 0 . 01 . Error bars represent s . e . m .

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A: Time course of TFEB phosphorylation in prion-infected C57BL/6 mice. Total TFEB was unchanged but reduced TFEB phosphorylation was evident at ≥90 days. Quantification: Each dot represents an individual biological replicate. Statistics: Unpaired t-test. *: p0.05; **: p<0.01. Error bars represent s.e.m.


B : TFEB - controlled transcripts in brains of terminally scrapie - sick C57BL / 6 mice were measured by qPCR . Prion infection led to upregulation of these genes , but not of STAT3 . Control : NBH - inoculated mice . ANOVA on independent triplicates . * * * : p < 0 . 001 . Error bars represent s . e . m .

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B: TFEB-controlled transcripts in brains of terminally scrapie-sick C57BL/6 mice were measured by qPCR. Prion infection led to upregulation of these genes, but not of STAT3. Control: NBH-inoculated mice. ANOVA on independent triplicates. ***: p<0.001. Error bars represent s.e.m.


E : Gt1 cells ( as in D ) were fixed and stained with anti - TFEB antibody . Prion - infected cells showed nuclear translocation of TFEB . DAPI : Blue . Quantification : Each dot represents an individual biological replicate ( 30 images were quantified per replicate ) . Statistics : Unpaired t - test . * * : p < 0 . 01 . Error bars represent s . e . m .

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E: Gt1 cells (as in D) were fixed and stained with anti-TFEB antibody. Prion-infected cells showed nuclear translocation of TFEB. DAPI: Blue. Quantification: Each dot represents an individual biological replicate (30 images were quantified per replicate). Statistics: Unpaired t-test. **: p<0.01. Error bars represent s.e.m.


F : TFEB responsive genes were upregulated in Gt1 cells chronically infected with RML prions ( 75 dpi ) . Panels depict independent triplicates . Statistics : ANOVA . * : p0 . 05 . Error bars represent s . e . m . G : TFEB responsive genes were upregulated in cells transfected with shPIKfyve for 5 days compared to shCtrl ( shRNA against luciferase ) . Panels depict independent triplicates . Statistics : ANOVA . * : p0 . 05 . Error bars represent s . e . m .

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F: TFEB responsive genes were upregulated in Gt1 cells chronically infected with RML prions (75 dpi). Panels depict independent triplicates. Statistics: ANOVA. *: p0.05. Error bars represent s.e.m. G: TFEB responsive genes were upregulated in cells transfected with shPIKfyve for 5 days compared to shCtrl (shRNA against luciferase). Panels depict independent triplicates. Statistics: ANOVA. *: p0.05. Error bars represent s.e.m.


C : Prion - infected Gt1 cells were lentivirally transduced with GADD34 at 75 dpi . Control : empty lentiviral vector . At 79 dpi , GADD34 overexpression had normalized eIF2α phosphorylation and restored PIKfyve levels . Right : Quantification of western blots ( n = 3 ; unpaired t - test ) . Each dot represents an individual experiment . * * : p < 0 . 01 . Error bars represent s . e . m .

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C: Prion-infected Gt1 cells were lentivirally transduced with GADD34 at 75 dpi. Control: empty lentiviral vector. At 79 dpi, GADD34 overexpression had normalized eIF2α phosphorylation and restored PIKfyve levels. Right: Quantification of western blots (n=3; unpaired t-test). Each dot represents an individual experiment. **: p<0.01. Error bars represent s.e.m.


E : tga20 COCS were infected with RML and optionally treated with bPIP ( 5µg / ml ) . At 45 dpi , NeuN morphometry revealed ablation of cerebellar granule layer ( CGN ) in prion - infected slices and its rescue by bPIP . Control : NBH treated COCS . Each dot represents an individual slice ( Statistics : ANOVA ) . * : p < 0 . 05 . * * * * p < 0 . 0001 .

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E: tga20 COCS were infected with RML and optionally treated with bPIP (5µg/ml). At 45 dpi, NeuN morphometry revealed ablation of cerebellar granule layer (CGN) in prion-infected slices and its rescue by bPIP. Control: NBH treated COCS. Each dot represents an individual slice (Statistics: ANOVA). *: p<0.05. **** p<0.0001.


F : tga20 COCS were treated with POM1 ( optionally pre - blocked with recPrP ) and treated with bPIP ( 5µg / ml ) . At 14 dpi NeuN morphometry revealed POM1 - induced ablation of cerebellar granule layer ( CGL ) and rescue by bPIP .

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F: tga20 COCS were treated with POM1 (optionally pre-blocked with recPrP) and treated with bPIP (5µg/ml). At 14 dpi NeuN morphometry revealed POM1-induced ablation of cerebellar granule layer (CGL) and rescue by bPIP.


B ) Binding affinity of CodB for cytosine as measured using the thermostability assay . Cytosine was titrated into detergent solubilised membranes from cells overexpressing CodB . The Kd was estimated to be 51 ± 9 µM . The measurements are the average of 4 independent titrations with error bars of the s . e . m .

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B) Binding affinity of CodB for cytosine as measured using the thermostability assay. Cytosine was titrated into detergent solubilised membranes from cells overexpressing CodB. The Kd was estimated to be 51 ± 9 µM. The measurements are the average of 4 independent titrations with error bars of the s.e.m.


A . Glioma tumour micro arrays ( grade II - IV gliomas ) were stained to visualise MDGI ( red ) . Samples from the epileptic brain served as a negative control . Scale bar 100 µm .

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A. Glioma tumour micro arrays (grade II-IV gliomas) were stained to visualise MDGI (red). Samples from the epileptic brain served as a negative control. Scale bar 100 µm.


B . Overall survival of grade II and III glioma patients ( n = 76 ) was significantly better when none / low MDGI ( blue line ) was detected compared to patients with moderate / high MDGI ( red line ) expression ( P = 0 . 007 ) . The cumulative survival rates were estimated by using the Kaplan - Meier method .

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B. Overall survival of grade II and III glioma patients (n = 76) was significantly better when none/low MDGI (blue line) was detected compared to patients with moderate/high MDGI (red line) expression (P = 0.007). The cumulative survival rates were estimated by using the Kaplan-Meier method.


C . Analysis of the association of MDGI mRNA expression with glioma patient survival using the GlioVis data portal . In the TCGA GBMLGG dataset ( n = 667 ) , patients with low MDGI expression ( green line ; n = 333 , events = 101 , median = 67 , 5 ) show significantly better survival than patients with high MDGI ( red line ; n = 334 , events = 138 , median = 41 . 5 ) expression ( P = 2e - 04 ) . HR = 0 . 62 ( 0 . 48 - 0 . 8 ) . The cumulative survival rates were estimated by using the Kaplan - Meier method .

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C. Analysis of the association of MDGI mRNA expression with glioma patient survival using the GlioVis data portal. In the TCGA GBMLGG dataset (n = 667), patients with low MDGI expression (green line; n = 333, events = 101, median = 67,5) show significantly better survival than patients with high MDGI (red line; n = 334, events = 138, median = 41.5) expression (P = 2e-04). HR = 0.62 (0.48 - 0.8). The cumulative survival rates were estimated by using the Kaplan-Meier method.


D . In glioblastoma samples , highest MDGI expression was observed in the mesenchymal subtype ( M ) compared to the classical ( Cl ) or pro - neural ( PN ) ones . However , it did not reach the statistical significance . GlioVis data portal , TCGA GBM dataset , n = 156 . Pairwise t - test with corrections for multiple testing , p - values with Bonferroni correction .

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D. In glioblastoma samples, highest MDGI expression was observed in the mesenchymal subtype (M) compared to the classical (Cl) or pro-neural (PN) ones. However, it did not reach the statistical significance. GlioVis data portal, TCGA GBM dataset, n = 156. Pairwise t-test with corrections for multiple testing, p-values with Bonferroni correction.


E . MDGI expression was high in the leading edge of the tumour ( LE ) and in infiltrating tumour cells ( IT ) in the IVY _ Gap dataset ( n = 122 ) where the gene expression profile in different anatomical structures of glioblastomas was analysed . LE , leading edge ; IT , infiltrating tumour cells ; CT , cellular tumour ( cells within the tumour mass ) ; PC , pseudopalisading cells ; MP , microvascular proliferation . Numbers in the graph depict the P values : Pairwise t - test with corrections for multiple testing , p - values with Bonferroni correction .

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E. MDGI expression was high in the leading edge of the tumour (LE) and in infiltrating tumour cells (IT) in the IVY_Gap dataset (n = 122) where the gene expression profile in different anatomical structures of glioblastomas was analysed. LE, leading edge; IT, infiltrating tumour cells; CT, cellular tumour (cells within the tumour mass); PC, pseudopalisading cells; MP, microvascular proliferation. Numbers in the graph depict the P values: Pairwise t-test with corrections for multiple testing, p-values with Bonferroni correction.


F . Western blot analysis shows MDGI expression in human glioma cells including patient - derived spheroids ( S24 , ZH161 , ZH305 , BT3 , BT11 , BT12 , BT13 ) , patient - derived adherent cells ( BT5 and BT5R ) , and long - term cell lines ( U87MG , LN229 , LN308 ) . Numbers above the lanes show the relative expression of MDGI compared to the levels of β - tubulin that served as loading control in each cell line . Representative Western blot image of 3 separate experiments is shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F. Western blot analysis shows MDGI expression in human glioma cells including patient-derived spheroids (S24, ZH161, ZH305, BT3, BT11, BT12, BT13), patient-derived adherent cells (BT5 and BT5R), and long-term cell lines (U87MG, LN229, LN308). Numbers above the lanes show the relative expression of MDGI compared to the levels of β-tubulin that served as loading control in each cell line. Representative Western blot image of 3 separate experiments is shown.


G . High MDGI and HIF - 1α expression in the patient - derived glioblastoma spheroids decreased significantly when cells were cultured in medium containing 10 % of serum ( FBS ) for seven days . Numbers show the relative expression of MDGI and HIF - 1α compared to the levels of β - tubulin . Expression in the serum - free medium was set as 1 . Representative Western blot image of 2 separate experiments is shown .

G. High MDGI and HIF-1α expression in the patient-derived glioblastoma spheroids decreased significantly when cells were cultured in medium containing 10 % of serum (FBS) for seven days. Numbers show the relative expression of MDGI and HIF-1α compared to the levels of β-tubulin. Expression in the serum-free medium was set as 1. Representative Western blot image of 2 separate experiments is shown.


H . MDGI expression in adherent glioma cell lines and in glioma cell spheroids after 24h culture under hypoxia ( 1 % O2 ) or normoxia ( 21 % O2 ) . Numbers show the relative MDGI expression compared to the levels of β - tubulin . Expression under normoxia was set as 1 . Representative Western blot image of 3 separate experiments is shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H. MDGI expression in adherent glioma cell lines and in glioma cell spheroids after 24h culture under hypoxia (1 % O2) or normoxia (21 % O2). Numbers show the relative MDGI expression compared to the levels of β-tubulin. Expression under normoxia was set as 1. Representative Western blot image of 3 separate experiments is shown.


A , B Representative whole coronal section micrographs of murine brain injected with GFP ( A ) or MDGI - GFP ( B ) expressing U87MG glioma cells . Xenografted human cells were visualised by using anti - human vimentin ( hVim ) antibodies ( red ) . Nuclei were visualised by using DAPI ( white ) . Scale bar : 1 mm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A, B Representative whole coronal section micrographs of murine brain injected with GFP (A) or MDGI-GFP (B) expressing U87MG glioma cells. Xenografted human cells were visualised by using anti-human vimentin (hVim) antibodies (red). Nuclei were visualised by using DAPI (white). Scale bar: 1 mm.


Micrographs of consecutive ( separating distance : 9 µm ) brain sections stained for human glioma cells using antibodies specific for human vimentin ( hVim red in C and D ) , Dashed lines separate the primary tumour mass and the normal brain . Nuclei were visualised by using DAPI ( blue ) . Arrows in D point to the angiotropic tumours . Scale bar : 50 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Micrographs of consecutive (separating distance: 9 µm) brain sections stained for human glioma cells using antibodies specific for human vimentin (hVim red in C and D), Dashed lines separate the primary tumour mass and the normal brain. Nuclei were visualised by using DAPI (blue). Arrows in D point to the angiotropic tumours. Scale bar: 50 µm.


Micrographs of consecutive ( separating distance : 9 µm ) brain sections stained for human glioma cells using antibodies specific for MDGI ( green in E and F ) , and CD31 ( red in E and F ) . Dashed lines separate the primary tumour mass and the normal brain . Nuclei were visualised by using DAPI ( blue ) . Arrows in F point to the MDGI - expressing angiotropic tumour cells . Scale bar : 50 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Micrographs of consecutive (separating distance: 9 µm) brain sections stained for human glioma cells using antibodies specific for MDGI (green in E and F), and CD31 (red in E and F). Dashed lines separate the primary tumour mass and the normal brain. Nuclei were visualised by using DAPI (blue). Arrows in F point to the MDGI-expressing angiotropic tumour cells. Scale bar: 50 µm.


G . Quantification of the number of U87MG secondary tumours ( diameter > 300 µm ) detected in the whole brain ( GFP : n = 5 , MDGI - GFP : n = 9 ) . Data are represented as mean ± SEM . * * P < 0 . 01 , two - tailed , nonparametric Mann - Whitney ' s U - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G. Quantification of the number of U87MG secondary tumours (diameter >300 µm) detected in the whole brain (GFP: n = 5, MDGI-GFP: n = 9). Data are represented as mean ± SEM. **P < 0.01, two-tailed, nonparametric Mann-Whitney's U-test.


H . Quantification of the number of invasive U87MG cells that grew next to brain blood vessels ( angiotropic co - opting tumour cells , diameter < 300 µm ) ( GFP : n = 5 , MDGI - GFP : n = 9 ) . Data are represented as mean ± SEM . * * * * P < 0 . 0001 , two - tailed , nonparametric Mann - Whitney ' s U - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H. Quantification of the number of invasive U87MG cells that grew next to brain blood vessels (angiotropic co-opting tumour cells, diameter <300 µm) (GFP: n = 5, MDGI-GFP: n = 9). Data are represented as mean ± SEM. ****P < 0.0001, two-tailed, nonparametric Mann-Whitney's U-test.


I , J Representative micrographs of whole murine brain coronal sections injected with GFP ( I ) or GFP - MDGI ( J ) expressing LN229 glioma cells . Xenografted human cells were visualised by using anti - human vimentin ( hVim ) antibodies ( red ) . Nuclei were visualised by using DAPI ( white ) . Scale bar : 1 mm .

I, J Representative micrographs of whole murine brain coronal sections injected with GFP (I) or GFP-MDGI (J) expressing LN229 glioma cells. Xenografted human cells were visualised by using anti-human vimentin (hVim) antibodies (red). Nuclei were visualised by using DAPI (white). Scale bar: 1 mm.


Micrographs of consecutive ( separating distance : 9 µm ) brain sections stained for human glioma cells using antibodies specific for human vimentin ( hVim red in K and L ) Dashed lines separate the primary tumour mass and the normal brain . Nuclei were visualised by using DAPI ( blue ) . ) . Arrows in L point to the angiotropic tumours . Scale bar : 50 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Micrographs of consecutive (separating distance: 9 µm) brain sections stained for human glioma cells using antibodies specific for human vimentin (hVim red in K and L) Dashed lines separate the primary tumour mass and the normal brain. Nuclei were visualised by using DAPI (blue). ). Arrows in L point to the angiotropic tumours. Scale bar: 50 µm.


Micrographs of consecutive ( separating distance : 9 µm ) brain sections stained for human glioma cells using antibodies specific for MDGI ( green in M and N ) , and CD31 ( red in M and N ) . Dashed lines separate the primary tumour mass and the normal brain . Nuclei were visualised by using DAPI ( blue ) . ) . Arrows in N point to the MDGI - expressing angiotropic tumour cells . Scale bar : 50 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Micrographs of consecutive (separating distance: 9 µm) brain sections stained for human glioma cells using antibodies specific for MDGI (green in M and N), and CD31 (red in M and N). Dashed lines separate the primary tumour mass and the normal brain. Nuclei were visualised by using DAPI (blue). ). Arrows in N point to the MDGI-expressing angiotropic tumour cells. Scale bar: 50 µm.


O . Quantification of the number of invasive LN229 cells detected in the brain ( GFP : n = 10 , MDGI - GFP : n = 10 ) . Data are represented as mean ± SEM . * * * * P < 0 . 0001 , two - tailed , nonparametric Mann - Whitney ' s U - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

O. Quantification of the number of invasive LN229 cells detected in the brain (GFP: n = 10, MDGI-GFP: n = 10). Data are represented as mean ± SEM. ****P < 0.0001, two-tailed, nonparametric Mann-Whitney's U-test.


P . Quantification of the number of invasive LN229 cells that grew next to brain blood vessels ( angiotropic co - opting tumour cells , diameter < 300 µm ) detected in the brain ( GFP : n = 10 , MDGI - GFP : n = 10 ) . Data are represented as mean ± SEM . Data are represented as mean ± SEM . * * * * P < 0 . 0001 , two - tailed , nonparametric Mann - Whitney ' s U - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

P. Quantification of the number of invasive LN229 cells that grew next to brain blood vessels (angiotropic co-opting tumour cells, diameter <300 µm) detected in the brain (GFP: n = 10, MDGI-GFP: n = 10). Data are represented as mean ± SEM. Data are represented as mean ± SEM. ****P < 0.0001, two-tailed, nonparametric Mann-Whitney's U-test.


A . Transmitted light micrographs of MDGI - silenced ( shMDGI1 and shMDGI2 ) and control ( Scr ) patient - derived BT12 and BT13 spheroids 11 days after gene silencing . Representative images of at least 3 independent experiments are shown . Scale bar : 200 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Transmitted light micrographs of MDGI-silenced (shMDGI1 and shMDGI2) and control (Scr) patient-derived BT12 and BT13 spheroids 11 days after gene silencing. Representative images of at least 3 independent experiments are shown. Scale bar: 200 μm.


B . Methylcellulose assay demonstrating the ability for self - renewal of control ( Scr ) and MDGI - silenced ( shMDGI1 and shMDGI2 ) patient - derived BT12 and BT13 cells after three weeks of culture . Representative images of at least 3 independent experiments are shown . Scale bar : 200 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. Methylcellulose assay demonstrating the ability for self-renewal of control (Scr) and MDGI-silenced (shMDGI1 and shMDGI2) patient-derived BT12 and BT13 cells after three weeks of culture. Representative images of at least 3 independent experiments are shown. Scale bar: 200 μm.


C . Quantification of the colony size in methylcellulose of MDGI - silenced ( sh1 and sh2 ) cells relative to the controls ( Scr ) that is set as 100 ( n = 3 ) . Data are represented as mean ± SD . * * * P < 0 . 001 , two - tailed , nonparametric Mann - Whitney ' s U - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. Quantification of the colony size in methylcellulose of MDGI-silenced (sh1 and sh2) cells relative to the controls (Scr) that is set as 100 (n = 3). Data are represented as mean ± SD. ***P < 0.001, two-tailed, nonparametric Mann-Whitney's U-test.


D . MTT - proliferation assay of control ( Scr ) and MDGI - silenced ( shMDGI1 ) BT12 and BT13 cells . The values of each cell line were normalized to the values at day 1 marked with a dashed line ( n = 3 ) . Data are represented as mean ± SD . * * * P < 0 . 001 , two - tailed , nonparametric Mann - Whitney ' s U - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. MTT-proliferation assay of control (Scr) and MDGI-silenced (shMDGI1) BT12 and BT13 cells. The values of each cell line were normalized to the values at day 1 marked with a dashed line (n = 3). Data are represented as mean ± SD. ***P < 0.001, two-tailed, nonparametric Mann-Whitney's U-test.


E , F Representative whole coronal section micrographs of murine brain injected intracranially with control ( BT12 Scr , n = 5 ) and MDGI - silenced ( BT12 sh1 , n = 5 ) BT12 cells . After four weeks animals were sacrificed , the brains were excised and prepared for immunohistochemistry . Control ( Scr ) cells grew very similarly to the parental BT12 cells forming a tumour mass with invasive tumour cells ( single cell invasion ) that eventually co - opted existing blood vessels ( angiotropism ) and formed secondary tumours ( upper panels ) . No tumour growth was observed when MDGI - silenced cells were implanted ( lower panels ) . Glioma cells were visualised by using anti - human vimentin ( hVim red ) and blood vessels using anti - podocalyxin antibodies ( green ) . Nuclei were visualised by using DAPI ( white ) . Scale bar : 1 mm ( E ) and 50 µm ( F ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E, F Representative whole coronal section micrographs of murine brain injected intracranially with control (BT12 Scr, n = 5) and MDGI-silenced (BT12 sh1, n = 5) BT12 cells. After four weeks animals were sacrificed, the brains were excised and prepared for immunohistochemistry. Control (Scr) cells grew very similarly to the parental BT12 cells forming a tumour mass with invasive tumour cells (single cell invasion) that eventually co-opted existing blood vessels (angiotropism) and formed secondary tumours (upper panels). No tumour growth was observed when MDGI-silenced cells were implanted (lower panels). Glioma cells were visualised by using anti-human vimentin (hVim red) and blood vessels using anti-podocalyxin antibodies (green). Nuclei were visualised by using DAPI (white). Scale bar: 1 mm (E) and 50 µm (F).


A . Binding of Annexin V Alexa Fluor 488 to control ( Scr ) and MDGI - silenced ( shMDGI1 ) BT12 cells was measured by flow cytometric analyses five days after silencing . The analysis was performed by gating the cells into two populations : P1 ( small , granular ) and P2 ( normal - sized cells ) ( 30 000 analysed events / cell line , n = 3 ) . B . Geometric mean fluorescence ( FL - 1 ) relative to the controls ( Scr ) that was set as 1 in P1 and P2 in MDGI - silenced ( shMDGI1 ) cells . Data are represented as mean ± SD . * P < 0 . 05 , two - tailed , nonparametric Mann - Whitney ' s U - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Binding of Annexin V Alexa Fluor 488 to control (Scr) and MDGI-silenced (shMDGI1) BT12 cells was measured by flow cytometric analyses five days after silencing. The analysis was performed by gating the cells into two populations: P1 (small, granular) and P2 (normal-sized cells) (30 000 analysed events/cell line, n = 3). B. Geometric mean fluorescence (FL-1) relative to the controls (Scr) that was set as 1 in P1 and P2 in MDGI-silenced (shMDGI1) cells. Data are represented as mean ± SD. *P < 0.05, two-tailed, nonparametric Mann-Whitney's U-test.


C , D Representative micrographs of non - treated control BT12 cells ( C ) and cells treated with 2 mM LLOMe for 4 h ( D ) before staining with the anti - galectin - 1 antibodies ( LGALS1 ) ( green ) . Nuclei were visualised by using DAPI ( blue ) . Scale bar : 10 µm .

O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C, D Representative micrographs of non-treated control BT12 cells (C) and cells treated with 2 mM LLOMe for 4 h (D) before staining with the anti-galectin-1 antibodies (LGALS1) (green). Nuclei were visualised by using DAPI (blue). Scale bar: 10 µm.


E , F Representative micrographs of LGALS1 - stained ( green ) BT12 control ( E ) and MDGI - silenced cells ( F ) six days after silencing . Nuclei were visualised by using DAPI ( blue ) . Scale bar : 10 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E, F Representative micrographs of LGALS1-stained (green) BT12 control (E) and MDGI-silenced cells (F) six days after silencing. Nuclei were visualised by using DAPI (blue). Scale bar: 10 µm.


G , H Representative micrographs of LGALS1 - stained ( green ) BT13 control ( G ) and MDGI - silenced cells ( H ) six days after silencing . Nuclei were visualised by using DAPI ( blue ) . Scale bar : 10 µm .

G, H Representative micrographs of LGALS1-stained (green) BT13 control (G) and MDGI-silenced cells (H) six days after silencing. Nuclei were visualised by using DAPI (blue). Scale bar: 10 µm.


I . Percentage of LGALS1 - positive puncta staining in the control ( Scr ) and MDGI - silenced ( sh1 ) cells . In total 0 . 4 - 1 . 2x104 LGALS1 - stained cells were analysed from 50 mm2 coverslip areas ( n = 6 ) . Data are represented as mean ± SD . * * P < 0 . 01 , two - tailed , nonparametric Mann - Whitney ' s U - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

I. Percentage of LGALS1-positive puncta staining in the control (Scr) and MDGI-silenced (sh1) cells. In total 0.4-1.2x104 LGALS1-stained cells were analysed from 50 mm2 coverslip areas (n = 6). Data are represented as mean ± SD. **P < 0.01, two-tailed, nonparametric Mann-Whitney's U-test.


J . Quantification of the percentage of dead cells six days after MDGI silencing and LGALS1 staining ( n = 100 ) in control ( Scr ) and MDGI - silenced ( sh1 ) BT12 and BT13 cells . Data are represented as mean ± SD . * * P < 0 . 01 , two - tailed , nonparametric Mann - Whitney ' s U - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

J. Quantification of the percentage of dead cells six days after MDGI silencing and LGALS1 staining (n = 100) in control (Scr) and MDGI-silenced (sh1) BT12 and BT13 cells. Data are represented as mean ± SD. **P < 0.01, two-tailed, nonparametric Mann-Whitney's U-test.


K . Cytoplasmic cathepsin B activity in the control ( Scr ) and MDGI - silenced ( sh1 ) BT12 and BT13 cells . Data are represented as mean ± SD . * * P < 0 . 01 ; * * P < 0 . 001 , two - tailed , nonparametric Mann - Whitney ' s U - test .

K. Cytoplasmic cathepsin B activity in the control (Scr) and MDGI-silenced (sh1) BT12 and BT13 cells. Data are represented as mean ± SD. **P < 0.01; **P < 0.001, two-tailed, nonparametric Mann-Whitney's U-test.


L . Number of live cells in MDGI - silenced BT12 and BT13 cells incubated without ( sh1 ) or with ( sh1 + K777 ) the pan - cathepsin inhibitor ( K777 ) after 1 , 3 and 5 days of incubation . Data shows the percentage of live cells compared to the control ( Scr ) cells set as 100 % . Data are represented as mean ± SD . * P < 0 . 05 ; * * P < 0 . 01 , two - tailed , nonparametric Mann - Whitney ' s U - test .

O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

L. Number of live cells in MDGI-silenced BT12 and BT13 cells incubated without (sh1) or with (sh1+K777) the pan-cathepsin inhibitor (K777) after 1, 3 and 5 days of incubation. Data shows the percentage of live cells compared to the control (Scr) cells set as 100 %. Data are represented as mean ± SD. *P < 0.05; **P < 0.01, two-tailed, nonparametric Mann-Whitney's U-test.


A . Proportions ( mol % ) of saturated ( Sat ) , monounsaturated ( Mono ) , diunsaturated ( Di ; total of 2 double bonds in the acyl chains ) , and polyunsaturated ( Poly ; total of 3 - 7 double bonds in the acyl chains ) lipid species from the two main phospholipid classes ( phosphatidylcholine PC and phosphatidylethanolamine PE ) of the lysosomal fractions of the BT12 and BT13 control ( Scr ) and MDGI - silenced ( shMDGI ) cells . A representative graph of two independent experiments is shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Proportions (mol %) of saturated (Sat), monounsaturated (Mono), diunsaturated (Di; total of 2 double bonds in the acyl chains), and polyunsaturated (Poly; total of 3-7 double bonds in the acyl chains) lipid species from the two main phospholipid classes (phosphatidylcholine PC and phosphatidylethanolamine PE) of the lysosomal fractions of the BT12 and BT13 control (Scr) and MDGI-silenced (shMDGI) cells. A representative graph of two independent experiments is shown.


B . Fold change of the main PC and PE species in the lysosomal membranes of MDGI - silenced ( shMDGI ) and control ( Scr ) cells ( Log2 , by mol % data ) . Abbreviations : [ Lipid class ] [ total number of acyl chain carbons ] : [ total number of double bonds in the chains ] . A representative heatmap of two independent experiments is shown .

O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. Fold change of the main PC and PE species in the lysosomal membranes of MDGI-silenced (shMDGI) and control (Scr) cells (Log2, by mol % data). Abbreviations: [Lipid class] [total number of acyl chain carbons]:[total number of double bonds in the chains]. A representative heatmap of two independent experiments is shown.


C . Profiles of ceramide ( Cer ) species ( mol % ) in the lysosomal membranes of MDGI - silenced ( shMDGI ) and control ( Scr ) BT12 and BT13 cells . Abbreviations : Cer [ number of acyl chain carbons ] : [ number of double bonds in the acyl chain ] ; all principal species containing a sphingosine chain . A representative graph of two independent experiments is shown .

O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O

C. Profiles of ceramide (Cer) species (mol %) in the lysosomal membranes of MDGI-silenced (shMDGI) and control (Scr) BT12 and BT13 cells. Abbreviations: Cer [number of acyl chain carbons]:[number of double bonds in the acyl chain]; all principal species containing a sphingosine chain. A representative graph of two independent experiments is shown.


D . Ratio of the 24 : 1 to 24 : 0 ( mol % . mol % ) Cer species in the lysosomal membranes of MDGI - silenced ( shMDGI ) and control ( Scr ) BT12 and BT13 cells . A representative graph of two independent experiments is shown .

O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. Ratio of the 24:1 to 24:0 (mol %.mol %) Cer species in the lysosomal membranes of MDGI-silenced (shMDGI) and control (Scr) BT12 and BT13 cells. A representative graph of two independent experiments is shown.


A . Measurement of the BT12 , BT13 , and ZH305 glioblastoma cell viability using the MTT - assay at the indicated clemastine concentrations and time points ( n = 12 ) . A dashed line marks the 50 % cell viability .

O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O

A. Measurement of the BT12, BT13, and ZH305 glioblastoma cell viability using the MTT-assay at the indicated clemastine concentrations and time points (n = 12). A dashed line marks the 50 % cell viability.


B . Measurement of the viability of normal human endothelial cells ( HuAR2T ) , normal human astrocytes ( NHA ) , and embryonic kidney ( HEK293T ) cells at the indicated clemastine concentrations and time points ( n = 12 ) . A dashed line marks the 50 % cell viability .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O

B. Measurement of the viability of normal human endothelial cells (HuAR2T), normal human astrocytes (NHA), and embryonic kidney (HEK293T) cells at the indicated clemastine concentrations and time points (n = 12). A dashed line marks the 50 % cell viability.


C . Representative micrographs of BT12 , BT13 , and ZH305 glioblastoma cells treated with 1 µg / ml of clemastine for 24 h and stained with anti - galectin - 1 ( LGALS1 ) antibody ( green ) . Non - treated cells served as control . Nuclei were visualised by using DAPI ( blue ) . Scale bar : 20 µm .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. Representative micrographs of BT12, BT13, and ZH305 glioblastoma cells treated with 1 µg/ml of clemastine for 24 h and stained with anti-galectin-1 (LGALS1) antibody (green). Non-treated cells served as control. Nuclei were visualised by using DAPI (blue). Scale bar: 20 µm.


D . Representative micrographs of normal human endothelial cells ( HuAR2T ) , normal human astrocytes ( NHA ) , and kidney cells ( HEK293T ) treated with 1 µg / ml clemastine for 24 h and stained with anti - LGALS1 antibody ( green ) . Non - treated cells served as control . Nuclei were visualised by using DAPI ( blue ) . Scale bar : 10 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. Representative micrographs of normal human endothelial cells (HuAR2T), normal human astrocytes (NHA), and kidney cells (HEK293T) treated with 1 µg/ml clemastine for 24 h and stained with anti-LGALS1 antibody (green). Non-treated cells served as control. Nuclei were visualised by using DAPI (blue). Scale bar: 10 µm.


A . Representative whole coronal section micrographs of murine brain intracranially implanted with various glioblastoma cells ) , and daily treated with saline vehicle ( Ve ) or 50 mg / kg clemastine ( Cle , 100 mg / kg single dose administered at day 1 ) for 12 days starting at day 15 after tumour implantation . BT12 ( parental ( wt , Ve n = 9 , Cle n = 12 ) and control shRNA infected ( Ve n = 5 , Cle n = 5 ) , ZH305 ( Ve n = 10 , Cle n = 10 ) , and BT13 ( Ve n = 10 , Cle n = 9 ) . Human glioma cells were visualised with an anti - human vimentin ( hVim , red ) . Nuclei were visualised by using DAPI ( white ) . Scale bar : 1 mm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Representative whole coronal section micrographs of murine brain intracranially implanted with various glioblastoma cells), and daily treated with saline vehicle (Ve) or 50 mg/kg clemastine (Cle, 100 mg/kg single dose administered at day 1) for 12 days starting at day 15 after tumour implantation. BT12 (parental (wt, Ve n = 9, Cle n = 12) and control shRNA infected (Ve n = 5, Cle n = 5), ZH305 (Ve n = 10, Cle n = 10), and BT13 (Ve n = 10, Cle n = 9). Human glioma cells were visualised with an anti-human vimentin (hVim, red). Nuclei were visualised by using DAPI (white). Scale bar: 1 mm.


B , C Quantification of the number of the invading single BT12 wt glioblastoma cells ( B ) and distance of the invaded cells from the primary tumour ( C ) of vehicle ( black bars , Ve ) and clemastine - treated ( white bars , Cle ) animals ( Ve n = 9 , Cle n = 12 ) . Data are represented as mean ± SEM . * * * * P < 0 . 0001 , two - tailed , nonparametric Mann - Whitney ' s U - test .

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B, C Quantification of the number of the invading single BT12 wt glioblastoma cells (B) and distance of the invaded cells from the primary tumour (C) of vehicle (black bars, Ve) and clemastine-treated (white bars, Cle) animals (Ve n = 9, Cle n = 12). Data are represented as mean ± SEM. ****P < 0.0001, two-tailed, nonparametric Mann-Whitney's U-test.


D . Quantification of the number of secondary tumours ( diameter > 300 µm ) in the murine brain for the indicated treatments and tumour models . BT12 ( parental ( wt , Ve n = 9 , Cle n = 12 ) and control shRNA infected ( Ve n = 5 , Cle n = 5 ) , and BT13 ( Ve n = 10 , Cle n = 9 ) . Data are represented as mean ± SEM . * * * * P < 0 . 0001 , two - tailed , nonparametric Mann - Whitney ' s U - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. Quantification of the number of secondary tumours (diameter >300 µm) in the murine brain for the indicated treatments and tumour models. BT12 (parental (wt, Ve n = 9, Cle n = 12) and control shRNA infected (Ve n = 5, Cle n = 5), and BT13 (Ve n = 10, Cle n = 9). Data are represented as mean ± SEM. ****P < 0.0001, two-tailed, nonparametric Mann-Whitney's U-test.


E . Quantification of the number of angiotropic BT12 cells that have co - opted existing blood vessels in the murine brain for the indicated treatments ( Ve n = 9 , Cle n = 12 ) . Data are represented as mean ± SEM . * * * * P < 0 . 0001 , two - tailed , nonparametric Mann - Whitney ' s U - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. Quantification of the number of angiotropic BT12 cells that have co-opted existing blood vessels in the murine brain for the indicated treatments (Ve n = 9, Cle n = 12). Data are represented as mean ± SEM. ****P < 0.0001, two-tailed, nonparametric Mann-Whitney's U-test.


Quantification of the number of TUNEL positive cells in the murine brain for the indicated treatments ( Ve n = 9 , Cle n = 12 ) . Data are represented as mean ± SEM . * * * * P < 0 . 0001 , two - tailed , nonparametric Mann - Whitney ' s U - test .

O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Quantification of the number of TUNEL positive cells in the murine brain for the indicated treatments (Ve n = 9, Cle n = 12). Data are represented as mean ± SEM. ****P < 0.0001, two-tailed, nonparametric Mann-Whitney's U-test.


G . Representative micrographs of consecutive brain sections ( separating distance : 9 µm ) showing xenografts treated with vehicle or clemastine and labelled with the anti - human vimentin ( red in left panels ) and TUNEL ( red in right panels ) . Nuclei were visualised by using DAPI ( white ) . Scale bar : 50 µm .

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G. Representative micrographs of consecutive brain sections (separating distance: 9 µm) showing xenografts treated with vehicle or clemastine and labelled with the anti-human vimentin (red in left panels) and TUNEL (red in right panels). Nuclei were visualised by using DAPI (white). Scale bar: 50 µm.


H . Kaplan - Meier survival curve of the BT12 xenografted animals treated with vehicle ( green line , n = 15 ) and clemastine ( black line , n = 12 ) . Survival of the animals has been measured and pooled from two independent experiments . P = 0 . 044 , Mantel - Cox Log - rank test .

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H. Kaplan-Meier survival curve of the BT12 xenografted animals treated with vehicle (green line, n = 15) and clemastine (black line, n = 12). Survival of the animals has been measured and pooled from two independent experiments. P = 0.044, Mantel-Cox Log-rank test.


MIP of cerebral vessels of 3dpf Tg ( kdrl : HRAS - mCherry ) s916 embryo ( grey LUT ; inverted ) . Higher magnification panel showing two kugeln ( arrowheads ) arising from the middle mesencephalic central artery ( MMCtA ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

MIP of cerebral vessels of 3dpf Tg(kdrl:HRAS-mCherry)s916 embryo (grey LUT; inverted). Higher magnification panel showing two kugeln (arrowheads) arising from the middle mesencephalic central artery (MMCtA).


Diameter of kugeln at 3dpf ( mean ± s . e . m . 10 . 13 ± 0 . 49 ; n = 93 kugeln from 32 3dpf embryos ; 3 experimental repeats ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Diameter of kugeln at 3dpf (mean ± s.e.m. 10.13 ± 0.49; n=93 kugeln from 32 3dpf embryos; 3 experimental repeats).


Number of kugeln per embryo in 3 , 4 and 5dpf animals was not statistically significantly different ( mean ± s . e . m . 3dpf 5 . 47 ± 1 . 09 , 4dpf 7 . 47 ± 1 . 46 , 5dpf 7 . 09 ± 1 . 55 ; p = 0 . 8571 ; 3dpf : 175 kugeln from 32 embryos ; 4dpf : 239 kugeln from 32 embryos , 5dpf : 227 kugeln from 32 embryos ; 4 experimental repeats ; Kruskal - Wallis test ) .

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Number of kugeln per embryo in 3, 4 and 5dpf animals was not statistically significantly different (mean ± s.e.m. 3dpf 5.47 ± 1.09, 4dpf 7.47 ± 1.46, 5dpf 7.09 ± 1.55; p = 0.8571; 3dpf: 175 kugeln from 32 embryos; 4dpf: 239 kugeln from 32 embryos, 5dpf: 227 kugeln from 32 embryos; 4 experimental repeats; Kruskal-Wallis test).


Kugeln ( arrowheads ) could be observed in 28dpf animals .

Kugeln (arrowheads) could be observed in 28dpf animals.


MIP of cerebral vessels of 4dpf Tg ( fli1aep : CAAX - eGFP ) embryo ( grey LUT ; inverted ) . Higher magnification panel shows a kugel ( arrowhead ) protruding from the posterior mesencephalic central artery ( PMCtA ) .

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MIP of cerebral vessels of 4dpf Tg(fli1aep:CAAX-eGFP) embryo (grey LUT; inverted). Higher magnification panel shows a kugel (arrowhead) protruding from the posterior mesencephalic central artery (PMCtA).


Single Z - plane micrograph of cerebral vessels of a 3dpf Tg ( gata1 : dsRed ) , injected with a Tol2 - fli1a : myr - Cherry construct showing two kugeln ( arrowheads ) in the higher magnification panel protruding from the MMCtA .

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Single Z-plane micrograph of cerebral vessels of a 3dpf Tg(gata1:dsRed), injected with a Tol2-fli1a:myr-Cherry construct showing two kugeln (arrowheads) in the higher magnification panel protruding from the MMCtA.


Locations and proportion of kugeln on cerebral vessels ( colour coded by vessel ; total n = 107 kugeln from 34 4dpf embryos ; 3 experimental repeats ) . AMCtA - anterior mesencephalic central artery , BCA - basal communicating artery , CaDI - caudal division of internal carotid artery , CMV - communicating vessel , CtA - central arteries , MMCtA - middle mesencephalic central artery , MtA - metencephalic artery , PCS - posterior communicating segment , PMBC - posterior midbrain channel , PMCtA - posterior mesencephalic central artery , PrA - prosencephalic artery .

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Locations and proportion of kugeln on cerebral vessels (colour coded by vessel; total n=107 kugeln from 34 4dpf embryos; 3 experimental repeats). AMCtA - anterior mesencephalic central artery, BCA - basal communicating artery, CaDI - caudal division of internal carotid artery, CMV - communicating vessel, CtA - central arteries, MMCtA - middle mesencephalic central artery, MtA - metencephalic artery, PCS - posterior communicating segment, PMBC - posterior midbrain channel, PMCtA - posterior mesencephalic central artery, PrA - prosencephalic artery.


MIPs of a time - series light sheet acquisition shows three different kugeln ( kugel 1 - black arrowhead ; kugel 2 - grey arrowhead ; kugel 3 - unfilled arrowhead ) protruding and retracting from parent vessels ( 2min intervals ; inverted LUT ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

MIPs of a time-series light sheet acquisition shows three different kugeln (kugel 1 - black arrowhead; kugel 2 - grey arrowhead; kugel 3 - unfilled arrowhead) protruding and retracting from parent vessels (2min intervals; inverted LUT).


Kugeln persisted for variable durations before regression into the parent vessel ( 43 kugeln from 9 4dpf embyros ; 4 experimental repeats ) .

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Kugeln persisted for variable durations before regression into the parent vessel (43 kugeln from 9 4dpf embyros; 4 experimental repeats).


MIPs taken 20min apart showing examples of different kugel behaviour including shape changes ( asterisk ) , expansion ( circle ) , retraction ( triangle ) , or little change ( pentagon ) .

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MIPs taken 20min apart showing examples of different kugel behaviour including shape changes (asterisk), expansion (circle), retraction (triangle), or little change (pentagon).


MIPs taken 1h apart show that kugeln may be observed at one time - point ( red arrowhead , 55 : 20 : 00 ) but not 1h later in the same animal ( 56 : 20 : 00 ) and to develop on other vessels 1h later ( 57 : 20 : 00 ) which again have regressed by 58 : 20 : 00 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

MIPs taken 1h apart show that kugeln may be observed at one time-point (red arrowhead, 55:20:00) but not 1h later in the same animal (56:20:00) and to develop on other vessels 1h later (57:20:00) which again have regressed by 58:20:00.


Kymographs generated by line - scanning across the diameter of a typical kugel shows that kugel diameter oscillated with a periodicity of minutes , while no such oscillations were observed in adjacent similarly sized cerebral vessels ( all images grey LUT ; inverted ) .

Kymographs generated by line-scanning across the diameter of a typical kugel shows that kugel diameter oscillated with a periodicity of minutes, while no such oscillations were observed in adjacent similarly sized cerebral vessels (all images grey LUT; inverted).


Double transgenic visualizing endothelial membrane ( red ) and endothelial nuclei ( green ) . Endothelial nuclei ( arrowhead ) were observed close to but never within the kugel ( unfilled arrowhead ) .

Double transgenic visualizing endothelial membrane (red) and endothelial nuclei (green). Endothelial nuclei (arrowhead) were observed close to but never within the kugel (unfilled arrowhead).


Double transgenic showing endothelial membrane ( red ) and endothelial F - actin ( green ) . F - actin was found to localize at the neck of the

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Double transgenic showing endothelial membrane (red) and endothelial F-actin (green). F-actin was found to localize at the neck of the kugeln


Double transgenic showing endothelial membrane ( red ) and endothelial cytoplasm ( green ) . The cytoplasmic reporter was visible in the parent vessel but not in the kugel ( unfilled arrowhead ) .

Double transgenic showing endothelial membrane (red) and endothelial cytoplasm (green). The cytoplasmic reporter was visible in the parent vessel but not in the kugel (unfilled arrowhead).


Triple transgenic showing endothelial membrane ( red ) , neurons ( green ) and erythrocytes ( red ) . Surrounding neurons were excluded from the volume of the kugel ( unfilled arrowhead ) and erythrocytes were not observed inside kugeln ( arrowhead ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Triple transgenic showing endothelial membrane (red), neurons (green) and erythrocytes (red). Surrounding neurons were excluded from the volume of the kugel (unfilled arrowhead) and erythrocytes were not observed inside kugeln (arrowhead).


Treatment with the inhibitor of actin polymerization Latrunculin B statistically significantly increased number of kugeln per embryo ( 100nM 1 hour ; * * p = 0 . 0041 ; control n = 21 embryos 8 . 05 ± 1 . 76 ( mean ± s . e . m . ) ; Latrunculin n = 21 embryos 17 . 19 ± 2 . 93 ( mean ± s . e . m . ) ; 4dpf ; 3 experimental repeats ; Mann - Whitney U test ) .

O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Treatment with the inhibitor of actin polymerization Latrunculin B statistically significantly increased number of kugeln per embryo (100nM 1 hour; **p = 0.0041; control n=21 embryos 8.05 ± 1.76 (mean ± s.e.m.); Latrunculin n=21 embryos 17.19 ± 2.93 (mean ± s.e.m.); 4dpf; 3 experimental repeats; Mann-Whitney U test).


Latrunculin B treatment statistically significantly reduced kugel diameter ( * p = 0 . 0164 ; control n = 169 kugeln from 21 embryos 7 . 56 ± 2 . 25 ( mean ± s . e . m . ) ; Latrunculin n = 361 kugeln from 21 embryos 5 . 91 ± 1 . 78 ( mean ± s . e . m . ) ; 4dpf ; 3 experimental repeats ; Student ' s t - test ) .

B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Latrunculin B treatment statistically significantly reduced kugel diameter (*p = 0.0164; control n=169 kugeln from 21 embryos 7.56 ± 2.25 (mean ± s.e.m.); Latrunculin n=361 kugeln from 21 embryos 5.91 ± 1.78 (mean ± s.e.m.); 4dpf; 3 experimental repeats; Student's t-test).


Treatment with the Myosin II inhibitor Blebbistatin statistically significantly reduced number of kugeln per embryo ( 25 µM 1h ; * * * * p < 0 . 0001 ; control n = 22 embryos 3 . 77 ± 0 . 56 ( mean ± s . e . m . ) , Blebbistatin n = 24 embryos 1 . 08 ± 0 . 22 ( mean ± s . e . m . ) ; 3dpf ; 3 experimental repeats ; Mann - Whitney U test ) .

O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Treatment with the Myosin II inhibitor Blebbistatin statistically significantly reduced number of kugeln per embryo (25 µM 1h; ****p <0.0001; control n=22 embryos 3.77 ± 0.56 (mean ± s.e.m.), Blebbistatin n=24 embryos 1.08 ± 0.22 (mean ± s.e.m.); 3dpf; 3 experimental repeats; Mann-Whitney U test).


Blebbistatin treatment had no effect on kugel diameter ( p = 0 . 3731 ; control n = 83 kugeln from 22 embryos 6 . 27 ± 0 . 72 ( mean ± s . e . m . ) , Blebbistatin n = 26 kugeln from 24 embryos 7 . 02 ± 0 . 89 ( mean ± s . e . m . ) ; 3dpf ; 3 experimental repeats ; Mann - Whitney U test ) .

B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Blebbistatin treatment had no effect on kugel diameter (p = 0.3731; control n=83 kugeln from 22 embryos 6.27 ± 0.72 (mean ± s.e.m.), Blebbistatin n=26 kugeln from 24 embryos 7.02 ± 0.89 (mean ± s.e.m.); 3dpf; 3 experimental repeats; Mann-Whitney U test).


MIP of the cerebral vessels of a 4dpf embryo before exsanguination ( grey LUT ; inverted ) . MIP of the same embryo after exsanguination , which did not alter kugel size .

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MIP of the cerebral vessels of a 4dpf embryo before exsanguination (grey LUT; inverted). MIP of the same embryo after exsanguination, which did not alter kugel size.


Time - lapse imaging of an embryo with transiently halted cardiac contraction ( using Tricaine ) . Despite absent blood flow kugeln still changed shape ( grey arrowhead ) , retained shape ( white arrowhead ) or protruded and retracted ( black arrowhead ; time post cessation of flow is indicated on micrographs ; 3dpf ; grey inverted LUT ) . Time - lapse of an embryo with transiently halted cardiac contraction as in ( C ) showed that kugel diameter still oscillates ( time post cessation of flow ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Time-lapse imaging of an embryo with transiently halted cardiac contraction (using Tricaine). Despite absent blood flow kugeln still changed shape (grey arrowhead), retained shape (white arrowhead) or protruded and retracted (black arrowhead; time post cessation of flow is indicated on micrographs; 3dpf; grey inverted LUT). Time-lapse of an embryo with transiently halted cardiac contraction as in (C) showed that kugel diameter still oscillates (time post cessation of flow).


Dextran microangiography filled perfused vessels with dextran ( arrowhead ) , while dextran was not observed to enter kugeln ( unfilled arrowhead ) .

Dextran microangiography filled perfused vessels with dextran (arrowhead), while dextran was not observed to enter kugeln (unfilled arrowhead).


Inhibition of cardiac contraction by tnnt2a morpholino ( MO ) knockdown statistically significantly reduced kugel number per embryo ( * * * * p < 0 . 0001 ; control n = 20 embryos 5 . 10 ± 1 . 47 ( mean ± s . e . m . ) , tnnt2a MO = 18 embryos 0 . 06 ± 0 . 06 ( mean ± s . e . m . ) ; 3dpf ; 3 experimental repeats ; Mann - Whitney U test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Inhibition of cardiac contraction by tnnt2a morpholino (MO) knockdown statistically significantly reduced kugel number per embryo (****p<0.0001; control n=20 embryos 5.10 ± 1.47 (mean ± s.e.m.), tnnt2a MO=18 embryos 0.06 ± 0.06 (mean ± s.e.m.); 3dpf; 3 experimental repeats; Mann-Whitney U test).


Kugeln were studied in the double - transgenic Tg ( fli1a : LifeAct - mClover ) sh467 , Tg ( kdrl : HRAS - mCherry ) s916 . BLECs were mClover positive and mCherry - negative ( BLECs - black arrowheads ; kugeln - white arrowheads ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Kugeln were studied in the double-transgenic Tg(fli1a:LifeAct-mClover)sh467, Tg(kdrl:HRAS-mCherry)s916. BLECs were mClover positive and mCherry-negative (BLECs - black arrowheads; kugeln - white arrowheads).


No direct physical interaction between kugeln ( BLECs - black arrowheads ; kugeln - white arrowheads ) and BLECs was observed ( n = 21 4dpf embryos ; 3 experimental repeats ) . Depth - coded MIPs showed that BLECs and kugeln are present on different anatomical planes ( depths ; purple ventral ( v ) , white dorsal ( d ) ; BLECs - black arrowheads ; kugeln - white arrowheads ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

No direct physical interaction between kugeln (BLECs - black arrowheads; kugeln - white arrowheads) and BLECs was observed (n=21 4dpf embryos; 3 experimental repeats). Depth-coded MIPs showed that BLECs and kugeln are present on different anatomical planes (depths; purple ventral (v), white dorsal (d); BLECs - black arrowheads; kugeln - white arrowheads).


Ccbe1 morpholino ( MO ) injection led to a loss of lymphatics ( white arrowhead ) .

Ccbe1 morpholino (MO) injection led to a loss of lymphatics (white arrowhead).


Kugel number was not statistically significantly altered by ccbe1 morpholino ( MO ) knockdown ( p = 0 . 3496 ; control MO n = 22 embryos 0 . 95 ± 0 . 28 ( mean ± s . e . m . ) , ccbe1 MO n = 23 embryos 0 . 57 ± 0 . 14 ( mean ± s . e . m . ) ; 3dpf ; 3 experimental repeats ; Mann - Whitney U test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Kugel number was not statistically significantly altered by ccbe1 morpholino (MO) knockdown (p = 0.3496; control MO n=22 embryos 0.95 ± 0.28 (mean ± s.e.m.), ccbe1 MO n=23 embryos 0.57 ± 0.14 (mean ± s.e.m.); 3dpf; 3 experimental repeats; Mann-Whitney U test).


Kugel diameter was not statistically significantly altered by ccbe1 MO knockdown ( p = 0 . 8783 ; control MO n = 21 kugeln from 22 embryos 7 . 75 ± 1 . 29 ( mean ± s . e . m . ) , ccbe1 MO n = 13 kugeln from 23 embryos 8 . 93 ± 2 . 35 ( mean ± s . e . m . ) ; 3dpf ; 3 experimental repeats ; Mann - Whitney U test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Kugel diameter was not statistically significantly altered by ccbe1 MO knockdown (p = 0.8783; control MO n=21 kugeln from 22 embryos 7.75 ± 1.29 (mean ± s.e.m.), ccbe1 MO n=13 kugeln from 23 embryos 8.93 ± 2.35 (mean ± s.e.m.); 3dpf; 3 experimental repeats; Mann-Whitney U test).


Injection of IgG - conjugated Alexa 647 into the tectum showed no uptake ( blue ; unfilled arrowhead ) by kugeln ( white arrowhead ; 140 kugeln from 17 4dpf embryos ; 2 experimental repeats ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Injection of IgG-conjugated Alexa 647 into the tectum showed no uptake (blue; unfilled arrowhead) by kugeln (white arrowhead; 140 kugeln from 17 4dpf embryos; 2 experimental repeats).


The possible interaction of macrophages with kugeln was studied in the transgenic Tg ( kdrl : HRAS - mCherry ) s916 , Tg ( fms : GAL4 . VP16 ) i186 , Tg ( UAS - E1b : nfsB . mCherry ) il149 , visualizing macrophages and the vasculature simultaneously . Coloured depth - coding indicates the different anatomical depths of kugeln and macrophages ( ventral purple , dorsal white ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

The possible interaction of macrophages with kugeln was studied in the transgenic Tg(kdrl:HRAS-mCherry)s916, Tg(fms:GAL4.VP16)i186, Tg(UAS-E1b:nfsB.mCherry)il149, visualizing macrophages and the vasculature simultaneously. Coloured depth-coding indicates the different anatomical depths of kugeln and macrophages (ventral purple, dorsal white).


Maximum intensity projections of z - stacks acquired every 10 minutes during a time - lapse showed no interaction of macrophages ( unfilled arrowhead ) with kugeln ( filled arrowhead ; n = 21 kugeln in 8 3dpf embryos ) . Macrophages are all white , indicating they are on a different z - plane to the vessels seen .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Maximum intensity projections of z-stacks acquired every 10 minutes during a time-lapse showed no interaction of macrophages (unfilled arrowhead) with kugeln (filled arrowhead; n=21 kugeln in 8 3dpf embryos). Macrophages are all white, indicating they are on a different z-plane to the vessels seen.


VEGF inhibition by 2h treatment with AV951 statistically significantly increased kugel number ( * * * * p < 0 . 0001 ; DMSO control n = 30 embryos 8 . 53 ± 2 . 62 ( mean ± s . e . m . ) ; AV951 n = 31 embryos 21 . 10 ± 2 . 71 ( mean ± s . e . m . ) ; 4dpf ; 4 experimental repeats ; Mann - Whitney U test ) .

O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

VEGF inhibition by 2h treatment with AV951 statistically significantly increased kugel number (****p<0.0001; DMSO control n=30 embryos 8.53 ± 2.62 (mean ± s.e.m.); AV951 n=31 embryos 21.10 ± 2.71 (mean ± s.e.m.); 4dpf; 4 experimental repeats; Mann-Whitney U test).


Mean kugel diameter was not statistically significantly different after AV951 treatment ( p = 0 . 7890 ; DMSO control n = 243 kugeln from 30 embryos 9 . 94 ± 0 . 76 ( mean ± s . e . m . ) ; AV951 n = 652 kugeln from 31 embryos 9 . 73 ± 0 . 32 ( mean ± s . e . m . ) ; 4dpf ; 4 experimental repeats ; Student ' s t - test ) .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Mean kugel diameter was not statistically significantly different after AV951 treatment (p = 0.7890; DMSO control n=243 kugeln from 30 embryos 9.94 ± 0.76 (mean ± s.e.m.); AV951 n=652 kugeln from 31 embryos 9.73 ± 0.32 (mean ± s.e.m.); 4dpf; 4 experimental repeats; Student's t-test).


DAF - FM staining , a vital dye for nitric oxide ( NO ) , showed that 57 . 56 % of kugeln were positive for nitric oxide reactivity ( 2 . 5µM incubation from 96 - 102hpf ; n = 22 4dpf embryos ; 118 of 205 kugeln filled ; 3 experimental repeats ) . Images show representative " filled " ( DAF - FM positive ) and " unfilled " ( DAF - FM negative ) kugeln .

O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

DAF-FM staining, a vital dye for nitric oxide (NO), showed that 57.56% of kugeln were positive for nitric oxide reactivity (2.5µM incubation from 96-102hpf; n=22 4dpf embryos; 118 of 205 kugeln filled; 3 experimental repeats). Images show representative "filled" (DAF-FM positive) and "unfilled" (DAF-FM negative) kugeln.


Time - lapse acquisition with DAF - FM revealed that kugeln contained NO early in their biogenesis ( grey LUT ; inverted ) .

O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O

Time-lapse acquisition with DAF-FM revealed that kugeln contained NO early in their biogenesis (grey LUT; inverted).


Application of LysoTracker , a vital dye that stains lysosomes or acidic compartments , showed that 17 . 08 % of kugeln contained acidic contents ( 8 . 33µM ; 96 - 101hpf ; n = 22 4dpf embryos ; 62 of 363 kugeln filled ; 3 experimental repeats ) . Images show representative " filled " ( Lysotracker positive ) and " unfilled " ( Lysotracker negative ) kugeln .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Application of LysoTracker, a vital dye that stains lysosomes or acidic compartments, showed that 17.08% of kugeln contained acidic contents (8.33µM; 96-101hpf; n=22 4dpf embryos; 62 of 363 kugeln filled; 3 experimental repeats). Images show representative "filled" (Lysotracker positive) and "unfilled" (Lysotracker negative) kugeln.


The nitric oxide synthase ( NOS ) inhibitor L - NAME had no statistically significant effect on kugel number per embryo ( 0 . 5mM L - NAME 18h ; p = 0 . 4870 ; control n = 22 embryos 14 . 27 ± 3 . 35 ( mean ± s . e . m . ) , L - NAME n = 24 embryos 7 . 46 ± 1 . 61 ( mean ± s . e . m . ) ; 4dpf ; 3 experimental repeats ; Mann - Whitney U test ) .

O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

The nitric oxide synthase (NOS) inhibitor L-NAME had no statistically significant effect on kugel number per embryo (0.5mM L-NAME 18h; p = 0.4870; control n=22 embryos 14.27 ± 3.35 (mean ± s.e.m.), L-NAME n=24 embryos 7.46 ± 1.61 (mean ± s.e.m.); 4dpf; 3 experimental repeats; Mann-Whitney U test).


Diameter of kugeln was not affected by L - NAME ( p = 0 . 4161 ; control n = 315 kugeln from 22 embryos 8 . 77 ± 0 . 49 ( mean ± s . e . m . ) , L - NAME n = 179 kugeln from 24 8 . 66 ± 0 . 71 ( mean ± s . e . m . ) ; 4dpf ; 3 experimental repeats ; Mann - Whitney U test ) .

O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Diameter of kugeln was not affected by L-NAME (p = 0.4161; control n=315 kugeln from 22 embryos 8.77 ± 0.49 (mean ± s.e.m.), L-NAME n=179 kugeln from 24 8.66 ± 0.71 (mean ± s.e.m.); 4dpf; 3 experimental repeats; Mann-Whitney U test).


Kugel number was significantly decreased by inhibition of Notch signalling by 12h treatment with 50µM DAPT ( * * * * p < 0 . 0001 ; control n = 24 embryos 9 . 38 ± 1 . 77 ( mean ± s . e . m . ) , DAPT n = 24 embryos 0 . 92 ± 0 . 28 ( mean ± s . e . m . ) ; 4dpf ; 3 experimental repeats ; Mann - Whitney U test ) .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Kugel number was significantly decreased by inhibition of Notch signalling by 12h treatment with 50µM DAPT (****p<0.0001; control n=24 embryos 9.38 ± 1.77 (mean ± s.e.m.), DAPT n=24 embryos 0.92 ± 0.28 (mean ± s.e.m.); 4dpf; 3 experimental repeats; Mann-Whitney U test).


Mean kugel diameter was not statistically significantly altered by DAPT treatment ( p = 0 . 0832 ; control n = 225 kugeln from 24 embryos 8 . 64 ± 0 . 54 ( mean ± s . e . m . ) ; DAPT n = 22 kugeln from 24 embryos 6 . 88 ± 0 . 86 ( mean ± s . e . m . ) ; 4dpf ; 3 experimental repeats ; Student ' s t - test ) .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Mean kugel diameter was not statistically significantly altered by DAPT treatment (p = 0.0832; control n=225 kugeln from 24 embryos 8.64 ± 0.54 (mean ± s.e.m.); DAPT n=22 kugeln from 24 embryos 6.88 ± 0.86 (mean ± s.e.m.); 4dpf; 3 experimental repeats; Student's t-test).


A transgenic dll4 reporter line showed no indication of different dll4 expression at the sites of kugeln ( white arrowheads pointing to kugeln ; n = 122 kugeln from 23 3dpf embryos ; 2 experimental repeats ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A transgenic dll4 reporter line showed no indication of different dll4 expression at the sites of kugeln (white arrowheads pointing to kugeln; n=122 kugeln from 23 3dpf embryos; 2 experimental repeats).


Studying Notch signalling in the Notch reporter line Tg ( TP1glob : venusPest ) s940 , showed high Notch levels in the mid - brain , but , again , no difference in expression at the sites of kugeln ( n = 45 kugeln from 19 4dpf embryos ; 2 experimental repeats ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Studying Notch signalling in the Notch reporter line Tg(TP1glob:venusPest)s940, showed high Notch levels in the mid-brain, but, again, no difference in expression at the sites of kugeln (n=45 kugeln from 19 4dpf embryos; 2 experimental repeats).


Kugel number was statistically significantly increased by inhibition of Wnt signalling by 4h treatment with 10µm XAV - 939 ( * * * p = 0 . 0003 ; control n = 22 embryos 1 . 32 ± 0 . 24 ( mean ± s . e . m . ) ; XAV - 939 n = 21 embryos 4 . 29 ± 0 . 71 ( mean ± s . e . m . ) ; 3dpf ; 3 experimental repeats ; Mann - Whitney U test ) .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Kugel number was statistically significantly increased by inhibition of Wnt signalling by 4h treatment with 10µm XAV-939 (***p = 0.0003; control n=22 embryos 1.32 ± 0.24 (mean ± s.e.m.); XAV-939 n=21 embryos 4.29 ± 0.71 (mean ± s.e.m.); 3dpf; 3 experimental repeats; Mann-Whitney U test).


Kugel diameter was not statistically significantly altered by XAV - 939 ( p = 0 . 4098 ; control n = 29 kugeln from 22 embryos 8 . 78 ± 0 . 76 ( mean ± s . e . m . ) ; XAV - 939 n = 90 kugeln from 21 embryos 7 . 99 ± 0 . 56 ( mean ± s . e . m . ) ; 3dpf ; 3 experimental repeats ; Student ' s t - test )

O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Kugel diameter was not statistically significantly altered by XAV-939 (p = 0.4098; control n=29 kugeln from 22 embryos 8.78 ± 0.76 (mean ± s.e.m.); XAV-939 n=90 kugeln from 21 embryos 7.99 ± 0.56 (mean ± s.e.m.); 3dpf; 3 experimental repeats; Student's t-test)


Kugel number was statistically significantly increased by activation of Wnt signalling by 4h treatment with 10µm GSK3 inhibition XV ( p 0 . 0359 ; control n = 22 embryos 1 . 04 ± 0 . 34 ( mean ± s . e . m . ) ; GSK3 inhibitor n = 21 embryos 4 . 24 ± 1 . 28 ( mean ± s . e . m . ) ; 3dpf ; 3 experimental repeats ; Mann - Whitney U test ) .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Kugel number was statistically significantly increased by activation of Wnt signalling by 4h treatment with 10µm GSK3 inhibition XV (p 0.0359; control n=22 embryos 1.04 ± 0.34 (mean ± s.e.m.); GSK3 inhibitor n=21 embryos 4.24 ± 1.28 (mean ± s.e.m.); 3dpf; 3 experimental repeats; Mann-Whitney U test).


The diameter of kugel was not statistically significantly altered by GSK3 inhibition ( p 0 . 5555 ; control n = 23 kugeln from 22 embryos 7 . 26 ± 0 . 89 ( mean ± s . e . m . ) ; GSK3 inhibitor n = 89 kugeln from 21 embryos 8 . 09 ± 0 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

The diameter of kugel was not statistically significantly altered by GSK3 inhibition (p 0.5555; control n=23 kugeln from 22 embryos 7.26 ± 0.89 (mean ± s.e.m.); GSK3 inhibitor n=89 kugeln from 21 embryos 8.09 ± 0.97


Double fluorescent whole mount in situ hybridization ( WM - ISH ) to detect her6 ( green ) and her9 ( magenta ) ; coronal view ( left panel ) and transversal section ( right panel ) , scale bar 20μm ; 30 - 32hpf ; annotations denote anterior ( A ) , posterior ( P ) , otic vesicle ( ov ) , dorsal ( D ) and ventral ( V ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Double fluorescent whole mount in situ hybridization (WM-ISH) to detect her6 (green) and her9 (magenta); coronal view (left panel) and transversal section (right panel), scale bar 20μm; 30-32hpf; annotations denote anterior (A), posterior (P), otic vesicle (ov), dorsal (D) and ventral (V).


Representative time series example of Her6 : : Venus expression during development , in the midbrain and hindbrain . Confocal images represented as 2D Maximum projection ; longitudinal view ; scale bar 50μm ; otic vesicle ( ov ) ; also included in Movie EV1 . r1 : rhombomere 1 , r2 : rhombomere 2 , r3 : rhombomere 3 , r4 : rhombomere 4 , r5 : rhombomere 5 , r6 : rhombomere 6 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative time series example of Her6::Venus expression during development, in the midbrain and hindbrain. Confocal images represented as 2D Maximum projection; longitudinal view; scale bar 50μm; otic vesicle (ov); also included in Movie EV1. r1: rhombomere 1, r2: rhombomere 2, r3: rhombomere 3, r4: rhombomere 4, r5: rhombomere 5, r6: rhombomere 6.


Intensity mean of Her6 : : Venus per rhombomere area over development grouped by expression level , related to the r1 - r6 regions in panel ( c ) : ( d ) r1 and r2 ; ( e ) r3 and r4 ; ( f ) r5 and r6 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Intensity mean of Her6::Venus per rhombomere area over development grouped by expression level, related to the r1-r6 regions in panel (c) : (d) r1 and r2; (e) r3 and r4; (f) r5 and r6.


Transversal view of r6 in Her6 : : Venus embryos over time ; Her6 : : Venus protein expression domains : a ventral domain ( arrows ) and a more dorsal lateral domain ( arrowheads ) ; the caax - mRFP was used as membrane marker ( magenta ) ; scale bars 20μm ; images at 30 - 40hpf are maximum projection of 4 z - stacks from Movie EV2 .

Transversal view of r6 in Her6::Venus embryos over time; Her6::Venus protein expression domains: a ventral domain (arrows) and a more dorsal lateral domain (arrowheads); the caax-mRFP was used as membrane marker (magenta); scale bars 20μm; images at 30-40hpf are maximum projection of 4 z-stacks from Movie EV2.


Quantification of Her6 : : Venus ( + ) cell number ( green ) compared to total cell number ( black ) over development .

O O O O O O O O O O O O O O O O O O O O O O O O O

Quantification of Her6::Venus(+) cell number (green) compared to total cell number (black) over development.


Proportional changes in Her6 : : Venus ( + ) cell numbers during development ; bars indicate median and interquartile range of counts collected from 3 different z - stacks per embryo and one embryo per condition ; one - way ANOVA with Bonferroni multiple comparison test with significance p < 0 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Proportional changes in Her6::Venus(+) cell numbers during development; bars indicate median and interquartile range of counts collected from 3 different z-stacks per embryo and one embryo per condition; one-way ANOVA with Bonferroni multiple comparison test with significance p<0.01


Nuclear abundance and nuclear concentration of Her6 : : Venus protein in homozygous embryos at different stages during development measured by fluorescence correlation spectroscopy ( FCS ) ; bars indicate median and interquartile range of ( 19hpf : 6 embryos , 50 cells ) , ( 29hpf : 6 embryos , 86 cells ) , ( 33hpf : 5 embryos , 76 cells ) , ( 35hpf : 6 embryos , 59 cells ) and ( 48hpf : 6 embryos , 89 cells ) ; Kruskal - Wallis with Dunn ' s multiple comparison test , significance p < 0 . 05 * , p < 0 . 001 * * * , p < 0 . 0001 * * * * .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Nuclear abundance and nuclear concentration of Her6::Venus protein in homozygous embryos at different stages during development measured by fluorescence correlation spectroscopy (FCS); bars indicate median and interquartile range of (19hpf: 6 embryos, 50 cells), (29hpf: 6 embryos, 86 cells), (33hpf: 5 embryos, 76 cells), (35hpf: 6 embryos, 59 cells) and (48hpf: 6 embryos, 89 cells); Kruskal-Wallis with Dunn's multiple comparison test, significance p<0.05*, p<0.001***, p<0.0001****.


3D confocal representative images of a single neural progenitor cell ( red arrow ) tracked over time , data starting from 34hpf ; scale bar 10μm , images from Movie EV3 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

3D confocal representative images of a single neural progenitor cell (red arrow) tracked over time, data starting from 34hpf; scale bar 10μm, images from Movie EV3.


Single cell time series of Her6 : : Venus , mKeima - H2B and Her6 : : Venus signal normalized by mKeima - H2B corresponding to the cell in ( c ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Single cell time series of Her6::Venus, mKeima-H2B and Her6::Venus signal normalized by mKeima-H2B corresponding to the cell in (c).


Chromogenic WM - ISH of miR - 9 using miR - 9 LNA 5 ' - Dig observed at different stages during development ; longitudinal view , anterior to the left .

Chromogenic WM-ISH of miR-9 using miR-9 LNA 5'-Dig observed at different stages during development; longitudinal view, anterior to the left.


Transverse section of double fluorescent WM - ISH for her6 ( green ) and mir - 9 - 4 ( magenta ) imaged in fixed embryo at 31hpf ; scale bar 30μm .

Transverse section of double fluorescent WM-ISH for her6 (green) and mir-9-4 (magenta) imaged in fixed embryo at 31hpf; scale bar 30μm.


Confocal imaging of CTRL and MBSm embryo showing Her6 : : Venus expression in the hindbrain , rhombomeres 3 to 6 ( r3 - r6 ) over the course of development ; longitudinal view , scale 30μm , otic vesicle ( ov ) . Images are representing 2D maximum projection .

Confocal imaging of CTRL and MBSm embryo showing Her6::Venus expression in the hindbrain, rhombomeres 3 to 6 (r3-r6) over the course of development; longitudinal view, scale 30μm, otic vesicle (ov). Images are representing 2D maximum projection.


Her6 : : Venus expression ( red arrows ) in hindbrain , rhombomere 6 of live CTRL and MBSm embryos at 52hpf ; edge of ventricular zone shown in yellow ; transversal view ; scale 30μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Her6::Venus expression (red arrows) in hindbrain, rhombomere 6 of live CTRL and MBSm embryos at 52hpf; edge of ventricular zone shown in yellow; transversal view; scale 30μm.