F . Thermal denaturation profiles of MB , in the absence ( solid line ) and presence ( dashed line ) of increasing target concentration . Each melting curve represents the average of three separate replicates . Yellow boxes indicate the range of working temperatures for ex vivo trafficking experiments . Abbreviations : MB , molecular beacon ; Tm , MB melting temperature .

O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Thermal denaturation profiles of MB, in the absence (solid line) and presence (dashed line) of increasing target concentration. Each melting curve represents the average of three separate replicates. Yellow boxes indicate the range of working temperatures for ex vivo trafficking experiments. Abbreviations: MB, molecular beacon; Tm, MB melting temperature.


H . Quantification of the expression levels of pre - mir - 181a - 1 using the 2 - ΔCt method and U6 as normalizer from small total RNA fraction ( < ~ 150 nt ) . Each data point corresponds to one independent experiment . n = 3 independent experiments . Values are mean ± SEM . Abbreviations : ns , not significant .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O 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 expression levels of pre-mir-181a-1 using the 2-ΔCt method and U6 as normalizer from small total RNA fraction (<~150 nt). Each data point corresponds to one independent experiment. n=3 independent experiments. Values are mean ± SEM. Abbreviations: ns, not significant.


I . Total number of MB puncta normalized to axon length ( µm ) . Each data point corresponds to one axon . Total number of puncta and axons analyzed : 928 puncta , 61 axons ( WT ) , 226 puncta , 15 axons ( co - MO ) , 208 puncta , 35 axons ( MO ) . n = 4 independent experiments . Values are mean ± SEM . Abbreviations : ns , not significant ; co - MO , control morpholino ; pri - miR - MO , morpholino blocking pre - miR - 181a - 1 processing by targeting the Drosha cleavage site .

O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O O B-SMALL_MOLECULE O O O O O O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O

I. Total number of MB puncta normalized to axon length (µm). Each data point corresponds to one axon. Total number of puncta and axons analyzed: 928 puncta, 61 axons (WT), 226 puncta, 15 axons (co-MO), 208 puncta, 35 axons (MO). n=4 independent experiments. Values are mean ± SEM. Abbreviations: ns, not significant; co-MO, control morpholino; pri-miR-MO, morpholino blocking pre-miR-181a-1 processing by targeting the Drosha cleavage site.


J . Representative axons . MB puncta are indicated ( white arrows ) . Dashed white lines delineate axons . Abbreviations : MB , molecular beacon . Scale bars : 5µm .

O O O B-SUBCELLULAR O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O

J. Representative axons. MB puncta are indicated (white arrows). Dashed white lines delineate axons. Abbreviations: MB, molecular beacon. Scale bars: 5µm.


M . Frequency ( in percentage ) of puncta colocalization between MB and cy5 - pre - miR - 181a - 1 ( # MB + / pre - miR + ) . Each data point corresponds to one axon . Total number of puncta and axons analyzed : 354 puncta , 32 axons ( MB ) , 337 puncta , 32 axons ( cy5 - pre - miR - 181a - 1 ) . n = 5 independent experiments . Values are mean ± SEM . Abbreviations : MB , molecular beacon .

O O O O O O O O O B-EXP_ASSAY O O O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O

M. Frequency (in percentage) of puncta colocalization between MB and cy5-pre-miR-181a-1 (#MB+/pre-miR+). Each data point corresponds to one axon. Total number of puncta and axons analyzed: 354 puncta, 32 axons (MB), 337 puncta, 32 axons (cy5-pre-miR-181a-1). n=5 independent experiments. Values are mean ± SEM. Abbreviations: MB, molecular beacon.


N . Representative image of RGC axons . White , red and blue arrows indicate , respectively , co - localized , single MB , and single pre - miR - 181a - 1 puncta . Scale bars : 5µm .

O O O O O B-CELL_TYPE B-SUBCELLULAR O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O

N. Representative image of RGC axons. White, red and blue arrows indicate, respectively, co-localized, single MB, and single pre-miR-181a-1 puncta. Scale bars: 5µm.


B . Representative image of a single distal RGC axon from MB - electroporated retina . Growth cone wrist and central domain are indicated with white arrow and star , respectively . Dashed white line delineates the axon . Abbreviations : MB , molecular beacon . Scale bars : 5µm . C . Illustrative kymograph . Scale bars : 5µm .

O O O O O O O O B-CELL_TYPE B-SUBCELLULAR O O O B-EXP_ASSAY B-TISSUE O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O

B. Representative image of a single distal RGC axon from MB-electroporated retina. Growth cone wrist and central domain are indicated with white arrow and star, respectively. Dashed white line delineates the axon. Abbreviations: MB, molecular beacon. Scale bars: 5µm. C. Illustrative kymograph. Scale bars: 5µm.


D . Frequency distribution ( in percentage ) of MB ( endo ) and cy3 - pre - miR - 181a - 1 ( exo ) puncta along the RGC axon shaft . Each data point corresponds to one independent experiment . Total number of puncta and axons analyzed : 353 puncta , 20 axons ( endo ) , 484 puncta , 29 axons ( exo ) . n = 3 ( endo ) , n = 4 ( exo ) independent experiments . Values are mean ± SEM . Abbreviations : MB , molecular beacon ; ns , not significant .

O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-CELL_TYPE B-SUBCELLULAR O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O 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. Frequency distribution (in percentage) of MB (endo) and cy3-pre-miR-181a-1 (exo) puncta along the RGC axon shaft. Each data point corresponds to one independent experiment. Total number of puncta and axons analyzed: 353 puncta, 20 axons (endo), 484 puncta, 29 axons (exo). n=3 (endo), n=4 (exo) independent experiments. Values are mean ± SEM. Abbreviations: MB, molecular beacon; ns, not significant.


E . Average velocity of MB ( endo ) and cy3 - pre - miR - 181a - 1 ( exo ) puncta . Each data point corresponds to one punctum . Total number of puncta and axons analyzed : 353 puncta , 20 axons ( endo ) , 484 puncta , 29 axons ( exo ) . n = 3 ( endo ) , n = 4 ( exo ) independent experiments . Values are median with interquartile range . Abbreviations : MB , molecular beacon ; ns , not significant ; antero , anterograde movement ; retro , retrograde movement .

O O O B-EXP_ASSAY O O O O O O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O 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. Average velocity of MB (endo) and cy3-pre-miR-181a-1 (exo) puncta. Each data point corresponds to one punctum. Total number of puncta and axons analyzed: 353 puncta, 20 axons (endo), 484 puncta, 29 axons (exo). n=3 (endo), n=4 (exo) independent experiments. Values are median with interquartile range. Abbreviations: MB, molecular beacon; ns, not significant; antero, anterograde movement; retro, retrograde movement.


F . MSD data for MB ( endo ) and cy3 - pre - miR - 181a - 1 ( exo ) tracked particles were fitted with an anomalous diffusion model and α thus calculated ( red ) . Total number of particles and axons analyzed : 67 particles , 20 axons ( endo ) , 82 particles , 29 axons ( exo ) . n = 3 ( endo ) , n = 4 ( exo ) independent experiment . Values are mean ± SEM . Abbreviations : MB , molecular beacon .

O O B-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O 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-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O O O O O O O O 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. MSD data for MB (endo) and cy3-pre-miR-181a-1 (exo) tracked particles were fitted with an anomalous diffusion model and α thus calculated (red). Total number of particles and axons analyzed: 67 particles, 20 axons (endo), 82 particles, 29 axons (exo). n=3 (endo), n=4 (exo) independent experiment. Values are mean ± SEM. Abbreviations: MB, molecular beacon.


G . MSD alpha coefficient distribution for each single MB ( endo ) and cy3 - pre - miR - 181a - 1 ( exo ) tracked particle . Each data point corresponds to one particle . Total number of particles and axons analyzed : 67 particles , 20 axons ( endo ) , 82 particles , 29 axons ( exo ) . n = 3 ( endo ) , n = 4 ( exo ) independent experiments . Values are median with interquartile range .

O O B-EXP_ASSAY O O O O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O O 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. MSD alpha coefficient distribution for each single MB (endo) and cy3-pre-miR-181a-1 (exo) tracked particle. Each data point corresponds to one particle. Total number of particles and axons analyzed: 67 particles, 20 axons (endo), 82 particles, 29 axons (exo). n=3 (endo), n=4 (exo) independent experiments. Values are median with interquartile range.


H . Relative frequency distribution ( percentage ) of MB ( endo ) and cy3 - pre - miR - 181a - 1 ( exo ) tracked particles . Each data point corresponds to one independent experiment . Total number of particles and axons analyzed : 67 particles , 20 axons ( endo ) , 82 particles , 29 axons ( exo ) . n = 3 ( endo ) , n = 4 ( exo ) independent experiments . Values are mean ± SEM . Abbreviations : MB , molecular beacon ; ns , not significant .

O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O 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. Relative frequency distribution (percentage) of MB (endo) and cy3-pre-miR-181a-1 (exo) tracked particles. Each data point corresponds to one independent experiment. Total number of particles and axons analyzed: 67 particles, 20 axons (endo), 82 particles, 29 axons (exo). n=3 (endo), n=4 (exo) independent experiments. Values are mean ± SEM. Abbreviations: MB, molecular beacon; ns, not significant.


J . Representative kymographs before Nocodazole ( top panel ; - Noco ) and 30 min after 2 . 4μM Noco bath - application ( bottom panel ; + Noco ) . Stationary puncta in both panels are indicated with black arrows . Scale bars : 5µm .

O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O

J. Representative kymographs before Nocodazole (top panel; - Noco) and 30 min after 2.4μM Noco bath-application (bottom panel; +Noco). Stationary puncta in both panels are indicated with black arrows. Scale bars: 5µm.


K . Frequency distribution ( in percentage ) of MB punctum speed . Each data point corresponds to one independent experiment . Total number of puncta and axons analyzed : 358 puncta , 25 axons ( - Noco ) ; 503 puncta , 34 axons ( + Noco ) . n = 3 independent experiments . Values are mean ± SEM . Abbreviations : MB , molecular beacon ; ns , not significant ; Noco , nocodazole .

O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O B-SMALL_MOLECULE O O O O O O B-SUBCELLULAR O O B-SMALL_MOLECULE O O 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-SMALL_MOLECULE O B-SMALL_MOLECULE O

K. Frequency distribution (in percentage) of MB punctum speed. Each data point corresponds to one independent experiment. Total number of puncta and axons analyzed: 358 puncta, 25 axons (-Noco); 503 puncta, 34 axons (+Noco). n=3 independent experiments. Values are mean ± SEM. Abbreviations: MB, molecular beacon; ns, not significant; Noco, nocodazole.


L . Frequency distribution of MB punctum speed . Each data point corresponds to one punctum . Total number of puncta and axons analyzed : 358 puncta , 25 axons ( - Noco ) , 503 puncta , 34 axons ( + Noco ) . n = 3 independent experiments . Values are median with interquartile range . Abbreviations : MB , molecular beacon ; ns , not significant ; Noco , nocodazole .

O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O B-SMALL_MOLECULE O O O O O O B-SUBCELLULAR O O B-SMALL_MOLECULE O O O 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-SMALL_MOLECULE O B-SMALL_MOLECULE O

L. Frequency distribution of MB punctum speed. Each data point corresponds to one punctum. Total number of puncta and axons analyzed: 358 puncta, 25 axons (-Noco), 503 puncta, 34 axons (+Noco). n=3 independent experiments. Values are median with interquartile range. Abbreviations: MB, molecular beacon; ns, not significant; Noco, nocodazole.


B . Representative axon where MB - labeled pre - miR - 181a - 1 ( red ) and CD63 - eGFP - labeled vesicles ( green ) are co - trafficked ( white arrows ) . Abbreviations : MB , molecular beacon . Scale bars : 5µm .

O O O B-SUBCELLULAR O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR 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. Representative axon where MB-labeled pre-miR-181a-1 (red) and CD63-eGFP-labeled vesicles (green) are co-trafficked (white arrows). Abbreviations: MB, molecular beacon. Scale bars: 5µm.


C . Representative kymographs . Abbreviations : CD63 , CD63 - eGFP ; MB , molecular beacon . Scale bars : 5µm .

O O O B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O

C. Representative kymographs. Abbreviations: CD63, CD63-eGFP; MB, molecular beacon. Scale bars: 5µm.


D . Composite kymograph shown in ( C ) where the individual traces where drawn and color coded . Yellow trajectories represent co - trafficking MB - labeled pre - miRNA ( red ) and CD63 - eGFP - labeled vesicle ( green ) . Abbreviations : MB , molecular beacon . Scale bars : 5µm .

O O O B-EXP_ASSAY O O O O O O O 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-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O O O O O O O

D. Composite kymograph shown in (C) where the individual traces where drawn and color coded. Yellow trajectories represent co-trafficking MB-labeled pre-miRNA (red) and CD63-eGFP-labeled vesicle (green). Abbreviations: MB, molecular beacon. Scale bars: 5µm.


E . Representative time - lapse depicting MB - labeled pre - miR - 181a - 1 ( red arrow ) and CD63 - eGFP - positive vesicle ( green arrow ) co - trafficked along the axon shaft to the growth cone ( delineated with dashed white lines ) wrist ( white arrowhead ) and central domain ( white star ) . Scale bars : 5µm .

O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR O 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. Representative time-lapse depicting MB-labeled pre-miR-181a-1 (red arrow) and CD63-eGFP-positive vesicle (green arrow) co-trafficked along the axon shaft to the growth cone (delineated with dashed white lines) wrist (white arrowhead) and central domain (white star). Scale bars: 5µm.


F . Frequency ( in percentage ) of MB - labeled pre - miR - 181a - 1 co - trafficked with CD63 - eGFP - positive vesicles . Each data point corresponds to one axon . Total number of puncta and axons analyzed : 253 puncta , 22 axons ( MB + ) , 306 puncta , 22 axons ( CD63 + ) . n = 5 independent experiments . Values are mean ± SEM . Abbreviations : CD63 , CD63 - eGFP ; MB , molecular beacon .

O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O

F. Frequency (in percentage) of MB-labeled pre-miR-181a-1 co-trafficked with CD63-eGFP-positive vesicles. Each data point corresponds to one axon. Total number of puncta and axons analyzed: 253 puncta, 22 axons (MB+), 306 puncta, 22 axons (CD63+). n=5 independent experiments. Values are mean ± SEM. Abbreviations: CD63, CD63-eGFP; MB, molecular beacon.


G . Frequency distribution ( in percentage ) of MB - labeled pre - miR - 181a - 1 co - trafficked with CD63 - eGFP - positive vesicles . Each data point corresponds to one axon . Total number of puncta and axons analyzed : 92 puncta , 22 axons ( anterograde ) , 78 puncta , 22 axons ( retrograde ) . n = 5 independent experiments . Values are mean ± SEM . Abbreviations : CD63 , CD63 - eGFP ; MB , molecular beacon .

O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O

G. Frequency distribution (in percentage) of MB-labeled pre-miR-181a-1 co-trafficked with CD63-eGFP-positive vesicles. Each data point corresponds to one axon. Total number of puncta and axons analyzed: 92 puncta, 22 axons (anterograde), 78 puncta, 22 axons (retrograde). n=5 independent experiments. Values are mean ± SEM. Abbreviations: CD63, CD63-eGFP; MB, molecular beacon.


I . Representative image of an electroporated and dissociated RGC . Abbreviations : CD63 , CD63 - eGFP . Scale bars : 5µm .

O O O O O O B-EXP_ASSAY O O B-CELL_TYPE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O

I. Representative image of an electroporated and dissociated RGC. Abbreviations: CD63, CD63-eGFP. Scale bars: 5µm.


K , L . Fluorescence intensity of MB ( K ) or CD63 - eGFP ( L ) measured in axons and growth cone central domains . Each data point corresponds to one axon or a growth cone central domain . Total number of axons analyzed : 16 . n = 5 independent experiments . Values are mean ± SEM . Abbreviations : CD63 , CD63 - eGFP ; MB , molecular beacon ; GC , growth cone .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O

K,L. Fluorescence intensity of MB (K) or CD63-eGFP (L) measured in axons and growth cone central domains. Each data point corresponds to one axon or a growth cone central domain. Total number of axons analyzed: 16. n=5 independent experiments. Values are mean ± SEM. Abbreviations: CD63, CD63-eGFP; MB, molecular beacon; GC, growth cone.


C . Representative images of co - electroporated axons . Dashed white lines delineate axons . White arrows indicate colocalized puncta . Colored arrows represent non - colocalized signal as follows : magenta for CD63 and yellow for Lamp1 or Rab5a . Abbreviations : CD63 , CD63 - eGFP or CD63 - mRFP ; Lamp1 , Lamp1 - eGFP ; Rab5a , Rab5a - eGFP or Rab5a - mRFP . Scale bars : 5μm .

O O O O O O O B-EXP_ASSAY B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O

C. Representative images of co-electroporated axons. Dashed white lines delineate axons. White arrows indicate colocalized puncta. Colored arrows represent non-colocalized signal as follows: magenta for CD63 and yellow for Lamp1 or Rab5a. Abbreviations: CD63, CD63-eGFP or CD63-mRFP; Lamp1, Lamp1-eGFP; Rab5a, Rab5a-eGFP or Rab5a-mRFP. Scale bars: 5μm.


D . Frequency ( in percentage ) of CD63 colocalization with specific organelle markers . Each data point corresponds to one axon . Total number of puncta , axons , and independent experiments analyzed : 540 puncta , 23 axons , n = 4 ( CD63 / Rab7a ) , 416 puncta , 18 axons , n = 3 ( CD63 / LysoTracker ) , 584 puncta , 17 axons , n = 2 ( CD63 / Lamp1 ) , 649 puncta , 19 axons , n = 3 ( CD63 / Rab5a ) . Values are mean ± SEM . Abbreviations : CD63 , CD63 - eGFP or CD63 - mRFP ; Lamp1 , Lamp1 - eGFP ; Rab5a , Rab5a - eGFP or Rab5a - mRFP ; Rab7a , Rab7a - eGFP or Rab7a - mRFP .

O O O O O O O O B-GENEPROD B-EXP_ASSAY O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O O O O B-SUBCELLULAR O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-SUBCELLULAR O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O B-SUBCELLULAR O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-SUBCELLULAR O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O

D. Frequency (in percentage) of CD63 colocalization with specific organelle markers. Each data point corresponds to one axon. Total number of puncta, axons, and independent experiments analyzed: 540 puncta, 23 axons, n=4 (CD63/Rab7a), 416 puncta, 18 axons, n=3 (CD63/LysoTracker), 584 puncta, 17 axons, n=2 (CD63/Lamp1), 649 puncta, 19 axons, n=3 (CD63/Rab5a). Values are mean ± SEM. Abbreviations: CD63, CD63-eGFP or CD63-mRFP; Lamp1, Lamp1-eGFP; Rab5a, Rab5a-eGFP or Rab5a-mRFP; Rab7a, Rab7a-eGFP or Rab7a-mRFP.


E . Representative images of co - electroporated axons . Dashed white lines delineate axons . White arrows indicate colocalized puncta . Colored arrows represent non - colocalized signal as follows : cyan for pre - miR - 181a - 1 and yellow for Lamp1 or Rab5a . Abbreviations : Lamp1 , Lamp1 - eGFP ; Rab5a , Rab5a - eGFP or Rab5a - mRFP ; pre - miR - 181a - 1 , cy3 - pre - miR - 181a - 1 or cy5 - pre - miR - 181a - 1 . Scale bars : 5μm .

O O O O O O O B-EXP_ASSAY B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O

E. Representative images of co-electroporated axons. Dashed white lines delineate axons. White arrows indicate colocalized puncta. Colored arrows represent non-colocalized signal as follows: cyan for pre-miR-181a-1 and yellow for Lamp1 or Rab5a. Abbreviations: Lamp1, Lamp1-eGFP; Rab5a, Rab5a-eGFP or Rab5a-mRFP; pre-miR-181a-1, cy3-pre-miR-181a-1 or cy5-pre-miR-181a-1. Scale bars: 5μm.


F . Frequency ( in percentage ) of pre - miR - 181a - 1 colocalization with specific organelle markers . Each data point corresponds to one axon . Total number of puncta , axons and independent experiments analyzed : 825 puncta , 41 axons , n = 8 ( pre - miR - 181a - 1 / CD63 ) , 732 puncta , 33 axons , n = 7 ( pre - miR - 181a - 1 / Rab7a ) , 681 puncta , 29 axons , n = 5 ( pre - miR - 181a - 1 / LysoTracker ) , 501 puncta , 17 axons , n = 2 ( pre - miR - 181a - 1 / Lamp1 ) , 791 puncta , 25 axons , n = 3 ( pre - miR - 181a - 1 / Rab5a ) . Values are median with interquartile range . Abbreviations : CD63 , CD63 - eGFP or CD63 - mRFP ; Rab7a , Rab7a - eGFP or Rab7a - mRFP , Lamp1 , Lamp1 - eGFP ; Rab5a , Rab5a - eGFP or Rab5a - mRFP ; pre - miR - 181a - 1 , cy3 - pre - miR - 181a - 1 or cy5 - pre - miR - 181a - 1 .

O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O B-SUBCELLULAR O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O B-SUBCELLULAR O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O O O B-SUBCELLULAR O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O B-SUBCELLULAR O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O

F. Frequency (in percentage) of pre-miR-181a-1 colocalization with specific organelle markers. Each data point corresponds to one axon. Total number of puncta, axons and independent experiments analyzed: 825 puncta, 41 axons, n=8 (pre-miR-181a-1/CD63), 732 puncta, 33 axons, n=7 (pre-miR-181a-1/Rab7a), 681 puncta, 29 axons, n=5 (pre-miR-181a-1/LysoTracker), 501 puncta, 17 axons, n=2 (pre-miR-181a-1/Lamp1), 791 puncta, 25 axons, n=3 (pre-miR-181a-1/Rab5a). Values are median with interquartile range. Abbreviations: CD63, CD63-eGFP or CD63-mRFP; Rab7a, Rab7a-eGFP or Rab7a-mRFP, Lamp1, Lamp1-eGFP; Rab5a, Rab5a-eGFP or Rab5a-mRFP; pre-miR-181a-1, cy3-pre-miR-181a-1 or cy5-pre-miR-181a-1.


G . Representative images of co - electroporated axons . Dashed white lines delineate axons . White arrows indicate colocalized puncta . Colored arrows represent non - colocalized signal as follows : magenta for CD63 , cyan for pre - miR - 181a - 1 , yellow for Rab7a and blue for LysoTracker . Abbreviations : CD63 , CD63 - eGFP or CD63 - mRFP ; Rab7a , Rab7a - eGFP or Rab7a - mRFP ; pre - miR - 181a - 1 , cy3 - pre - miR - 181a - 1 or cy5 - pre - miR - 181a - 1 . Scale bar : 5μm .

O O O O O O O B-EXP_ASSAY B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O

G. Representative images of co-electroporated axons. Dashed white lines delineate axons. White arrows indicate colocalized puncta. Colored arrows represent non-colocalized signal as follows: magenta for CD63, cyan for pre-miR-181a-1, yellow for Rab7a and blue for LysoTracker. Abbreviations: CD63, CD63-eGFP or CD63-mRFP; Rab7a, Rab7a-eGFP or Rab7a-mRFP; pre-miR-181a-1, cy3-pre-miR-181a-1 or cy5-pre-miR-181a-1. Scale bar: 5μm.


H . Frequency ( in percentage ) of pre - miR - 181a - 1 colocalization with vesicles double positive for organelle markers . Each data point corresponds to one axon . Total number of puncta , axons and independent experiments analyzed : 745 puncta , 23 axons , n = 5 ( CD63 / pre - miR - 181a - 1 / Rab7a ) , 577 puncta , 18 axons , n = 3 ( CD63 / pre - miR - 181a - 1 / LysoTracker ) . Values are median with interquartile range . Abbreviations : ns , not significant ; CD63 , CD63 - eGFP or CD63 - mRFP ; Rab7a , Rab7a - eGFP or Rab7a - mRFP ; pre - miR - 181a - 1 , cy3 - pre - miR - 181a - 1 or cy5 - pre - miR - 181a - 1 .

O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O B-SUBCELLULAR O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O

H. Frequency (in percentage) of pre-miR-181a-1 colocalization with vesicles double positive for organelle markers. Each data point corresponds to one axon. Total number of puncta, axons and independent experiments analyzed: 745 puncta, 23 axons, n=5 (CD63/pre-miR-181a-1/Rab7a), 577 puncta, 18 axons, n=3 (CD63/pre-miR-181a-1/LysoTracker). Values are median with interquartile range. Abbreviations: ns, not significant; CD63, CD63-eGFP or CD63-mRFP; Rab7a, Rab7a-eGFP or Rab7a-mRFP; pre-miR-181a-1, cy3-pre-miR-181a-1 or cy5-pre-miR-181a-1.


I . Representative 3D - STED super - resolution image . Total number of puncta , axons and growth cones analyzed : 99 puncta , 16 axons and 10 growth cones ( MB ) , 291 puncta , 36 axons and 15 growth cones ( cy3 - pre - miR - 181a - 1 ) . n = 2 ( MB ) , n = 3 ( cy3 - pre - miR - 181a - 1 ) independent experiments . Abbreviations : CD63 , CD63 - eGFP ; pre - miR - 181a - 1 , cy3 - pre - miR - 181a - 1 ; MB ; molecular beacon .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR I-SUBCELLULAR O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O

I. Representative 3D-STED super-resolution image. Total number of puncta, axons and growth cones analyzed: 99 puncta, 16 axons and 10 growth cones (MB), 291 puncta, 36 axons and 15 growth cones (cy3-pre-miR-181a-1). n=2 (MB), n=3 (cy3-pre-miR-181a-1) independent experiments. Abbreviations: CD63, CD63-eGFP; pre-miR-181a-1, cy3-pre-miR-181a-1; MB; molecular beacon.


A . Schematic representation of the experimental protocol . B . Representative Xenopus growth cones stained with anti - Dicer and anti - Ago2 . White dashed lines delineate growth cones . Scale bars : 5µm .

O O O O O O O O O O O O B-ORGANISM B-SUBCELLULAR I-SUBCELLULAR B-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O

A. Schematic representation of the experimental protocol. B. Representative Xenopus growth cones stained with anti-Dicer and anti-Ago2. White dashed lines delineate growth cones. Scale bars: 5µm.


C . Representative E13 . 5 mice brain cross - section ( dashed red line in the schematic ) comprising the optic nerve ( ON ) stained with anti - neurofilament and anti - HA antibodies to detect RGC axons and ( FLAG - HA2 ) - Dicer , respectively . Note the absence of HA signal in wild type ( WT ) mice . The white dashed line delineates the ON . Zoom of the triple stained ON ( right panel ) : Dicer signal is detected inside ON cells surrounding axon bundles but not in axons per se ( arrowheads ) . n = 3 independent experiments . Abbreviations : WT , wild type ; HA , ( FLAG - HA2 ) - Dicer ; E13 . 5 , embryonic day 13 . 5 ; ONH , optic nerve head ; ON , optic nerve . Scale bars : 30µm .

O O O O O O B-ORGANISM B-TISSUE O O O O O O O O O O O O O B-TISSUE I-TISSUE O B-TISSUE O B-EXP_ASSAY O O O B-SUBCELLULAR O O O B-GENEPROD O O O B-CELL_TYPE B-SUBCELLULAR O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O B-ORGANISM O O O O O O O B-TISSUE O O O O O B-EXP_ASSAY B-TISSUE O O O O O B-GENEPROD O O O O B-TISSUE O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O B-TISSUE O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O O O O O O

C. Representative E13.5 mice brain cross-section (dashed red line in the schematic) comprising the optic nerve (ON) stained with anti-neurofilament and anti-HA antibodies to detect RGC axons and (FLAG-HA2)-Dicer, respectively. Note the absence of HA signal in wild type (WT) mice. The white dashed line delineates the ON. Zoom of the triple stained ON (right panel): Dicer signal is detected inside ON cells surrounding axon bundles but not in axons per se (arrowheads). n=3 independent experiments. Abbreviations: WT, wild type; HA, (FLAG-HA2)-Dicer; E13.5, embryonic day 13.5; ONH, optic nerve head; ON, optic nerve. Scale bars: 30µm.


D . Representative P0 superior colliculus stratum griseum superficiale ( SGS ) ( sagittal sectioning of P0 brains along the dashed red line in the schematic , maximal projection of 5 µm depth ) . Note the presence of Dicer within the SGS marked with neurofilament . n = 3 independent experiments . Abbreviations : WT , wild type ; P0 , postnatal day 0 . Scale bars : 30µm .

O O O O B-TISSUE I-TISSUE I-TISSUE I-TISSUE I-TISSUE O B-TISSUE O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-TISSUE O O B-SUBCELLULAR O 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 P0 superior colliculus stratum griseum superficiale (SGS) (sagittal sectioning of P0 brains along the dashed red line in the schematic, maximal projection of 5 µm depth). Note the presence of Dicer within the SGS marked with neurofilament. n=3 independent experiments. Abbreviations: WT, wild type; P0, postnatal day 0. Scale bars: 30µm.


Quantification of miRNA and pre - miRNA expression levels using the 2 - ΔCt method and U6 as normalizer , upon Sema3A ( B ) stimulation . Data are normalized to PBS control . Each data point represents a single RT - qPCR reaction . n = 3 ( B ) independent experiments . Values are mean ± SEM . Abbreviations : ns , not significant .

O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY 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 miRNA and pre-miRNA expression levels using the 2-ΔCt method and U6 as normalizer, upon Sema3A (B) stimulation. Data are normalized to PBS control. Each data point represents a single RT-qPCR reaction. n=3 (B) independent experiments. Values are mean ± SEM. Abbreviations: ns, not significant.


Quantification of miRNA and pre - miRNA expression levels using the 2 - ΔCt method and U6 as normalizer , upon Slit2 ( C ) stimulation . Data are normalized to PBS control . Each data point represents a single RT - qPCR reaction . n = 4 ( C ) independent experiments . Values are mean ± SEM . Abbreviations : ns , not significant .

Quantification of miRNA and pre-miRNA expression levels using the 2-ΔCt method and U6 as normalizer, upon Slit2 (C) stimulation. Data are normalized to PBS control. Each data point represents a single RT-qPCR reaction. n=4 (C) independent experiments. Values are mean ± SEM. Abbreviations: ns, not significant.


Frequency ( in percentage ) of collapsed growth cones from stage 37 / 38 embryos , following a 10 min Sema3A bath application . Concentration used : 200ng / mL ( Sema3A ) , 2µM ( MOs - 5p ) ( E ) Total number of counted growth cones is reported in the column . Each data point represents one independent experiment . n = 3 independent experiments . Values are mean ± SEM . Abbreviations : ns , not significant ; co - MO , control morpholino .

O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O 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-SMALL_MOLECULE O O B-SMALL_MOLECULE O

Frequency (in percentage) of collapsed growth cones from stage 37/38 embryos, following a 10 min Sema3A bath application. Concentration used: 200ng/mL (Sema3A), 2µM (MOs-5p) (E) Total number of counted growth cones is reported in the column. Each data point represents one independent experiment. n=3 independent experiments. Values are mean ± SEM. Abbreviations: ns, not significant; co-MO, control morpholino.


Frequency ( in percentage ) of collapsed growth cones from stage 37 / 38 embryos , following a 10 min Sema3A bath application . Concentration used : 200ng / mL ( Sema3A ) , 2µM ( MOs - 3p ) ( F ) . Total number of counted growth cones is reported in the column . Each data point represents one independent experiment . n = 3 independent experiments . Values are mean ± SEM . Abbreviations : ns , not significant ; co - MO , control morpholino .

O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O 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-SMALL_MOLECULE O O B-SMALL_MOLECULE O

Frequency (in percentage) of collapsed growth cones from stage 37/38 embryos, following a 10 min Sema3A bath application. Concentration used: 200ng/mL (Sema3A), 2µM (MOs-3p) (F). Total number of counted growth cones is reported in the column. Each data point represents one independent experiment. n=3 independent experiments. Values are mean ± SEM. Abbreviations: ns, not significant; co-MO, control morpholino.


B . Representative images of RGC axons within the optic tectum . A subset of aberrantly projecting axons are indicated ( red arrows ) . Note that a few straying axons are always observed within the wild type tectum . Abbreviations : co - MO , control morpholino . Scale bars : 20µm . C . Quantification of misprojecting axons . Each data point corresponds to one brain . Total number of brains analyzed : 45 brains ( co - MO ) , 52 brains ( miR - 181 - MO ) . n = 4 independent experiments . Values are mean ± SEM . Abbreviations : co - MO , control morpholino .

O O O O O B-CELL_TYPE B-SUBCELLULAR O O B-TISSUE I-TISSUE O O O O O O B-SUBCELLULAR O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O B-TISSUE O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O B-SUBCELLULAR O O O O O O O B-TISSUE O O O O B-TISSUE O O O B-TISSUE O O O B-SMALL_MOLECULE O O O B-TISSUE O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O

B. Representative images of RGC axons within the optic tectum. A subset of aberrantly projecting axons are indicated (red arrows). Note that a few straying axons are always observed within the wild type tectum. Abbreviations: co-MO, control morpholino. Scale bars: 20µm. C. Quantification of misprojecting axons. Each data point corresponds to one brain. Total number of brains analyzed: 45 brains (co-MO), 52 brains (miR-181-MO). n=4 independent experiments. Values are mean ± SEM. Abbreviations: co-MO, control morpholino.


D . Schematic representation of the experimental paradigm . Concentrations used : 0 . 5μg / μL pCS2 - mCherry or pCS2 - eGFP plasmids ; 250μM miR - 181 - MO or control MO cocktail ; 50μM miR - 181 or control mimics ( top ) . Representative images of RGC axons within the optic tectum . White arrows indicate axons targeted both by MO ( green ) and miRNA mimics ( red ) ( bottom ) . Abbreviations : co - MO , control morpholino . Scale bars : 20µm . E . Quantification of misprojecting axons . The number reported on the bars are the total number of co - electroporated axons . Note how miR - 181 mimics rescued aberrant misprojection of morphant axons in vivo . Each data point corresponds to one independent experiment . Total number of axons and brains analyzed : 96 axons , 4 brains ( co - MO + ctrl mimics ) , 101 axons , 4 brains ( miR - 181 - MO + ctrl mimics ) , 134 axons , 4 brains ( miR - 181 - MO + miR - 181 mimics ) . n = 4 independent experiments . Values are mean ± SEM . Abbreviations : co - MO , control morpholino ; ns , not significant .

O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-CELL_TYPE B-SUBCELLULAR O O B-TISSUE I-TISSUE O O O O B-SUBCELLULAR O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O B-EXP_ASSAY B-SUBCELLULAR O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O B-SUBCELLULAR O B-TISSUE O O O B-SUBCELLULAR O O B-TISSUE O O O B-SMALL_MOLECULE O O O O O O B-SUBCELLULAR O O B-TISSUE O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O O O B-SUBCELLULAR O O B-TISSUE O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O

D. Schematic representation of the experimental paradigm. Concentrations used: 0.5μg/μL pCS2-mCherry or pCS2-eGFP plasmids; 250μM miR-181-MO or control MO cocktail; 50μM miR-181 or control mimics (top). Representative images of RGC axons within the optic tectum. White arrows indicate axons targeted both by MO (green) and miRNA mimics (red) (bottom). Abbreviations: co-MO, control morpholino. Scale bars: 20µm. E. Quantification of misprojecting axons. The number reported on the bars are the total number of co-electroporated axons. Note how miR-181 mimics rescued aberrant misprojection of morphant axons in vivo. Each data point corresponds to one independent experiment. Total number of axons and brains analyzed: 96 axons, 4 brains (co-MO+ctrl mimics), 101 axons, 4 brains (miR-181-MO + ctrl mimics), 134 axons, 4 brains (miR-181-MO+miR-181 mimics). n=4 independent experiments. Values are mean ± SEM. Abbreviations: co-MO, control morpholino; ns, not significant.


F . Representative images of RGC axons within the optic tectum . A subset of aberrantly projecting axons are indicated ( red arrows ) . Note that a few straying axons are always observed within the wild type tectum . Abbreviations : co - MO , control morpholino . Scale bars : 20µm . G . Quantification of misprojecting axons . Each data point corresponds to one brain . Total number of brains analyzed : 21 brains ( co - MO ) , 31 brains ( miR - 181 - MO ) . n = 3 independent experiments . Values are mean ± SEM . Abbreviations : co - MO , control morpholino .

F. Representative images of RGC axons within the optic tectum. A subset of aberrantly projecting axons are indicated (red arrows). Note that a few straying axons are always observed within the wild type tectum. Abbreviations: co-MO, control morpholino. Scale bars: 20µm. G. Quantification of misprojecting axons. Each data point corresponds to one brain. Total number of brains analyzed: 21 brains (co-MO), 31 brains (miR-181-MO). n=3 independent experiments. Values are mean ± SEM. Abbreviations: co-MO, control morpholino.


I . Frequency ( in percentage ) of the amount of time embryos spent on black background . Each data point corresponds to one embryo . Total number of embryos analyzed : 43 embryos ( co - MO ) , 45 embryos ( miR - 181 - MO ) . n = 4 independent experiments . Values are mean ± SEM . Abbreviations : co - MO , control morpholino .

O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O

I. Frequency (in percentage) of the amount of time embryos spent on black background. Each data point corresponds to one embryo. Total number of embryos analyzed: 43 embryos (co-MO), 45 embryos (miR-181-MO). n=4 independent experiments. Values are mean ± SEM. Abbreviations: co-MO, control morpholino.


Quantification ( in percentage ) of the axonal fluorescence recovery after photobleaching ( FRAP ) of different Venus - 3 ' UTR constructs ex vivo and in vivo . Concentrations used : 200ng / mL Sema3A ; 100µM cycloheximide Growth cones expressing Venus alone ( no 3 ' UTR ) displayed a minimal amount of recovery after photobleaching corresponding to Venus diffusion from adjacent , non - bleached regions to the bleached growth cone ( Wong et al , 2017 ) . ( B ) 200ng / mL Sema3A was bath applied with or without 100µM cycloheximide ( CHX , a translational blocker ) . Mutating TUBB3 3 ' UTR did not affect fluorescence recovery in basal conditions compared to WT indicating that mature miRNAs do not regulate the constitutive TUBB3 expression in distal axons .

O O O O O O O B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O B-SMALL_MOLECULE B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O B-SUBCELLULAR O

Quantification (in percentage) of the axonal fluorescence recovery after photobleaching (FRAP) of different Venus-3'UTR constructs ex vivo and in vivo. Concentrations used: 200ng/mL Sema3A; 100µM cycloheximide Growth cones expressing Venus alone (no 3'UTR) displayed a minimal amount of recovery after photobleaching corresponding to Venus diffusion from adjacent, non-bleached regions to the bleached growth cone (Wong et al, 2017). (B) 200ng/mL Sema3A was bath applied with or without 100µM cycloheximide (CHX, a translational blocker). Mutating TUBB3 3'UTR did not affect fluorescence recovery in basal conditions compared to WT indicating that mature miRNAs do not regulate the constitutive TUBB3 expression in distal axons.


Quantification ( in percentage ) of the axonal fluorescence recovery after photobleaching ( FRAP ) of different Venus - 3 ' UTR constructs ex vivo and in vivo . Concentrations used : 200ng / mL Sema3A ; Growth cones expressing Venus alone ( no 3 ' UTR ) displayed a minimal amount of recovery after photobleaching corresponding to Venus diffusion from adjacent , non - bleached regions to the bleached growth cone ( Wong et al , 2017 ) . , isolated axons ( C ) were used for FRAP analysis .

O O O O O O O B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O B-SUBCELLULAR O O O O O O B-EXP_ASSAY O O

Quantification (in percentage) of the axonal fluorescence recovery after photobleaching (FRAP) of different Venus-3'UTR constructs ex vivo and in vivo. Concentrations used: 200ng/mL Sema3A; Growth cones expressing Venus alone (no 3'UTR) displayed a minimal amount of recovery after photobleaching corresponding to Venus diffusion from adjacent, non-bleached regions to the bleached growth cone (Wong et al, 2017). , isolated axons (C) were used for FRAP analysis.


Quantification ( in percentage ) of the axonal fluorescence recovery after photobleaching ( FRAP ) of different Venus - 3 ' UTR constructs ex vivo and in vivo . Concentrations used : 200ng / mL Sema3A ; 2µM co - MO or MOs - 3p . Growth cones expressing Venus alone ( no 3 ' UTR ) displayed a minimal amount of recovery after photobleaching corresponding to Venus diffusion from adjacent , non - bleached regions to the bleached growth cone ( Wong et al , 2017 ) . isolated transfected axons ( D ) were used for FRAP analysis .

O O O O O O O B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O B-SUBCELLULAR O O O O O O B-EXP_ASSAY O O

Quantification (in percentage) of the axonal fluorescence recovery after photobleaching (FRAP) of different Venus-3'UTR constructs ex vivo and in vivo. Concentrations used: 200ng/mL Sema3A; 2µM co-MO or MOs-3p. Growth cones expressing Venus alone (no 3'UTR) displayed a minimal amount of recovery after photobleaching corresponding to Venus diffusion from adjacent, non-bleached regions to the bleached growth cone (Wong et al, 2017). isolated transfected axons (D) were used for FRAP analysis.


Quantification ( in percentage ) of the axonal fluorescence recovery after photobleaching ( FRAP ) of different Venus - 3 ' UTR constructs ex vivo and in vivo . Growth cones expressing Venus alone ( no 3 ' UTR ) displayed a minimal amount of recovery after photobleaching corresponding to Venus diffusion from adjacent , non - bleached regions to the bleached growth cone ( Wong et al , 2017 ) . In vivo ( E ) , whole embryos with exposed brain were used . The electroporated eye was removed prior to mounting the embryo to eliminate somatic contribution . ( E ) The red area on the brain schematic indicate Sema3A expressing territories . Numbers of axons analyzed are reported between brackets .

O O O O O O O B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O B-TISSUE O O O O B-EXP_ASSAY B-TISSUE O O O O O O O O O O O O O O O O O O O O B-TISSUE O O B-GENEPROD O O O O O B-SUBCELLULAR O O O O O O

Quantification (in percentage) of the axonal fluorescence recovery after photobleaching (FRAP) of different Venus-3'UTR constructs ex vivo and in vivo. Growth cones expressing Venus alone (no 3'UTR) displayed a minimal amount of recovery after photobleaching corresponding to Venus diffusion from adjacent, non-bleached regions to the bleached growth cone (Wong et al, 2017). In vivo (E), whole embryos with exposed brain were used. The electroporated eye was removed prior to mounting the embryo to eliminate somatic contribution. (E) The red area on the brain schematic indicate Sema3A expressing territories. Numbers of axons analyzed are reported between brackets.


Quantification ( in percentage ) of the axonal fluorescence recovery after photobleaching ( FRAP ) of different Venus - 3 ' UTR constructs ex vivo and in vivo . Concentrations used : 200ng / mL Sema3A Growth cones expressing Venus alone ( no 3 ' UTR ) displayed a minimal amount of recovery after photobleaching corresponding to Venus diffusion from adjacent , non - bleached regions to the bleached growth cone ( Wong et al , 2017 ) . Ex vivo , whole explants were used for FRAP analysis . APP , Amyloid beta precursor

O O O O O O O B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD

Quantification (in percentage) of the axonal fluorescence recovery after photobleaching (FRAP) of different Venus-3'UTR constructs ex vivo and in vivo. Concentrations used: 200ng/mL Sema3A Growth cones expressing Venus alone (no 3'UTR) displayed a minimal amount of recovery after photobleaching corresponding to Venus diffusion from adjacent, non-bleached regions to the bleached growth cone (Wong et al, 2017). Ex vivo, whole explants were used for FRAP analysis. APP, Amyloid beta precursor protein


Quantification ( in percentage ) of the axonal fluorescence recovery after photobleaching ( FRAP ) of different Venus - 3 ' UTR constructs ex vivo and in vivo . Concentrations used : 200ng / mL Sema3A Growth cones expressing Venus alone ( no 3 ' UTR ) displayed a minimal amount of recovery after photobleaching corresponding to Venus diffusion from adjacent , non - bleached regions to the bleached growth cone ( Wong et al , 2017 ) . Ex vivo , whole explants were used for FRAP analysis . THBS1 , Thrombospondin

O O O O O O O B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD

Quantification (in percentage) of the axonal fluorescence recovery after photobleaching (FRAP) of different Venus-3'UTR constructs ex vivo and in vivo. Concentrations used: 200ng/mL Sema3A Growth cones expressing Venus alone (no 3'UTR) displayed a minimal amount of recovery after photobleaching corresponding to Venus diffusion from adjacent, non-bleached regions to the bleached growth cone (Wong et al, 2017). Ex vivo, whole explants were used for FRAP analysis. THBS1, Thrombospondin 1


H , I . Representative growth cones depicting Venus fluorescence intensity as a heatmap . Dashed white lines delineate the growth cones . Abbreviations : wt , wild type ; mut , miR - 181 - 5p responsive elements mutated ; CHX , cycloheximide ; TUBB3 , tubulin beta 3 class III . Scale bars : 10µm .

O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O

H,I. Representative growth cones depicting Venus fluorescence intensity as a heatmap. Dashed white lines delineate the growth cones. Abbreviations: wt, wild type; mut, miR-181-5p responsive elements mutated; CHX, cycloheximide; TUBB3, tubulin beta 3 class III. Scale bars: 10µm.


B . Schematic representation of the experimental paradigm . Representative images of local translation events of TUBB3 in the growth cone . Dashed white line delineates the growth cone . Each white arrow points to a puro - PLA puncta . Abbreviations : TUBB3 , tubulin beta 3 class III ; Puro , puromycin . Scale bars : 5 μm . Quantification of number of puncta per growth cone . Each data point corresponds to one growth cone . Total number of growth cones analyzed : 30 ( PBS ) , 36 ( Sema3A ) . n = 2 independent experiments . Values are median with interquartile range .

O O O O O O O O O O O O O O O O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O

B. Schematic representation of the experimental paradigm. Representative images of local translation events of TUBB3 in the growth cone. Dashed white line delineates the growth cone. Each white arrow points to a puro-PLA puncta. Abbreviations: TUBB3, tubulin beta 3 class III; Puro, puromycin. Scale bars: 5 μm. Quantification of number of puncta per growth cone. Each data point corresponds to one growth cone. Total number of growth cones analyzed: 30 (PBS), 36 (Sema3A). n= 2 independent experiments. Values are median with interquartile range.


D . Schematic representation of the experimental paradigm . Concentrations used : 2 μM co - MO ; 2 μM MOs - 3p ; 200ng / mL Sema3A . Representative images of local translation events of TUBB3 in the growth cone of transfected axons . Dashed white line delineates the growth cone . White arrows represent number of puro - PLA puncta per growth cone . Abbreviations : co - MO , control morpholino . Scale bars : 5 μm . E . Quantification of number of puncta per growth cone . Each data point corresponds to one growth cone . Total number of growth cones analyzed : 32 ( co - MO + PBS ) , 45 ( co - MO + Sema3A ) , 27 ( MOs - 3p + Sema3A ) . n = 2 independent experiments . Values are median with interquartile range . Abbreviations : co - MO , control morpholino ; ns , not significant .

O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY O O B-SUBCELLULAR I-SUBCELLULAR O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O B-SMALL_MOLECULE O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O

D. Schematic representation of the experimental paradigm. Concentrations used: 2 μM co-MO; 2 μM MOs-3p; 200ng/mL Sema3A. Representative images of local translation events of TUBB3 in the growth cone of transfected axons. Dashed white line delineates the growth cone. White arrows represent number of puro-PLA puncta per growth cone. Abbreviations: co-MO, control morpholino. Scale bars: 5 μm. E. Quantification of number of puncta per growth cone. Each data point corresponds to one growth cone. Total number of growth cones analyzed: 32 (co-MO + PBS), 45 (co-MO + Sema3A), 27 (MOs-3p + Sema3A). n=2 independent experiments. Values are median with interquartile range. Abbreviations: co-MO, control morpholino; ns, not significant.


G . Percentage of collapsed growth cones in axons transfected with co - MO , MOs - 3p + co - MO and MOs - 3p + MOs - TUBB3 . Each data point corresponds to one independent experiment . Total number of growth cones counted : 152 ( co - MO ) , 155 ( MOs - 3p + coMO ) , 179 ( MOs - 3p + MOs - TUBB3 ) . n = 3 independent experiments . Values are mean ± SEM . Abbreviations : ns , not significant ; co - MO , control morpholino ; TUBB3 , tubulin beta 3 class III .

O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR B-EXP_ASSAY O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O

G. Percentage of collapsed growth cones in axons transfected with co-MO, MOs-3p + co-MO and MOs-3p + MOs-TUBB3. Each data point corresponds to one independent experiment. Total number of growth cones counted: 152 (co-MO), 155 (MOs-3p + coMO), 179 (MOs-3p + MOs-TUBB3). n= 3 independent experiments. Values are mean ± SEM. Abbreviations: ns, not significant; co-MO, control morpholino; TUBB3, tubulin beta 3 class III.


B . Blood stage growth rate of mutated parasite lines . A single parasite was injected i . v . into a mouse and the growth rate was calculated from parasitemia at day 6 - 9 for each mouse ( black dots ) . Bars represent the mean of 4 - 7 mice . Significance for ( B ) determined by One - way analysis of variance with Tukeys Multiple Comparison test .

O O B-TISSUE O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O B-ORGANISM O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O B-ORGANISM O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O

B. Blood stage growth rate of mutated parasite lines. A single parasite was injected i.v. into a mouse and the growth rate was calculated from parasitemia at day 6-9 for each mouse (black dots). Bars represent the mean of 4-7 mice. Significance for (B) determined by One-way analysis of variance with Tukeys Multiple Comparison test.


C . Infected midgut showing oocysts stained with mercurochrome ; scale bar , 100 μm . The graph shows oocyst numbers per infected mosquito . Numbers of mosquitoes that were analyzed are indicated above the graph . Data derived from at least two independent cage feeds originating from independently infected mice . Box ‐ and ‐ whisker plots depict the 25 % quantile , median , 75 % quantile , and nearest observations within 1 . 5 times the interquartile range ( whiskers ) . Significance for ( C ) determined by Kruskal - Wallis test with Bonferroni ' s Multiple Comparison test .

O O O B-TISSUE O B-ORGANISM B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O B-ORGANISM O O O B-ORGANISM O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Infected midgut showing oocysts stained with mercurochrome; scale bar, 100 μm. The graph shows oocyst numbers per infected mosquito. Numbers of mosquitoes that were analyzed are indicated above the graph. Data derived from at least two independent cage feeds originating from independently infected mice. Box‐and‐whisker plots depict the 25% quantile, median, 75% quantile, and nearest observations within 1.5 times the interquartile range (whiskers). Significance for (C) determined by Kruskal-Wallis test with Bonferroni's Multiple Comparison test.


D . Ratio of salivary gland to midgut sporozoites of the indicated parasite lines . Black dots : individual experiments with 10 - 20 mosquitoes . Bars : mean . Significance for , ( D ) determined by One - way analysis of variance with Tukeys Multiple Comparison test .

O O O O B-TISSUE I-TISSUE O B-TISSUE B-ORGANISM O O O O O O O O O O O O O O O B-ORGANISM O 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 salivary gland to midgut sporozoites of the indicated parasite lines. Black dots: individual experiments with 10-20 mosquitoes. Bars: mean. Significance for , (D) determined by One-way analysis of variance with Tukeys Multiple Comparison test.


E . Hemolymph sporozoites were isolated and observed on glass slides by live cell imaging . Arrow indicates direction of a motile sporozoite . The graph shows the fraction of motile hemolymph sporozoites . Black dots : individual experiments with 10 - 20 mosquitoes . Bars : mean . Significance for ( E ) determined by One - way analysis of variance with Tukeys Multiple Comparison test .

O O B-TISSUE B-ORGANISM O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY B-ORGANISM O O O O O O O B-EXP_ASSAY B-TISSUE B-ORGANISM O O O O O O O O O O B-ORGANISM 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. Hemolymph sporozoites were isolated and observed on glass slides by live cell imaging. Arrow indicates direction of a motile sporozoite. The graph shows the fraction of motile hemolymph sporozoites. Black dots: individual experiments with 10-20 mosquitoes. Bars: mean. Significance for (E) determined by One-way analysis of variance with Tukeys Multiple Comparison test.


F . qRT - PCR analysis of schizonts and oocysts of the PbMyoA 3 ' dhfs parasite line . Individual data points correspond to the mean of three technical replicates .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-ORGANISM O B-ORGANISM O O B-ORGANISM O O O O O O O O O O O O O O O O O O

F. qRT-PCR analysis of schizonts and oocysts of the PbMyoA 3'dhfs parasite line. Individual data points correspond to the mean of three technical replicates.


C . Blood stage growth rate of clonal parasite lines . A single parasite or 100 iRBCs were injected i . v . into a mouse and the growth rate was calculated from parasitemia at day 6 - 9 . Black dots : individual mice ; bars : mean . Statistical analysis : ( C ) One - way analysis of variance with Tukeys multiple comparison test .

O O B-TISSUE O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-CELL_TYPE O O O O O O O O B-ORGANISM O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O

C. Blood stage growth rate of clonal parasite lines. A single parasite or 100 iRBCs were injected i.v. into a mouse and the growth rate was calculated from parasitemia at day 6-9. Black dots: individual mice; bars: mean. Statistical analysis: (C) One-way analysis of variance with Tukeys multiple comparison test.


D . Proportion of motile ookinetes . Black dots : Individual movie ; bars : mean . Data pooled from three independent biological replicates with 1 - 3 movies taken per experiment . Statistical analysis : ( D ) Kruskal - Wallis test with ( D ) Dunn ' s multiple comparisons

O O O O B-EXP_ASSAY B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O 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. Proportion of motile ookinetes. Black dots: Individual movie; bars: mean. Data pooled from three independent biological replicates with 1 - 3 movies taken per experiment. Statistical analysis: (D) Kruskal-Wallis test with (D) Dunn's multiple comparisons test


E . Speed of ookinetes of the different lines . Numbers of analysed parasites are shown above the graph . Data pooled from three independent experiments ( biological replicates ) . Violin plots show median ( line ) and quartiles ( dashed lines ) .

O O O O B-ORGANISM O O O O O O O O O O O O O O O O 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. Speed of ookinetes of the different lines. Numbers of analysed parasites are shown above the graph. Data pooled from three independent experiments (biological replicates). Violin plots show median (line) and quartiles (dashed lines).


F . Number of oocysts per infected mosquito . Numbers of analyzed mosquitoes are shown above the graph . Data pooled from at least three independent cage feeds originating from independently infected mice . Box ‐ and ‐ whisker plots depict the 25 % quantile , median , 75 % quantile , and nearest observations within 1 . 5 times the interquartile range ( whiskers ) . Statistical analysis : ( F ) Kruskal - Wallis test with ( F ) Bonferroni ' s Multiple Comparison test .

O O O O B-ORGANISM O O B-ORGANISM O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Number of oocysts per infected mosquito. Numbers of analyzed mosquitoes are shown above the graph. Data pooled from at least three independent cage feeds originating from independently infected mice. Box‐and‐whisker plots depict the 25% quantile, median, 75% quantile, and nearest observations within 1.5 times the interquartile range (whiskers). Statistical analysis: (F) Kruskal-Wallis test with (F) Bonferroni's Multiple Comparison test.


A . Ratio of hemolymph to midgut sporozoites . Black dots : individual experiments with 10 - 20 mosquitoes . Bars : mean . B . Ratio of salivary gland to midgut sporozoites . Black dots : individual experiments with 10 - 20 mosquitoes . Bars : mean . Statistical analysis : ( A ) and ( B ) One - way analysis of variance with Tukeys Multiple Comparison test .

O O O O B-TISSUE O B-TISSUE B-ORGANISM O O O O O O O O O O B-ORGANISM O O O O O O O O O B-TISSUE I-TISSUE O B-TISSUE B-ORGANISM O O O O O O O O O O B-ORGANISM O O O 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. Ratio of hemolymph to midgut sporozoites. Black dots: individual experiments with 10-20 mosquitoes. Bars: mean. B. Ratio of salivary gland to midgut sporozoites. Black dots: individual experiments with 10-20 mosquitoes. Bars: mean. Statistical analysis: (A) and (B) One-way analysis of variance with Tukeys Multiple Comparison test.


C . Blood stage infection of mice after transmission by mosquito bite . Shown is the mean ± standard deviation . D . Blood stage infection of mice after i . v . injection of 1000 sporozoites .

O O B-TISSUE O B-EXP_ASSAY O B-ORGANISM O O O B-ORGANISM O O O O O O O O O O O O B-TISSUE O B-EXP_ASSAY O B-ORGANISM O O O O O O O O B-ORGANISM O

C. Blood stage infection of mice after transmission by mosquito bite. Shown is the mean ± standard deviation. D. Blood stage infection of mice after i.v. injection of 1000 sporozoites.


AFraction ( left ) and speed ( right ) of motile hemolymph sporozoites on glass . Dots correspond to individual experiments and bars indicate the mean . Box ‐ and ‐ whisker plots depict the 25 % quantile , median , 75 % quantile , and nearest observations within 1 . 5 times the interquartile range ( whiskers ) . Significance for ( A left ) and ( B left ) determined by One - way analysis of variance with Tukeys Multiple Comparison test . Significance for ( A right ) and ( B right ) determined by Kruskal - Wallis test with Bonferroni ' s Multiple Comparison test .

O O O O O O O O O O B-EXP_ASSAY B-TISSUE B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

AFraction (left) and speed (right) of motile hemolymph sporozoites on glass . Dots correspond to individual experiments and bars indicate the mean. Box‐and‐whisker plots depict the 25% quantile, median, 75% quantile, and nearest observations within 1.5 times the interquartile range (whiskers). Significance for (A left) and (B left) determined by One-way analysis of variance with Tukeys Multiple Comparison test. Significance for (A right) and (B right) determined by Kruskal-Wallis test with Bonferroni's Multiple Comparison test.


C . Selected trajectories of 20 manually tracked sporozoites isolated from hemolymph and moving over a period of 3 min . Scale bar , 10 μm . D . Selected trajectories of 20 manually tracked sporozoites isolated from salivary glands and moving over a period of 3 min . Scale bar , 10 μm .

O O O O O O O O B-ORGANISM O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O

C. Selected trajectories of 20 manually tracked sporozoites isolated from hemolymph and moving over a period of 3 min. Scale bar, 10 μm. D. Selected trajectories of 20 manually tracked sporozoites isolated from salivary glands and moving over a period of 3 min. Scale bar, 10 μm.


A . Scheme depicting the setup of optical tweezers for measuring the forces that a sporozoite can generate to pull a bead out of an optical trap . The bead is placed at the apical end of the sporozoite . The graph shows the fraction of sporozoites that pulled the bead out of the optical trap . Black dots : individual experiments . Bars : mean . 73 - 131 sporozoites were probed for each line . Significance determined by One - way analysis of variance with Tukeys Multiple Comparison test .

O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O B-ORGANISM O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O

A. Scheme depicting the setup of optical tweezers for measuring the forces that a sporozoite can generate to pull a bead out of an optical trap. The bead is placed at the apical end of the sporozoite. The graph shows the fraction of sporozoites that pulled the bead out of the optical trap. Black dots: individual experiments. Bars: mean. 73-131 sporozoites were probed for each line. Significance determined by One-way analysis of variance with Tukeys Multiple Comparison test.


B . Scheme showing a sporozoite moving through a polymeric network in 3D . The graph shows sporozoite speeds as measured by manual tracking from imaging of sporozoites in a 3D hydrogel . Box ‐ and ‐ whisker plots depict the 25 % quantile , median , 75 % quantile , and nearest observations within 1 . 5 times the interquartile range ( whiskers ) . Significance determined by Kruskal - Wallis test with Bonferroni ' s Multiple Comparison test .

O O O O O B-ORGANISM O O O O O O O O O O O B-ORGANISM O O O O O O O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O 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. Scheme showing a sporozoite moving through a polymeric network in 3D. The graph shows sporozoite speeds as measured by manual tracking from imaging of sporozoites in a 3D hydrogel. Box‐and‐whisker plots depict the 25% quantile, median, 75% quantile, and nearest observations within 1.5 times the interquartile range (whiskers). Significance determined by Kruskal-Wallis test with Bonferroni's Multiple Comparison test.


C . Selected trajectories of 20 moving sporozoites observed over a period of 3 min . Scale bar , 10 μm .

O O O O O O O B-ORGANISM O O O O O O O O O O O O O O

C. Selected trajectories of 20 moving sporozoites observed over a period of 3 min. Scale bar, 10 μm.


( A ) Representative transmission electron microscope ( TEM ) image showing the presence of EVs in the CSF in two independent experiments .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O B-SUBCELLULAR O O B-TISSUE O O O O O

(A) Representative transmission electron microscope (TEM) image showing the presence of EVs in the CSF in two independent experiments.


( B ) NanoSightquantification of the amount of particles in the CSF 0 , 1 , 2 , 4 and 6 hours after i . p . LPS injection ( n = 3 - 5 ) .

O O O B-EXP_ASSAY O O O O O O O B-TISSUE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O

(B) NanoSightquantification of the amount of particles in the CSF 0, 1, 2, 4 and 6 hours after i.p. LPS injection (n=3-5).


( C ) Size distribution of the EVsin vivo in the CSF before ( black ; n = 5 ) and 6 h after ( grey ; n = 3 ) LPS treatment determined by NanoSight analysis .

O O O O O O O B-SUBCELLULAR O O O B-TISSUE O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY O O

(C) Size distribution of the EVsin vivo in the CSF before (black; n=5) and 6 h after (grey; n=3) LPS treatment determined by NanoSight analysis.


( D - G ) Quantitative real - time polymerase chain reaction analysis of miR - 1a ( D ) , miR - 9 ( E ) , miR - 146a ( F ) , and miR - 155 ( G ) ( n = 4 ) . RNA was isolated from pooled CSF ( 50 µl ) from different mice ( n = 3 ) . Data are displayed as mean ± SEM and analyzed by Student ' s t - test . Significance levels are indicated on the graphs : * , 0 . 01 ≤ P < 0 . 05 ; * * , 0 . 001 ≤ P < 0 . 01 .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-TISSUE O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-G) Quantitative real-time polymerase chain reaction analysis of miR-1a (D), miR-9 (E), miR-146a (F), and miR-155 (G) (n=4). RNA was isolated from pooled CSF (50 µl) from different mice (n=3). Data are displayed as mean ± SEM and analyzed by Student's t-test. Significance levels are indicated on the graphs: *, 0.01 ≤ P < 0.05; **, 0.001 ≤ P < 0.01.


( A - B ) In vitro quantification ( A ) and size distribution ( B ) of EVs isolated from conditioned medium of primary CPE cells grown in transwell system after 12 h in the absence ( black ) or presence ( grey ) of LPS ( n = 5 ) determined by NanoSight analysis .

O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O O

(A-B) In vitro quantification (A) and size distribution (B) of EVs isolated from conditioned medium of primary CPE cells grown in transwell system after 12 h in the absence (black) or presence (grey) of LPS (n=5) determined by NanoSight analysis.


( C - E ) TaqMan qPCR assay for the quantification of miR - 9 ( C ) , miR - 146a ( D ) and miR - 155 ( E ) on the exosomal pellet isolated from conditioned medium of primary CPE cells grown in a transwell system and stimulated for 12 h with LPS ( n = 3 ) .

O O O O O O B-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-SUBCELLULAR O O O O O O O B-CELL_TYPE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O

(C-E) TaqMan qPCR assay for the quantification of miR-9 (C), miR-146a (D) and miR-155 (E) on the exosomal pellet isolated from conditioned medium of primary CPE cells grown in a transwell system and stimulated for 12 h with LPS (n=3).


( F - I ) Quantification of the miRNAs miR - 1a ( F ) , miR - 9 ( G ) , miR - 146a ( H ) and miR - 155 ( I ) by TaqMan qPCR assay from primary CPE cells grown in a transwell system without or with LPS stimulation ( n = 3 ) . Significance levels are indicated on the graphs : * , 0 . 01 ≤ P < 0 . 05 ; * * , 0 . 001 ≤ P < 0 . 01 .

O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY O O O B-CELL_TYPE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O 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-I) Quantification of the miRNAs miR-1a (F), miR-9 (G), miR-146a (H) and miR-155 (I) by TaqMan qPCR assay from primary CPE cells grown in a transwell system without or with LPS stimulation (n=3). Significance levels are indicated on the graphs: *, 0.01 ≤ P < 0.05; **, 0.001 ≤ P < 0.01.


( A ) In vitro quantification of EVs isolated from conditioned medium of LPS - stimulated primary CPE cells grown in a transwell system in the absence or presence of the exosome inhibitor GW4869 ( n = 3 ) .

O O O O O O O B-SUBCELLULAR O O O O O B-SMALL_MOLECULE O O O B-CELL_TYPE O O O O O O O O O O O O O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O O O

(A) In vitro quantification of EVs isolated from conditioned medium of LPS-stimulated primary CPE cells grown in a transwell system in the absence or presence of the exosome inhibitor GW4869 (n=3).


( B - D ) TaqMan assay quantification of the miRNAs miR - 9 ( B ) , miR - 146a ( C ) and miR - 155 ( D ) in supernatant of LPS - stimulated primary CPE cells grown in a transwell system and either left untreated or pretreated with GW4869 to inhibit exosome secretion ( n = 3 ) . miR - 1a levels were below detection limit .

O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-SMALL_MOLECULE O O O B-CELL_TYPE O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SUBCELLULAR O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O

(B-D) TaqMan assay quantification of the miRNAs miR-9 (B), miR-146a (C) and miR-155 (D) in supernatant of LPS-stimulated primary CPE cells grown in a transwell system and either left untreated or pretreated with GW4869 to inhibit exosome secretion (n=3). miR-1a levels were below detection limit.


( E - H ) TaqMan assay quantification of the miRNAs miR - 1a ( E ) , miR - 9 ( F ) , miR - 146a ( G ) and miR - 155 ( H ) in cell lysate of LPS - stimulated primary CPE cells grown in a transwell system left untreated or treated with GW4869 to inhibit exosome secretion ( n = 3 ) . Data are displayed as mean ± SEM and analyzed by Student ' s t - test . Significance levels are indicated on the graphs : * , 0 . 01 ≤ P < 0 . 05 ; * * , 0 . 001 ≤ P < 0 . 01 .

O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O B-CELL_TYPE O O O O O O O O O O O B-SMALL_MOLECULE O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-H) TaqMan assay quantification of the miRNAs miR-1a (E), miR-9 (F), miR-146a (G) and miR-155 (H) in cell lysate of LPS-stimulated primary CPE cells grown in a transwell system left untreated or treated with GW4869 to inhibit exosome secretion (n=3). Data are displayed as mean ± SEM and analyzed by Student's t-test. Significance levels are indicated on the graphs: *, 0.01 ≤ P < 0.05; **, 0.001 ≤ P < 0.01.


( A ) Representative confocal images of CD63 , RAB5 and ANXA2 ( red ) in the choroid plexus ( CP ) 0 , 4 and 8 h after LPS treatment . Hoechst ( blue ) was used to stain the nucleus . The dotted line indicates the ependymal cells that line the ventricle and the square boxes indicate the zoomed insert images displayed at the right corner of each image . Scalebar = 100 µm .

O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-TISSUE I-TISSUE O O O O O O O O O O B-SMALL_MOLECULE O O B-EXP_ASSAY O O O O O O O O B-SUBCELLULAR O O O O O O B-CELL_TYPE I-CELL_TYPE O O O B-TISSUE 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 confocal images of CD63, RAB5 and ANXA2 (red) in the choroid plexus (CP) 0, 4 and 8 h after LPS treatment. Hoechst (blue) was used to stain the nucleus. The dotted line indicates the ependymal cells that line the ventricle and the square boxes indicate the zoomed insert images displayed at the right corner of each image. Scalebar = 100 µm.


( B - C ) Representative TEM images showing the presence of MVBs in the CPE cells before ( B ) and 6 hours after ( C ) LPS administration in vivo . White arrow heads point to exosomes present in MVBs ( n = 3 ) . Scale bar = 9 µm .

O O O O O O B-EXP_ASSAY O O O O O B-SUBCELLULAR O O B-CELL_TYPE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O O O O O

(B-C) Representative TEM images showing the presence of MVBs in the CPE cells before (B) and 6 hours after (C) LPS administration in vivo. White arrow heads point to exosomes present in MVBs (n=3). Scale bar = 9 µm.


( D - F ) Quantification of amount of MVBs per cell section ( D ) , amount of exosomes per MVB ( E ) , and amount of exosomes per cell section ( F ) , based on TEM analysis .

O O O O O O O O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O O O O O O O B-EXP_ASSAY O O

(D-F) Quantification of amount of MVBs per cell section (D), amount of exosomes per MVB (E), and amount of exosomes per cell section (F), based on TEM analysis.


( G - J ) Quantitative real - time polymerase chain reaction ( qPCR ) analysis of miR - 1a ( G ) , miR - 9 ( H ) , miR - 146a ( I ) , and miR - 155 ( J ) . Data are presented as relative expression normalized with housekeeping miRs by TaqMan qPCR assay ( 0 h , n = 4 ; 1 h , n = 5 ; 6 h , n = 5 ; 24 h , n = 3 ) .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O 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-J) Quantitative real-time polymerase chain reaction (qPCR) analysis of miR-1a (G), miR-9 (H), miR-146a (I), and miR-155 (J). Data are presented as relative expression normalized with housekeeping miRs by TaqMan qPCR assay (0 h, n=4; 1 h, n=5; 6 h, n=5; 24 h, n=3).


( K ) NanoSight analysis of CSF isolated from LPS - injected mice followed by icv injection of vehicle or GW4869 , a neutral sphingomyelinase inhibitor that inhibits exosome secretion ( n = 8 ) .

O O O B-EXP_ASSAY O O B-TISSUE O O B-SMALL_MOLECULE O O B-ORGANISM O O O O O O O B-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD O O O B-SUBCELLULAR O O O O O O O

(K) NanoSight analysis of CSF isolated from LPS-injected mice followed by icv injection of vehicle or GW4869, a neutral sphingomyelinase inhibitor that inhibits exosome secretion (n = 8).


( L ) qPCR analysis of the expression of miR - 1a , miR - 9 , miR - 146a and miR - 155 in the choroid plexus of mice injected with LPS and then icv injected with vehicle ( black ) or GW4869 ( grey ) , a neutral sphingomyelinase inhibitor that inhibits exosome secretion ( n = 4 ) .

O O O B-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-TISSUE I-TISSUE O B-ORGANISM O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD I-GENEPROD O O O B-SUBCELLULAR O O O O O O O

(L) qPCR analysis of the expression of miR-1a, miR-9, miR-146a and miR-155 in the choroid plexus of mice injected with LPS and then icv injected with vehicle (black) or GW4869 (grey), a neutral sphingomyelinase inhibitor that inhibits exosome secretion (n = 4).


( M ) NanoSight analysis of supernatant of choroid plexus explants from PBS - or LPS - injected mice ( n = 6 ) . Data are displayed as mean ± SEM and analyzed by Student ' s t - test . Significance levels are indicated on the graphs : * , 0 . 01 ≤ P < 0 . 05 ; * * , 0 . 001 ≤ P < 0 . 01 ; * * * , 0 . 0001 ≤ P < 0 . 001 .

O O O B-EXP_ASSAY O O O O B-TISSUE I-TISSUE O O O O O B-SMALL_MOLECULE O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(M) NanoSight analysis of supernatant of choroid plexus explants from PBS- or LPS-injected mice (n = 6). Data are displayed as mean ± SEM and analyzed by Student's t-test. Significance levels are indicated on the graphs: *, 0.01 ≤ P < 0.05; **, 0.001 ≤ P < 0.01; ***, 0.0001 ≤ P < 0.001.


( A - B ) Representative confocal images of choroid plexus ( CP ) on brain sections from naive mice and four hours after LPS injection ( n = 3 ) . Brain sections were stained for TLR4 ( A ) and TNFR1 ( B ) ( red ) , pan - cytokeratine ( panCK ; green ) and the nuclei were stained with Hoechst ( blue ) . The ependymal cells aligning the ventricles are marked with a dotted line . Scalebar = 100 µm .

O O O O O O O O O B-TISSUE I-TISSUE O O O O B-TISSUE O O O B-ORGANISM O O O O B-SMALL_MOLECULE O O O O O O O B-TISSUE O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O B-SUBCELLULAR O O O B-EXP_ASSAY O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O

(A-B) Representative confocal images of choroid plexus (CP) on brain sections from naive mice and four hours after LPS injection (n=3). Brain sections were stained for TLR4 (A) and TNFR1 (B) (red), pan-cytokeratine (panCK; green) and the nuclei were stained with Hoechst (blue). The ependymal cells aligning the ventricles are marked with a dotted line. Scalebar = 100 µm.


( C ) NanoSight analysis of EVs in the CSF in sham - operated ( black ) relative to CLP - treated ( grey ) mice 10 hours after surgery .

O O O B-EXP_ASSAY O O B-SUBCELLULAR O O B-TISSUE O O O O O O O O O O O O O O O B-ORGANISM O O O O O

(C) NanoSight analysis of EVs in the CSF in sham-operated (black) relative to CLP-treated (grey) mice 10 hours after surgery.


( D ) qPCR analysis of the expression of miR - 1a , miR - 9 , miR - 146a and miR - 155 in the choroid plexus of sham - operated mice ( black ) and mice subjected to CLP ( grey ) 10 hours after surgery . ( E ) qPCR analysis of the expression of miR - 1a , miR - 9 , miR - 146a and miR - 155 in the CSF of sham - operated mice ( black ) and mice subjected to CLP ( grey ) 10 hours after surgery .

O O O B-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-TISSUE I-TISSUE O O O O B-ORGANISM O O O O B-ORGANISM O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-TISSUE O O O O B-ORGANISM O O O O B-ORGANISM O O O O O O O O O O O

(D) qPCR analysis of the expression of miR-1a, miR-9, miR-146a and miR-155 in the choroid plexus of sham-operated mice (black) and mice subjected to CLP (grey) 10 hours after surgery. (E) qPCR analysis of the expression of miR-1a, miR-9, miR-146a and miR-155 in the CSF of sham-operated mice (black) and mice subjected to CLP (grey) 10 hours after surgery.


( F ) NanoSight analysis of EVs in CSF of control ( black ) and TNF - injected ( 25 µg / 20 g ; grey ) mice 6 hours after TNF challenge .

O O O B-EXP_ASSAY O O B-SUBCELLULAR O B-TISSUE O O O O O O B-GENEPROD O O O O O O O O O O O B-ORGANISM O O O B-GENEPROD O O

(F) NanoSight analysis of EVs in CSF of control (black) and TNF-injected (25 µg/20 g; grey) mice 6 hours after TNF challenge.


( G ) qPCR analysis of the expression of miR - 1a , miR - 9 , miR - 146a and miR - 155 in the choroid plexus of control mice ( black ) and in mice injected with TNF ( grey ) 6 hours after injection . ( H ) qPCR analysis of the expression of miR - 1a , miR - 9 , miR - 146a and miR - 155 in the CSF of control mice ( black ) and on mice 6 hours after TNF injection ( grey ) . Data are displayed as mean ± SEM and analyzed by Student ' s t - test . Scalebar 100 µm . Significance levels are indicated on the graphs : * , 0 . 01 ≤ P < 0 . 05 ; * * , 0 . 001 ≤ P < 0 . 01 ; * * * , 0 . 0001 ≤ P < 0 . 001 .

O O O B-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-TISSUE I-TISSUE O O B-ORGANISM O O O O O B-ORGANISM O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-TISSUE O O B-ORGANISM O O O O O B-ORGANISM O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) qPCR analysis of the expression of miR-1a, miR-9, miR-146a and miR-155 in the choroid plexus of control mice (black) and in mice injected with TNF (grey) 6 hours after injection. (H) qPCR analysis of the expression of miR-1a, miR-9, miR-146a and miR-155 in the CSF of control mice (black) and on mice 6 hours after TNF injection (grey). Data are displayed as mean ± SEM and analyzed by Student's t-test. Scalebar 100 µm. Significance levels are indicated on the graphs: *, 0.01 ≤ P < 0.05; **, 0.001 ≤ P < 0.01; ***, 0.0001 ≤ P < 0.001.


( A ) Representative confocal image of brain parenchyma , four hours after intracerebroventricular injection of PKH26 labelled EVs ( red ) . Astrocytes are stained with GFAP ( white ) , nuclei with Hoechst ( blue ) , and pan - cytokeratin ( panCK ; green ) was used to label the choroid plexus . The dotted line shows the ventricular border and the white arrow heads point to EVs that crossed the ependymal cell layer .

O O O O O O O B-TISSUE O O O O O O O O B-EXP_ASSAY O B-SUBCELLULAR O O O O B-CELL_TYPE O O O B-GENEPROD O O O O B-SUBCELLULAR O B-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O B-SUBCELLULAR O O O B-CELL_TYPE I-CELL_TYPE O O

(A) Representative confocal image of brain parenchyma, four hours after intracerebroventricular injection of PKH26 labelled EVs (red). Astrocytes are stained with GFAP (white), nuclei with Hoechst (blue), and pan-cytokeratin (panCK; green) was used to label the choroid plexus. The dotted line shows the ventricular border and the white arrow heads point to EVs that crossed the ependymal cell layer.


( B - C ) Representative confocal images showing the uptake of double - labelled EVs ( Ribogreen ; green , PKH26 , red ) by astrocytes stained with GFAP ( white ) ( B ) and microglia cells stained with IBA1 ( white ) ( C ) incubated on mixed cortical cultures . Boxed areas are shown as zoomed images on right . Cell nuclei are stained with Hoechst ( blue ) . The in vivo and in vitro uptake experiments were performed 3 and 2 times ( n = 3 ) , respectively . Scalebar 100 = µm .

O O O O O O O O O O O O O O O B-SUBCELLULAR O B-EXP_ASSAY O O O B-EXP_ASSAY O O O O B-CELL_TYPE O O B-GENEPROD O O O O O O O B-CELL_TYPE O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O B-EXP_ASSAY O O O O O O O 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-C) Representative confocal images showing the uptake of double-labelled EVs (Ribogreen; green, PKH26, red) by astrocytes stained with GFAP (white) (B) and microglia cells stained with IBA1 (white) (C) incubated on mixed cortical cultures. Boxed areas are shown as zoomed images on right. Cell nuclei are stained with Hoechst (blue). The in vivo and in vitro uptake experiments were performed 3 and 2 times (n=3), respectively. Scalebar 100 = µm.


( A - B ) qPCR gene expression analysis of miRNA target genes Mapk3 , Notch1 , Dicer , Tab2 , Sox2 , Calm2 , Smad2 , Smad5 , Dnmt3 and Irak1 in mixed cortical cultures incubated with EVs isolated from CSF from untreated ( black ) or LPS treated ( grey ) mice ( A ) and in brain tissue ( B ) before ( black ) and 8 h after LPS injection ( grey ) ( n = 3 ) .

O O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-SUBCELLULAR O O B-TISSUE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-TISSUE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O

(A-B) qPCR gene expression analysis of miRNA target genes Mapk3, Notch1, Dicer, Tab2, Sox2, Calm2, Smad2, Smad5, Dnmt3 and Irak1 in mixed cortical cultures incubated with EVs isolated from CSF from untreated (black) or LPS treated (grey) mice (A) and in brain tissue (B) before (black) and 8 h after LPS injection (grey) (n=3).


( C ) qPCR gene expression analysis of inflammatory genes Il - 1β , Tnf , Il - 6 , iNos and iκbα by qPCR in mixed cortical cultures ( left ; in vitro ) incubated with EVs isolated from CSF from untreated ( black ) or LPS treated ( grey ) mice and in brain tissue ( right ; in vivo ) before ( black ) and 8 h after LPS injection ( grey ) ( n = 3 ) .

O O O B-EXP_ASSAY O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY O O O O O O O O O O O O B-SUBCELLULAR O O B-TISSUE O O O O O O B-SMALL_MOLECULE O O O O B-ORGANISM O O B-TISSUE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O

(C) qPCR gene expression analysis of inflammatory genes Il-1β, Tnf, Il-6, iNos and iκbα by qPCR in mixed cortical cultures (left; in vitro) incubated with EVs isolated from CSF from untreated (black) or LPS treated (grey) mice and in brain tissue (right; in vivo) before (black) and 8 h after LPS injection (grey) (n=3).


( D ) Cytokine analysis ( IL - 6 , IL - 1β and TNF ) of supernatant of mixed cortical cultures ( left ; in vitro ) incubated with EVs isolated from untreated ( black ; n = 3 ) or LPS treated ( grey ; n = 3 ) mice and of CSF ( right ; in vivo ) from untreated ( black ; n = 3 ) and LPS treated mice ( grey ; n = 6 ) .

O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-ORGANISM O O B-TISSUE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-ORGANISM O O O O O O O O

(D) Cytokine analysis (IL-6, IL-1β and TNF) of supernatant of mixed cortical cultures (left; in vitro) incubated with EVs isolated from untreated (black; n=3) or LPS treated (grey; n=3) mice and of CSF (right; in vivo) from untreated (black; n=3) and LPS treated mice (grey; n=6).


( E - F ) qPCR gene expression analysis of inflammatory genes Il - 1β , Tnf , Il - 6 , iNos and iκbα ( E ) and miRNA target genes Mapk3 , Notch1 , Dicer , Tab2 , Sox2 , Calm2 , Smad2 , Smad5 , Dnmt3 and Irak1 ( F ) in mixed cortical cultures incubated with CD63 - depleted EVs isolated from untreated mice ( black ) and mice treated with LPS for 6 h ( grey ) ( n = 3 ) .

O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O O B-SUBCELLULAR O O O B-ORGANISM O O O O B-ORGANISM O O B-SMALL_MOLECULE O O O O O O O O O O O O

(E-F) qPCR gene expression analysis of inflammatory genes Il-1β, Tnf, Il-6, iNos and iκbα (E) and miRNA target genes Mapk3, Notch1, Dicer, Tab2, Sox2, Calm2, Smad2, Smad5, Dnmt3 and Irak1 (F) in mixed cortical cultures incubated with CD63-depleted EVs isolated from untreated mice (black) and mice treated with LPS for 6 h (grey) (n=3).