RT - PCR in P30 mice showing presence of Clrn2 transcripts in inner ear , eye , but not in brain or muscle . β - actin was used as a positive control .

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RT-PCR analysis in P30 mice showing the presence of Clrn2 transcripts in the inner ear, eye, but not in brain or muscle. β-actin was used as a positive control.


Clrn2 transcripts could be detected in both inner ( IHCs ) outer ( OHCs ) hair of P15 wild - type mice . Otoferlin ( Otof ) oncomodulin ( Ocm ) transcripts were used as positive controls for IHCs OHCs , respectively . Ocm transcripts were only present in OHC lysate , demonstrating that sample had not been contaminated with OHCs .

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Clrn2 transcripts could be detected in both inner (IHCs) and outer (OHCs) hair cells of P15 wild-type mice. Otoferlin (Otof) and oncomodulin (Ocm) transcripts were used as positive controls for IHCs and OHCs, respectively. Ocm transcripts were only present in the OHC lysate, demonstrating that the IHC sample had not been contaminated with OHCs.


Auditory phenotyping of clarinet mice at P16 ( C ) , P21 ( D ) ABR threshold measurements show that Clrn2clarinet / clarinet mice ( red ) exhibit a severe - to - profound hearing loss affecting frequencies tested . At 16 kHz in Clrn2clarinet / clarinet mice , mean ABR hearing thresholds vary from 55 - 65 dB SPL at P16 , 60 - 90 dB SPL at P21 , to 80 - 100 dB SPL at P28 P42 . - matched Clrn2 + / + ( black ) Clrn2clarinet / + ( grey ) controls display thresholds within expected range ( 15 - 45 dB SPL ) at frequencies time - points tested . At P16 , eight Clrn2clarinet / clarinet mice exhibited recordable ABR responses for each frequency tested click stimulus . For longitudinal ABR study , at P21 P28 three of seven Clrn2clarinet / clarinet mice were found to not respond at highest stimulus ( 90 dB SPL ) for at least one frequency - specific / click stimulus . By P42 , five of Clrn2clarinet / clarinet mice were found to not respond at highest stimulus ( 90 dB SPL ) for at least two frequency - specific / click stimuli . ABR data shown are mean ± s . d . * * * p < 0 . 001 , one - way ANOVA .

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Auditory phenotyping of clarinet mice at P16 (C), P21 (D) ABR threshold measurements show that Clrn2clarinet/clarinet mice (red) exhibit a severe-to-profound hearing loss affecting all frequencies tested. At 16 kHz in Clrn2clarinet/clarinet mice, the mean ABR hearing thresholds vary from 55-65 dB SPL at P16, 60-90 dB SPL at P21, and to 80-100 dB SPL at P28 and P42. Age-matched Clrn2+/+ (black) and Clrn2clarinet/+ (grey) controls display thresholds within the expected range (15-45 dB SPL) at all frequencies and time-points tested. At P16, all eight Clrn2clarinet/clarinet mice exhibited recordable ABR responses for each frequency tested and the click stimulus. For the longitudinal ABR study, at P21 and P28 three of the seven Clrn2clarinet/clarinet mice were found to not respond at the highest intensity stimulus (90 dB SPL) for at least one frequency-specific/click stimulus. By P42, five of the Clrn2clarinet/clarinet mice were found to not respond at the highest intensity stimulus (90 dB SPL) for at least two frequency-specific/click stimuli. ABR data shown are mean ± s.d. ***p<0.001, one-way ANOVA.


Auditory phenotyping of clarinet mice at P28 ( E ) P42 ( F ) . ABR threshold measurements show that Clrn2clarinet / clarinet mice ( red ) exhibit a severe - to - profound hearing loss affecting frequencies tested . At 16 kHz in Clrn2clarinet / clarinet mice , mean ABR hearing thresholds vary from 55 - 65 dB SPL at P16 , 60 - 90 dB SPL at P21 , to 80 - 100 dB SPL at P28 P42 . - matched Clrn2 + / + ( black ) Clrn2clarinet / + ( grey ) controls display thresholds within expected range ( 15 - 45 dB SPL ) at frequencies time - points tested . At P16 , eight Clrn2clarinet / clarinet mice exhibited recordable ABR responses for each frequency tested click stimulus . For longitudinal ABR study , at P21 P28 three of seven Clrn2clarinet / clarinet mice were found to not respond at highest stimulus ( 90 dB SPL ) for at least one frequency - specific / click stimulus . By P42 , five of Clrn2clarinet / clarinet mice were found to not respond at highest stimulus ( 90 dB SPL ) for at least two frequency - specific / click stimuli . ABR data shown are mean ± s . d . * * * p < 0 . 001 , one - way ANOVA .

Auditory phenotyping of clarinet mice at P28 (E) and P42 (F). ABR threshold measurements show that Clrn2clarinet/clarinet mice (red) exhibit a severe-to-profound hearing loss affecting all frequencies tested. At 16 kHz in Clrn2clarinet/clarinet mice, the mean ABR hearing thresholds vary from 55-65 dB SPL at P16, 60-90 dB SPL at P21, and to 80-100 dB SPL at P28 and P42. Age-matched Clrn2+/+ (black) and Clrn2clarinet/+ (grey) controls display thresholds within the expected range (15-45 dB SPL) at all frequencies and time-points tested. At P16, all eight Clrn2clarinet/clarinet mice exhibited recordable ABR responses for each frequency tested and the click stimulus. For the longitudinal ABR study, at P21 and P28 three of the seven Clrn2clarinet/clarinet mice were found to not respond at the highest intensity stimulus (90 dB SPL) for at least one frequency-specific/click stimulus. By P42, five of the Clrn2clarinet/clarinet mice were found to not respond at the highest intensity stimulus (90 dB SPL) for at least two frequency-specific/click stimuli. ABR data shown are mean ± s.d. ***p<0.001, one-way ANOVA.


Averaged DPOAE responses for clarinet mice at P28 , showing significantly reduced responses in Clrn2clarinet / clarinet mutants at frequencies tested . DPOAE data shown are mean ± s . d . * p < 0 . 02 , * * p < 0 . 01 , one - way ANOVA . Please see Appendix supplemental table 1 for exact P - values .

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Averaged DPOAE responses for clarinet mice at P28, showing significantly reduced responses in Clrn2clarinet/clarinet mutants at all frequencies tested. DPOAE data shown are mean ± s.d. *p<0.02, **p<0.01, one-way ANOVA. Please see Appendix supplemental table 1 for exact P-values.


Vestibular behavioural ( swim , contact - righting , trunk - curl , platform ) . Clrn2clarinet / clarinet mice ( red , P28 , n = 5 , P60 , n = 7 ) have no vestibular dysfunction , displaying similar performances to - matched control Clrn2clarinet / + mice ( grey , P28 , n = 3 , P60 , n = 7 ) . Being similar , values at P28 P60 were combined . Electroretinogram ( ERG ) measurements from control Clrn2clarinet / + ( grey ) mutant Clrn2clarinet / clarinet ( red ) mice . Each trace in B C is representative of an ERG response from eye of - matched Clrn2clarinet / + Clrn2clarinet / clarinet mice , showing no significant difference in a - or b - wave amplitudes . ( D - F ) lack of change in ERG amplitude responses in Clrn2clarinet / clarinet mice ( aged 3 or 6 - 7 months ) , regardless of conditions : scotopic ( D , E ) , or photopic ( F ) , indicate normal photoreceptor kinetics no change in sensitivity of photoreceptor .

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Vestibular behavioural tests (swim tests, contact-righting, trunk-curl, and platform). The Clrn2clarinet/clarinet mice (red, P28, n=5, and P60, n=7) have no vestibular dysfunction, displaying similar performances to age-matched control Clrn2clarinet/+ mice (grey, P28, n=3, and P60, n=7). Being similar, the values at P28 and P60 were combined. Electroretinogram (ERG) measurements from control Clrn2clarinet/+ (grey) and mutant Clrn2clarinet/clarinet (red) mice. Each trace in B and C is representative of an ERG response from the eye of age-matched Clrn2clarinet/+ and Clrn2clarinet/clarinet mice, showing no significant difference in a- or b-wave amplitudes. (D-F) The lack of change in ERG amplitude responses in Clrn2clarinet/clarinet mice (aged 3 or 6-7 months), regardless of the test conditions: scotopic (D,E), or photopic (F), indicate normal photoreceptor kinetics and no change in the sensitivity of photoreceptor cells.


Electroretinogram ( ERG ) measurements from control Clrn2clarinet / + ( grey ) mutant Clrn2clarinet / clarinet ( red ) mice . Each trace in B C is representative of an ERG response from eye of - matched Clrn2clarinet / + Clrn2clarinet / clarinet mice , showing no significant difference in a - or b - wave amplitudes . ( D - F ) lack of change in ERG amplitude responses in Clrn2clarinet / clarinet mice ( aged 3 or 6 - 7 months ) , regardless of conditions : scotopic ( D , E ) , or photopic ( F ) , indicate normal photoreceptor kinetics no change in sensitivity of photoreceptor .

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Electroretinogram (ERG) measurements from control Clrn2clarinet/+ (grey) and mutant Clrn2clarinet/clarinet (red) mice. Each trace in B and C is representative of an ERG response from the eye of age-matched Clrn2clarinet/+ and Clrn2clarinet/clarinet mice, showing no significant difference in a- or b-wave amplitudes. (D-F) The lack of change in ERG amplitude responses in Clrn2clarinet/clarinet mice (aged 3 or 6-7 months), regardless of the test conditions: scotopic (D,E), or photopic (F), indicate normal photoreceptor kinetics and no change in the sensitivity of photoreceptor cells.


Confocal microscopy of whole - mount preparations of mid - basal cochlear sensory epithelia from Clrn2clarinet / + ( A ) Clrn2clarinet / clarinet ( B ) P6 mice immunostained for actin . Despite lack of clarin - 2 , developing sensory epithelium of mutants is similar to that of heterozygous controls .

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Confocal microscopy images of whole-mount preparations of mid-basal cochlear sensory epithelia from Clrn2clarinet/+ (A) and Clrn2clarinet/clarinet (B) P6 mice immunostained for actin. Despite lack of clarin-2, the developing sensory epithelium of mutants is similar to that of heterozygous controls.


Confocal microscopy of whole - mount preparations of cochlear sensory epithelia from Clrn2clarinet / + ( C ) , Clrn1 - / - ( D ) , Clrn2clarinet / clarinet ( E ) P6 mice immunostained for stereocilin ( green ) actin ( red ) . Unlike fragmented immunostaining in Clrn1 - / - mice ( mirroring fragmentation of hair bundle ) , stereocilin immunostaining in Clrn2clarinet / clarinet mice reflects normally V - shaped bundles of OHCs , similar to Clrn2clarinet / + OHCs .

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Confocal microscopy images of whole-mount preparations of cochlear sensory epithelia from Clrn2clarinet/+ (C), Clrn1-/- (D), and Clrn2clarinet/clarinet (E) P6 mice immunostained for stereocilin (green) and actin (red). Unlike the fragmented immunostaining in Clrn1-/- mice (mirroring the fragmentation of the hair bundle), stereocilin immunostaining in Clrn2clarinet/clarinet mice reflects the normally V-shaped bundles of OHCs, similar to Clrn2clarinet/+ OHCs.


Representative scanning electron of sensory epithelium of Clrn2clarinet / + ( F ) Clrn2clainet / clarinet ( G ) P8 mice , showing no apparent differences in gross patterning of IHCs OHCs . Arrowheads in a , b ( ) , f g ( black ) illustrate convex shape of apical circumference of OHCs .

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Representative scanning electron micrographs of the sensory epithelium of Clrn2clarinet/+ (F) and Clrn2clainet/clarinet (G) P8 mice, showing no apparent differences in the gross patterning of IHCs and OHCs. Arrowheads in a,b (white),f and g (black) illustrate the convex shape of the apical circumference of OHCs.


Pseudo - coloured scanning electron of outer inner hair bundles from clarinet mice at P8 P16 , showing that gross of OHC bundles is similar between Clrn2 + / + Clrn2clarinet / clarinet mice . Representative from mid region of cochlear spiral are shown close - up views illustrate three full rows , tallest ( red ) , middle ( blue ) short ( yellow ) , of stereocilia in OHC hair bundles . At P8 P16 ( upper panels in A B ) , three stereocilia rows are observed in Clrn2clarinet / clarinet OHCs ( A ) IHCs ( B ) , although shortest middle row of stereocilia appear less prolate compared to controls . ( C , D ) Distribution of stereocilia measures across genotypes at P8 P16 .

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Pseudo-coloured scanning electron micrographs of individual outer and inner hair cell bundles from clarinet mice at P8 and P16, showing that gross morphology of OHC and IHC bundles is similar between Clrn2+/+ and Clrn2clarinet/clarinet mice. Representative images from the mid region of the cochlear spiral are shown and close-up views illustrate the three full rows, tallest (red), middle (blue) and short (yellow), of stereocilia in IHC and OHC hair bundles. At P8 and P16 (upper panels in A and B), the three stereocilia rows are observed in Clrn2clarinet/clarinet OHCs (A) and IHCs (B), although the shortest and middle row of IHC stereocilia appear less prolate compared to controls. (C,D) Distribution of individual stereocilia measures across genotypes at P8 and P16.


OHC stereocilia measurements at both P8 P16 ( C ) show a significant difference in height of Clrn2 + / + stereocilia compared to Clrn2clarinet / clarinet in : e . g . for shortest row at P8 : Clrn2 + / + : 0 . 6188 ± 0 . 0944 p . µm ( n = 27 ) ; Clrn2clarinet / clarinet : 0 . 2948 ± 0 . 1029 p . µm ( n = 27 ) ( p < 0 . 0001 , Mann - Whitney ranks comparison ) , at P16 : Clrn2 + / + : 0 . 579 ± 0 . 06179 p . µm ( n = 27 ) ; Clrn2clarinet / clarinet : 0 . 07282 ± 0 . 1626 p . µm ( n = 27 ) ( p < 0 . 0001 , Mann - Whitney ranks comparison ) . Note amount of value points equal to zero at P16 . ( D ) Conversely , stereocilia measurements at P8 P16 did not show a difference between Clrn2 + t / + Clrn2clarinet / clarinet in : for shortest row at P8 : Clrn2 + / + : 0 . 8808 ± 0 . 1753 p . µm ( n = 27 ) ; Clrn2clarinet / clarinet : 0 . 9313 ± 0 . 1217 p . µm ( n = 27 ) ( p = 0 . 2244 , unpaired t - ) ; at P16 : Clrn2 + / + : 0 . 6784 ± 0 . 1171 p . µm ( n = 23 ) ; Clrn2clarinet / clarinet : 0 . 6063 ± 0 . 1855 p . µm ( n = 26 ) ( p = 0 . 2436 , Mann - Whitney ranks comparison ) .

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The OHC stereocilia measurements at both P8 and P16 (C) show a significant difference in height of Clrn2+/+ stereocilia compared to Clrn2clarinet/clarinet in: e.g. for the shortest row at P8: Clrn2+/+: 0.6188 ± 0.0944 p.µm (n=27); Clrn2clarinet/clarinet: 0.2948 ± 0.1029 p.µm (n=27) (p<0.0001, Mann-Whitney ranks comparison), and at P16: Clrn2+/+: 0.579 ± 0.06179 p.µm (n=27); Clrn2clarinet/clarinet: 0.07282 ± 0.1626 p.µm (n=27) (p<0.0001, Mann-Whitney ranks comparison). Note the amount of value points equal to zero at P16. (D) Conversely, IHC stereocilia measurements at P8 and P16 did not show a difference between Clrn2+t/+ and Clrn2clarinet/clarinet in: for the shortest row at P8: Clrn2+/+: 0.8808 ± 0.1753 p.µm (n=27); Clrn2clarinet/clarinet: 0.9313 ± 0.1217 p.µm (n=27) (p=0.2244, unpaired t-test); and at P16: Clrn2+/+: 0.6784 ± 0.1171 p.µm (n=23); Clrn2clarinet/clarinet: 0.6063 ± 0.1855 p.µm (n=26) (p=0.2436, Mann-Whitney ranks comparison).


Sensory hair bundle patterning measurements in clarinet mice Pseudo - coloured scanning electron of OHC ( left panels ) ( right panels ) hair bundles from clarinet mice at P28 . Clrn2clarinet / clarinet mutants have only two rows of OHC stereocilia , middle row of stereocilia is less uniform in height compared to controls . middle short rows of stereocilia in Clrn2clarinet / clarinet bundles appear fewer in number , heterogeneous in height . Scale - 2 µm .

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Sensory hair cell bundle patterning and measurements in clarinet mice Pseudo-coloured scanning electron micrographs of individual OHC (left panels) and IHC (right panels) hair bundles from clarinet mice at P28. Clrn2clarinet/clarinet mutants have only two rows of OHC stereocilia, and the middle row of stereocilia is less uniform in height compared to controls. The middle and short rows of stereocilia in Clrn2clarinet/clarinet IHC bundles appear fewer in number, and heterogeneous in height. Scale - 2 µm.


Schematic representation of auditory sensory organ , illustrating positioning of electrode used for injectoporation of GFP - tagged clarin - 2 construct into cochlear supporting hair . ( B - F ) Top side views of representative of supporting ( asterisk ) hair expressing clarin - 2 . In supporting , GFP - tagged clarin - 2 ( green ) was distributed diffusely throughout cytoplasm ( A , B ) . By contrast , in injectoporated hair , majority of clarin - 2 was observed in apical stereocilia ( C - E ) . Scale - 2 µm .

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Schematic representation of the auditory sensory organ, illustrating the positioning of the electrode used for injectoporation of GFP-tagged clarin-2 construct into cochlear supporting and hair cells. (B-F) Top and side views of representative images of supporting cells (asterisk) and hair cells expressing clarin-2. In supporting cells, GFP-tagged clarin-2 (green) was distributed diffusely throughout the cytoplasm (A,B). By contrast, in all injectoporated hair cells, the majority of clarin-2 was observed in the apical stereocilia (C-E). Scale - 2 µm.


Confocal of OHC hair bundles of Clrn2clarinet / clarinet mice heterozygous at P6 immunostained for PDZD7 ( A , green ) whirlin ( B , green ) actin ( red in both figures ) . PDZD7 immunostaining is normally restricted to base of stereocilia in both Clrn2clarinet / + Clrn2clarinet / clarinet P6 mice ( A ) . Whirlin immunostaining is properly located at stereocilia tips of OHC hair bundles ( B ) .

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Confocal images of IHC and OHC hair bundles of Clrn2clarinet/clarinet mice and heterozygous littermates at P6 immunostained for PDZD7 (A, green) and whirlin (B, green) and actin (red in both figures). The PDZD7 immunostaining is normally restricted to the base of stereocilia in both Clrn2clarinet/+ and Clrn2clarinet/clarinet P6 mice (A). Whirlin immunostaining is properly located at the stereocilia tips of IHC and OHC hair bundles (B).


Harmonin - b immunostaining in hair bundles . In Clrn2clarinet / clarinet mice ( D ) , harmonin - b immunoreactive ( green ) were still observable on stereocilia , but unlike in - matched heterozygote ( C ) , were located much closer to tip of stereocilia . two diagrams in ( C ) ( D ) illustrate position of harmonin - b immunostaining ( green ) corresponding to site of upper attachment point of tip link ( UAPTL ) , facing tip link . bright green signal outside stereociliary bundles are non - specific . change of harmonin - b localization along stereocilium in Clrn2clarinet / clarinet mice is illustrated further by scan ( E ) ( F ) analyses . insets in ( E ) show of stereocilia used for scan signal . harmonin - b immunoreactive were located within 850 ± 28 nm from tip of tallest stereocilia in Clrn2clarine / + mice ( n = 31 hair bundles from 5 mice ) ( F ) , within 575 ± 23 nm in Clrn2clarinet / clarinet mice ( n = 35 hair bundles from 5 mice ) .

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Harmonin-b immunostaining in IHC hair bundles. In Clrn2clarinet/clarinet mice (D), the harmonin-b immunoreactive puncta (green) were still observable on the stereocilia, but unlike in age-matched heterozygote littermates (C), were located much closer to the tip of stereocilia. The two diagrams in (C) and (D) illustrate the position of harmonin-b immunostaining (green) corresponding to the site of the upper attachment point of the tip link (UAPTL), facing the tip link. The bright green signal outside stereociliary bundles are non-specific. The change of harmonin-b localization along the stereocilium in Clrn2clarinet/clarinet mice is illustrated further by line scan (E) and quantification (F) analyses. The insets in (E) show images of individual stereocilia used for the line scan signal analysis. The harmonin-b immunoreactive puncta were located within 850 ± 28 nm distance from the tip of the tallest stereocilia in Clrn2clarine/+ mice (n=31 hair bundles from 5 mice) (F), and within 575 ± 23 nm in Clrn2clarinet/clarinet mice (n=35 hair bundles from 5 mice).


Cochlear whole - mounts from P21 clarinet mice , labelled with pre - synaptic ribbon marker Ribeye ( red ) post - synaptic density marker GluR2 ( green ) , showing a similar number of total matched Ribeye - positive GluR2 - postive in Clrn2clarinet / clarinet mutant cochleae compared to Clrn2clarinet / + , which is reflected in counted per five hair ( H ) n = 4 per genotype .

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Cochlear whole-mounts from P21 clarinet mice, labelled with the IHC pre-synaptic ribbon marker Ribeye (red) and the post-synaptic density marker GluR2 (green), showing a similar number of total and matched Ribeye-positive and GluR2-postive puncta in Clrn2clarinet/clarinet mutant cochleae compared to Clrn2clarinet/+ littermates, which is reflected in puncta counted per five hair cells (H) n = 4 per genotype.


Saturating MET currents recorded from P6 Clrn2clarinet / + ( A ) Clrn2clarinet / clarinet ( B ) apical - coil OHCs by applying sinusoidal force stimuli of 50 Hz to hair bundles at − 121 mV + 99 mV . driver voltage ( DV ) signal of ± 40 V to fluid jet is shown above traces ( positive deflections of DV are excitatory ) . holding potential was − 81 mV . Extracellular Ca2 + was 1 . 3 mM . Arrows arrowheads indicate closure of MET currents ( i . e . resting MET current ) elicited during inhibitory bundle displacements at hyperpolarized depolarized potentials , respectively . Dashed lines indicate holding current , which is current at holding potential .

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Saturating MET currents recorded from P6 Clrn2clarinet/+ (A) and Clrn2clarinet/clarinet (B) apical-coil OHCs by applying sinusoidal force stimuli of 50 Hz to the hair bundles at −121 mV and +99 mV. The driver voltage (DV) signal of ± 40 V to the fluid jet is shown above the traces (positive deflections of the DV are excitatory). The holding potential was −81 mV. Extracellular Ca2+ concentration was 1.3 mM. Arrows and arrowheads indicate the closure of the MET currents (i.e. resting MET current) elicited during inhibitory bundle displacements at hyperpolarized and depolarized membrane potentials, respectively. Dashed lines indicate the holding current, which is the current at the holding membrane potential.


Average peak - to - peak current - voltage curves recorded from Clrn2clarinet / + ( grey , P6 , n = 7 ) Clrn2clarinet / clarinet ( red , P6 - 7 , n = 9 ) apical coil OHCs .

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Average peak-to-peak current-voltage curves recorded from Clrn2clarinet/+ (grey, P6, n=7) and Clrn2clarinet/clarinet (red, P6-7, n=9) apical coil OHCs.


Resting open probability ( Po ) of MET current at potential of − 121 mV + 99 mV from apical - ( D ) basal - coil ( E ) OHCs . Number of OHCs investigated is shown above columns .

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Resting open probability (Po) of the MET current at the membrane potential of −121 mV and + 99 mV from apical- (D) and basal-coil (E) OHCs. Number of OHCs investigated is shown above the columns.


Saturating MET currents recorded from a P7 Clrn2clarinet / + ( F ) a P8 Clrn2clarinet / clarinet ( G ) apical - coil using same experimental protocol described above .

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Saturating MET currents recorded from a P7 Clrn2clarinet/+ (F) and a P8 Clrn2clarinet/clarinet (G) apical-coil IHC using the same experimental protocol described above.


Average Po of MET current measured in apical coil IHCs at potential of − 121 mV + 99 mV from Clrn2clarinet / + ( P7 , n = 3 ) Clrn2clarinet / clarinet ( P7 - 8 , n = 6 ) apical - coil IHCs .

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Average Po of the MET current measured in apical coil IHCs at the membrane potential of −121 mV and + 99 mV from Clrn2clarinet/+ (P7, n=3) and Clrn2clarinet/clarinet (P7-8, n=6) apical-coil IHCs.


Potassium currents recorded from mature Clrn2clarinet / + ( A , P15 ) Clrn2clarinet / clarinet ( B , P16 ) apical - coil OHCs . Currents were elicited by depolarizing voltage steps ( 10 mV nominal increments ) from - 144 mV to more depolarized values from holding potential of - 84 mV . Note that current characteristic of mature OHCs , of IK , n , measured in isolation as deactivating tail currents ( difference between instantaneous steady - state inward currents ) for voltage steps from holding potential to − 124 mV , was 545 ± 115 pA , ( n = 5 ) in Clrn2clarinet / + 595 ± 67 pA , ( n = 7 ) in Clrn2clarinet / clarinet OHCs .

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Potassium currents recorded from mature Clrn2clarinet/+ (A, P15) and Clrn2clarinet/clarinet (B, P16) apical-coil OHCs. Currents were elicited by depolarizing voltage steps (10 mV nominal increments) from -144 mV to more depolarized values from the holding potential of -84 mV. Note that the current characteristic of mature OHCs, The size of IK,n, measured in isolation as the deactivating tail currents (difference between instantaneous and steady-state inward currents) for voltage steps from the holding potential to −124 mV, was 545 ± 115 pA, (n=5) in Clrn2clarinet/+ and 595 ± 67 pA, (n=7) in Clrn2clarinet/clarinet OHCs.


Average peak current - voltage relation for total K + current recorded from Clrn2clarinet / + ( P15 - 16 , n = 7 ) Clrn2clarinet / clarinet ( P15 - 16 , n = 5 ) OHCs .

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Average peak current-voltage relation for the total K+ current recorded from Clrn2clarinet/+ (P15-16, n=7) and Clrn2clarinet/clarinet (P15-16, n=5) OHCs.


Potassium currents recorded from P22 mature Clrn2clarinet / + Clrn2clarinet / clarinet apical - coil IHCs , respectively , using same voltage protocol described above . Holding potential of - 64 mV .

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Potassium currents recorded from P22 mature Clrn2clarinet/+ and Clrn2clarinet/clarinet apical-coil IHCs, respectively, using the same voltage protocol described above. Holding potential of -64 mV.


Peak current - voltage relation for total K + current recorded from

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Peak current-voltage relation for the total K+ current recorded from the IHCs


Voltage responses from P22 Clrn2clarinet / + ( G ) Clrn2clarinet / clarinet ( H , I ) IHCs . Note that Ca2 + - dependent action potentials could be induced in mature IHCs ( I ) .

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Voltage responses from P22 Clrn2clarinet/+ (G) and Clrn2clarinet/clarinet (H,I) IHCs. Note that Ca2+-dependent action potentials could be induced in mature IHCs (I).


( A ) BH3 ‐ only BIK displaces a subset of BCL ‐ 2 complexes at ER . Purified LM from H1299 HA ‐ BCL ‐ 2b5 either mock infected or infected with Ad ‐ BIKb5 was subjected to cross ‐ linking with BMH visualized by immunoblot . Asterisks ( * ) denote BCL ‐ 2 cross ‐ linked products , which are displaced by BIK . Bold arrow denotes region containing cross ‐ linked NAF ‐ 1 / BCL ‐ 2 .

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(A) BH3‐only BIK displaces a subset of BCL‐2 complexes at the ER. Purified LM from H1299 HA‐BCL‐2b5 cells either mock infected or infected with Ad‐BIKb5 was subjected to cross‐linking with BMH and visualized by immunoblot. Asterisks (*) denote BCL‐2 cross‐linked products, which are displaced by BIK. Bold arrow denotes region containing cross‐linked NAF‐1/BCL‐2.


( D ) Endogenous NAF ‐ 1 cross ‐ links with BCL ‐ 2 at ER . LM purified from H1299 neo HA ‐ BCL ‐ 2b5 were subjected to cross ‐ linking by BMH immunoprecipitation with anti ‐ NAF ‐ 1 antibody . Precipitates were analysed by immunoblot with anti ‐ BCL ‐ 2 .

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(D) Endogenous NAF‐1 cross‐links with BCL‐2 at the ER. LM purified from H1299 neo and HA‐BCL‐2b5 cells were subjected to cross‐linking by BMH and immunoprecipitation with anti‐NAF‐1 antibody. Precipitates were analysed by immunoblot with anti‐BCL‐2.


( E ) NAF ‐ 1 is displaced from BCL ‐ 2 by BH3 ‐ only BIK . H1299 HA ‐ BCL ‐ 2b5 were either mock infected or infected with Ad ‐ BIK . Purified LM was treated as in ( D ) .

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(E) NAF‐1 is displaced from BCL‐2 by BH3‐only BIK. H1299 HA‐BCL‐2b5 cells were either mock infected or infected with Ad‐BIK. Purified LM was treated as in (D).


( A ) Co ‐ immunoprecipitation of endogenous NAF ‐ 1 BCL ‐ 2 . Lysates from SK ‐ Mel5 were collected immunoprecipitation was performed with anti ‐ NAF ‐ 1 antibody . precipitate was subjected to immunoblotting with anti ‐ BCL ‐ 2 anti ‐ NAF ‐ 1 .

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(A) Co‐immunoprecipitation of endogenous NAF‐1 and BCL‐2. Lysates from SK‐Mel5 cells were collected and immunoprecipitation was performed with anti‐NAF‐1 antibody. The precipitate was subjected to immunoblotting with anti‐BCL‐2 and anti‐NAF‐1.


( B ) H1299 were fixed double stained with anti ‐ NAF ‐ 1 anti ‐ Calnexin or anti ‐ Cytochrome c antibodies . Scale bar represents 10 μm .

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(B) H1299 cells were fixed and double stained with anti‐NAF‐1 and anti‐Calnexin or anti‐Cytochrome c antibodies. Scale bar represents 10 μm.


( C ) Co ‐ immunoprecipitation of NAF ‐ 1 ‐ Flag HA ‐ BCL ‐ 2b5 . Lysates from H1299 HA ‐ BCL ‐ 2b5 infected with Ad ‐ NAF ‐ 1 ‐ Flag were collected treated as in ( A ) .

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(C) Co‐immunoprecipitation of NAF‐1‐Flag and HA‐BCL‐2b5. Lysates from H1299 HA‐BCL‐2b5 cells infected with Ad‐NAF‐1‐Flag were collected and treated as in (A).


( D ) Mutations in CDGSH iron ‐ binding domain of NAF ‐ 1 interfere with NAF ‐ 1 binding to BCL ‐ 2 . H1299 HA ‐ BCL ‐ 2b5 were infected with either Ad ‐ rtTA , Ad ‐ NAF ‐ 1 ‐ Flag , or Ad ‐ NAF ‐ 1 ‐ mut ‐ Flag ( C99S C101S C110S H114Q ) . Lysates were treated as in ( A ) . Densitometric was performed using Scion Image software to quantify expression co ‐ precipitated of NAF ‐ 1 ‐ Flag NAF ‐ 1 ‐ mut ‐ Flag . Graph depicts of co ‐ precipitated to expression level .

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(D) Mutations in the CDGSH iron‐binding domain of NAF‐1 interfere with NAF‐1 binding to BCL‐2. H1299 HA‐BCL‐2b5 cells were infected with either Ad‐rtTA, Ad‐NAF‐1‐Flag, or Ad‐NAF‐1‐mut‐Flag (C99S C101S C110S H114Q). Lysates were treated as in (A). Densitometric analysis was performed using Scion Image software to quantify expression and co‐precipitated levels of NAF‐1‐Flag and NAF‐1‐mut‐Flag. Graph depicts the ratio of co‐precipitated protein to expression level.


( E ) A functional CDGSH iron ‐ binding domain is necessary , but not sufficient for interaction between domains of NAF ‐ 1 BCL ‐ 2 . HA ‐ BCL ‐ 2 ΔTM was in vitro translated in rabbit reticulocyte lysate equivalent aliquots were added to each GST pull ‐ down reaction . GST ‐ fusion used were GST alone , GST ‐ NAF1 ‐ C , GST ‐ NAF1 ‐ C ‐ mut ( C99S C101S C110S H114Q ) , GST ‐ MitoNEET ‐ C . were detected using anti ‐ HA anti ‐

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(E) A functional CDGSH iron‐binding domain is necessary, but not sufficient for the interaction between the cytosolic domains of NAF‐1 and BCL‐2. HA‐BCL‐2 ΔTM was in vitro translated in rabbit reticulocyte lysate and equivalent aliquots were added to each GST pull‐down reaction. GST‐fusion proteins used were GST alone, GST‐NAF1‐C, GST‐NAF1‐C‐mut (C99S C101S C110S H114Q), and GST‐MitoNEET‐C. The proteins were detected using anti‐HA and anti‐GST


( A ) H1299 neo HA ‐ BCL ‐ 2b5 treated with control ( CTRL ) or NAF ‐ 1 shRNA were either mock infected or infected with Ad ‐ BIK in absence or presence of 50 μM zVAD ‐ fmk . lysates were analysed by immunoblot .

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(A) H1299 neo and HA‐BCL‐2b5 cells treated with control (CTRL) or NAF‐1 shRNA were either mock infected or infected with Ad‐BIK in the absence or presence of 50 μM zVAD‐fmk. Cell lysates were analysed by immunoblot.


( B ) Caspase activity was measured using substrate DEVD ‐ AMC . results represent average ± s . d . of three independent experiments .

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(B) Caspase activity was measured using the fluorescent substrate DEVD‐AMC. The results represent the average±s.d. of three independent experiments.


( C ) Prolonged BIK expression caspase inhibition induces autophagy , which is enhanced by knockdown of NAF ‐ 1 . H1299 neo HA ‐ BCL ‐ 2b5 treated with CTRL or NAF ‐ 1 shRNA were infected with Ad ‐ BIK in presence of zVAD ‐ fmk for indicated periods of time . lysates were analysed by immunoblot . of LC3 II were normalized to actin by densitometry . Graph depicts normalized LC3 II of each lane .

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(C) Prolonged BIK expression and caspase inhibition induces autophagy, which is enhanced by knockdown of NAF‐1. H1299 neo and HA‐BCL‐2b5 cells treated with CTRL or NAF‐1 shRNA were infected with Ad‐BIK in the presence of zVAD‐fmk for the indicated periods of time. Cell lysates were analysed by immunoblot. Levels of LC3 II were normalized to actin levels by densitometry analysis. Graph depicts normalized LC3 II levels of each lane.


( A ) Effect of NAF ‐ 1 knockdown on starvation ‐ induced autophagy . H1299 infected with CTRL or NAF ‐ 1 shRNA were starved for 4 h in EBSS with DMSO ( vehicle ) or Baf A1 ( 100 nM ) . lysates were analysed by immunoblot .

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(A) Effect of NAF‐1 knockdown on starvation‐induced autophagy. H1299 cells infected with CTRL or NAF‐1 shRNA were starved for 4 h in EBSS with DMSO (vehicle) or Baf A1 (100 nM). Cell lysates were analysed by immunoblot.


( B ) Representative of GFP ‐ LC3 in H1299 GFP ‐ LC3 transfected with LUC or NAF ‐ 1 siRNA , untreated starved . Scale bar represents 10 μm .

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(B) Representative images of GFP‐LC3 staining in H1299 GFP‐LC3 cells transfected with LUC or NAF‐1 siRNA, untreated and starved. Scale bar represents 10 μm.


( C ) of autophagy is expressed as of GFP ‐ LC3 ‐ expressing displaying punctate GFP ‐ LC3 . A minimum of 100 per sample were counted ; results represent average ± s . d . of three independent experiments . lysates were analysed by immunoblot .

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(C) Quantification of autophagy is expressed as the percentage of GFP‐LC3‐expressing cells displaying punctate GFP‐LC3. A minimum of 100 cells per sample were counted; results represent the average±s.d. of three independent experiments. Cell lysates were analysed by immunoblot.


( A ) NAF ‐ 1 contributes to interaction between BCL ‐ 2 Beclin 1 . H1299 HA ‐ BCL ‐ 2b5 were transfected with Flag ‐ Beclin 1 either LUC or NAF ‐ 1 siRNA . were lysed subjected to immunoprecipitation with anti ‐ BCL ‐ 2 antibody . Precipitates were subjected to by immunoblot using anti ‐ Beclin 1 anti ‐ BCL ‐ 2 . lanes are derived from same gel of same exposure . Thin lines indicate where lanes have been removed .

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(A) NAF‐1 contributes to the interaction between BCL‐2 and Beclin 1. H1299 HA‐BCL‐2b5 cells were transfected with Flag‐Beclin 1 and either LUC or NAF‐1 siRNA. Cells were lysed and subjected to immunoprecipitation with anti‐BCL‐2 antibody. Precipitates were subjected to analysis by immunoblot using anti‐Beclin 1 and anti‐BCL‐2. All lanes are derived from the same gel and of the same exposure. Thin white lines indicate where lanes have been removed.


( B ) Loss of NAF ‐ 1 prevents BCL ‐ 2b5 from antagonizing starvation ‐ induced autophagy . H1299 GFP ‐ LC3 BCL ‐ 2b5 / GFP ‐ LC3 were transfected with either LUC or NAF ‐ 1 siRNA starved for 4 h . were analysed as in Figure 4C .

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(B) Loss of NAF‐1 prevents BCL‐2b5 from antagonizing starvation‐induced autophagy. H1299 GFP‐LC3 and BCL‐2b5/GFP‐LC3 cells were transfected with either LUC or NAF‐1 siRNA and starved for 4 h. Cells were analysed as in Figure 4C.


( C ) Representative electron of H1299 neo HA ‐ BCL ‐ 2b5 treated with either LUC or NAF ‐ 1 siRNA , with or without subsequent starvation . Scale bar represents 10 μm . lysates were analysed by immunoblot . lanes are derived from same gel of same exposure . Thin indicates where lanes have been removed . Autophagy observed by electron microscopy ( C ) were quantified expressed as either of containing autophagic vacuoles ( D ) or number of autophagosomes per ( E ) . A minimum of 100 per sample were counted ; results represent average ± s . e . m .

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(C) Representative electron micrographs of H1299 neo and HA‐BCL‐2b5 cells treated with either LUC or NAF‐1 siRNA, with or without subsequent starvation. Scale bar represents 10 μm. Cell lysates were analysed by immunoblot. All lanes are derived from the same gel and of the same exposure. Thin white line indicates where lanes have been removed. Autophagy levels observed by electron microscopy (C) were quantified and expressed as either the percentage of cells containing autophagic vacuoles (D) or the number of autophagosomes per cell (E). A minimum of 100 cells per sample were counted; results represent the average±s.e.m.


( A ) H1299 neo HA ‐ BCL ‐ 2b5 treated with either CTRL or NAF ‐ 1 shRNA were loaded with Fura ‐ 2AM , ER Ca2 + stores were measured as difference in Ca2 + before after addition of TG ( 2 μM ) . Shown are representative traces of Fura ‐ 2AM measured at 340 / 380 nm excitation wavelength at 510 nm wavelength emission . Arrow indicates time at which TG was added , delta values indicate TG ‐ sensitive ER Ca2 + stores .

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

(A) H1299 neo and HA‐BCL‐2b5 cells treated with either CTRL or NAF‐1 shRNA were loaded with Fura‐2AM, and ER Ca2+ stores were measured as the difference in cytoplasmic Ca2+ concentration before and after addition of TG (2 μM). Shown are representative traces of Fura‐2AM fluorescence measured at 340/380 nm excitation wavelength ratio at 510 nm wavelength emission. Arrow indicates time at which TG was added, delta values indicate TG‐sensitive ER Ca2+ stores.


( B ) Differences in ER Ca2 + stores are shown as average ± s . e . m . of three independent experiments as described in ( A ) .

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

(B) Differences in ER Ca2+ stores are shown as the average±s.e.m. of three independent experiments as described in (A).


( C ) Co ‐ immunoprecipitation of endogenous NAF ‐ 1 endogenous IP3 receptor type I . Lysate from H1299 HA ‐ BCL ‐ 2b5 was collected immunoprecipitation was performed with anti ‐ IP3R1 antibody . resulting precipitate was analysed by immunoblot .

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

(C) Co‐immunoprecipitation of endogenous NAF‐1 and endogenous IP3 receptor type I. Lysate from H1299 HA‐BCL‐2b5 cells was collected and immunoprecipitation was performed with anti‐IP3R1 antibody. The resulting precipitate was analysed by immunoblot.


( A - B ' ' ) Nr2f1 ( red ) Pax6 ( green , retinal progenitors ) immunofluorescences ( IF ) on sagittal sections of E10 . 5 E12 . 5 mouse optic vesicles . Note high - ventral to low - dorsal Nr2f1 gradient in presumptive neural retina ( pNR ) , high Nr2f1 expression in both presumptive dorsal ventral optic stalk ( pvOS pdOS ) . Cr : crystal lens ; Di , distal ; Pr , proximal ; presumptive retinal pigmented epithelium ( pRPE ) . Scale bars : 50µm for mouse

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

(A-B'') Nr2f1 (red) and Pax6 (green, retinal progenitors) immunofluorescences (IF) on sagittal sections of E10.5 and E12.5 mouse optic vesicles. Note the high-ventral to low-dorsal Nr2f1 gradient in the presumptive neural retina (pNR), and high Nr2f1 expression in both the presumptive dorsal and ventral optic stalk (pvOS and pdOS). Cr: crystal lens; Di, distal; Pr, proximal; presumptive retinal pigmented epithelium (pRPE). Scale bars: 50µm for mouse sections


( C - D " ) IF on E13 . 5 mouse eye sagittal sections with Nr2f1 ( red ) Sox2 ( green , progenitors in C , C ' ) or Brn3a ( green , RGCs in D , D ' ) showing Nr2f1 expression in NR progenitors post - mitotic RGCs ( insets in C ' - D " ) . Scale bars : 50µm for mouse

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

(C-D") IF on E13.5 mouse eye sagittal sections with Nr2f1 (red) and Sox2 (green, progenitors in C,C') or Brn3a (green, RGCs in D,D') showing Nr2f1 expression in NR progenitors and post-mitotic RGCs (insets in C'-D"). Scale bars: 50µm for mouse sections


( E - F " ) Nr2f1 ( red ) Brn3a ( green ) IF on E18 . 5 P7 mouse eyes depicting Nr2f1 expression in virtually RGCs in ganglion layer ( GCL ) . Scale bars : 50µm for mouse

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(E-F") Nr2f1 (red) and Brn3a (green) IF on E18.5 and P7 mouse eyes depicting Nr2f1 expression in virtually all RGCs in the ganglion cell layer (GCL). Scale bars: 50µm for mouse sections


( G , G ' ) Nr2f1 ( red ) Sox2 ( green ) IF on P7 mouse retina illustrating low Nr2f1 expression in inner layer ( INL ) progenitors ( NPs ) , high in GCL ciliary marginal zone ( CMZ ) . No expression in outer layer ( ONL ) . Scale bars : 50µm for mouse

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(G,G') Nr2f1 (red) and Sox2 (green) IF on P7 mouse retina illustrating low Nr2f1 expression in the inner nuclear layer (INL) and progenitors (NPs), and high levels in the GCL and ciliary marginal zone (CMZ). No expression in the outer nuclear layer (ONL). Scale bars: 50µm for mouse sections


( H - K " ) IF on cross - sections of E13 . 5 optic stalks ( OS ) , E18 . 5 P7 optic nerves ( ONs ) with Nr2f1 ( red ) Glast ( green , astrocytic progenitors in H - H ' ' ) , NF1A ( green , astrocytes in I - I ' ' ) or Sox10 ( green , oligodendrocyte precursors in J - K ' ' ) showing Nr2f1 expression in both astrocytic oligodendrocytic lineages . Arrowheads point to double labeled . Scale bars : 50µm for mouse

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(H-K") IF on cross-sections of E13.5 optic stalks (OS), and E18.5 and P7 optic nerves (ONs) with Nr2f1 (red) and Glast (green, astrocytic progenitors in H-H''), NF1A (green, astrocytes in I-I'') or Sox10 (green, oligodendrocyte precursors in J-K'') showing Nr2f1 expression in both astrocytic and oligodendrocytic lineages. Arrowheads point to double labeled cells. Scale bars: 50µm for mouse sections


( L - L " ) NR2F1 ( red ) PAX6 ( green ; NR domain ) IF on sagittal sections of human eye primordia at gestational week ( GW ) 11 illustrating high NR2F1 expression in both ON NR . Scale bars : 100µm for human

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(L-L") NR2F1 (red) and PAX6 (green; NR domain) IF on sagittal sections of human eye primordia at gestational week (GW) 11 illustrating high NR2F1 expression in both ON and NR cells. Scale bars: 100µm for human sections


( M - N " ) NR2F1 ( red ) S100β ( green , astrocytes ) IF on cross - sections of human GW11 GW14 ONs . Higher magnifications ( M ' - N " ) show that most of S100β + astrocytes co - express NR2F1 . Scale bars : 100µm for human sections ,

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(M-N") NR2F1 (red) and S100β (green, astrocytes) IF on cross-sections of human GW11 and GW14 ONs. Higher magnifications (M'-N") show that most of S100β+ astrocytes co-express NR2F1. Scale bars: 100µm for human sections,


( O - Q " ' ) NR2F1 ( red ) SOX2 ( green in O - P ' ) or BRN3a ( green in Q - Q ' ' ' ) IF on sagittal sections of human GW14 eyes indicating high NR2F1 expression in virtually NR progenitors ( O ' ' ' , P , P ' ) , differentiating RGCs ( Q - Q ' ' ' ) in majority of ON astrocytic progenitors ( O ' ) . Nuclei counterstaining ( blue ) was obtained with DAPI . Scale bars 100µm for human

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

(O-Q"') NR2F1 (red) and SOX2 (green in O-P') or BRN3a (green in Q-Q''') IF on sagittal sections of human GW14 eyes indicating high NR2F1 expression in virtually all NR progenitors (O''',P,P'), differentiating RGCs (Q-Q''') and in the majority of ON astrocytic progenitors (O'). Nuclei counterstaining (blue) was obtained with DAPI. Scale bars 100µm for human sections


( A - A ' ' ) Pax2 ( red , OD ) TujI ( green , axons ) IF on E12 . 5 optic cup cross - sections in wild - type ( WT ) , Heterozygous ( HET ) knock - out ( KO ) showing abnormal ODs ( dotted lines arrowhead ) .

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(A-A'') Pax2 (red, OD) and TujI (green, axons) IF on E12.5 optic cup cross-sections in wild-type (WT), Heterozygous (HET) and knock-out (KO) embryos showing abnormal ODs (white dotted lines and arrowhead).


( B - B ' ' ) Pax6 ( red , retinal progenitors ) Tuj1 ( green ) IF indicating ectopic Pax6 + retinal tissue in HET ( B ' ) KO ( B " , arrowhead ) .

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

(B-B'') Pax6 (red, retinal progenitors) and Tuj1 (green) IF indicating ectopic Pax6+ retinal tissue in HET (B') and KO embryos (B", arrowhead).


( C - D " ) High magnification views highlight a morphological displacement of Pax2 - ( C - C " ) Pax6 + ( D - D " ) . Arrowheads in ( D ' , D " ) point to ectopic Tuj1 / Pax6 + neurons .

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(C-D") High magnification views highlight a morphological displacement of Pax2- (C-C") and Pax6+ (D-D") cells. Arrowheads in (D',D") point to ectopic Tuj1/Pax6+ neurons.


( E , E ' ) Pax2 ( red ) Tuj1 ( green ) IF on tangential sections of E13 . 5 optic cups revealing strong OD reduction in KO ( dotted arrowhead in E ' ) , compared to WT ( surrounded by a dotted in E ) .

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

(E,E') Pax2 (red) and Tuj1 (green) IF on tangential sections of E13.5 optic cups revealing strong OD reduction in KO embryos (dotted line and arrowhead in E'), compared to WT (surrounded by a white dotted line in E).


( F ) Histogram quantifying gradual reduction of Pax2 + OD in HET KO at different ages . data are represented as mean ± ; N = 3 - 4 Student t - ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . Scale bars : 50µm .

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(F) Histogram quantifying the gradual reduction of Pax2+ OD cells in HET and KO at different ages. data are represented as mean ± SEM; N=3-4 Student t-test (*P<0.05, **P<0.01, ***P<0.001). Scale bars: 50µm.


( G - G ' ' ' ) Low ( G ) high ( G ' - G ' ' ' ) magnifications of E15 . 5 optic cups stained for Pax2 ( red ) Tuj1 ( green ) confirming Pax2 + reduction in HET KO ODs severe morphological malformations in KO ( arrowheads in G ' ' ' ) .

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(G-G''') Low (G) and high (G'-G''') magnifications of E15.5 optic cups stained for Pax2 (red) and Tuj1 (green) confirming Pax2+ reduction in HET and KO ODs and severe morphological malformations in KO (arrowheads in G''').


( H - I ' ) Tangential ( H , H ' ) sagittal ( I , I ' ) sections of OD in E18 . 5 WT KO retinae stained for Brn3a ( red ; RGCs ) Tuj1 ( green ; axons ) highlighting malformed OD disorganized arrangement of RGCs in KO . In ( H , H ' ) dotted delineate borders of OD in ( I , I ' ) RGC layer ; red dotted surrounds mesodermal forming hyaloid vessel .

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(H-I') Tangential (H,H') and sagittal (I,I') sections of the OD in E18.5 WT and KO retinae stained for Brn3a (red; RGCs) and Tuj1 (green; axons) highlighting the malformed OD and disorganized arrangement of RGCs in KO. In (H,H') white dotted line delineate the borders of the OD and in (I,I') the RGC layer; red dotted line surrounds mesodermal cells forming the hyaloid vessel.


( J - K ' ) IF on E13 . 5 optic cup cross - sections in WT KO showing increased Pax6 ( green , retinal progenitors ) decreased Tuj1 + ( red , differentiating ) in ventral retina of HET KO . Arrowhead in ( J ' ) points to increased thickness bending of ventral retina , while arrowheads in ( K , K ' ) indicate Tuj1 + axons exiting OD .

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

(J-K') IF on E13.5 optic cup cross-sections in WT and KO embryos showing increased Pax6 (green, retinal progenitors) and decreased Tuj1+ (red, differentiating cells) in the ventral retina of HET and KO embryos. Arrowhead in (J') points to increased thickness and bending of the ventral retina, while arrowheads in (K,K') indicate Tuj1+ axons exiting the OD.


( L ) Histogram confirming decreased number of Tuj1 + neurons in ventral , but not dorsal , retina of Nr2f1 mutants . data are represented as mean ± ; N = 3 - 4 Student t - ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . Scale bars : 50µm .

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(L) Histogram confirming the decreased number of Tuj1+ neurons in the ventral, but not dorsal, retina of Nr2f1 mutants. data are represented as mean ± SEM; N=3-4 Student t-test (*P<0.05, **P<0.01, ***P<0.001). Scale bars: 50µm.


( M - N ' ) Double Tuj1 / Brn3a IF on E13 . 5 WT KO optic cup sections revealing decreased numbers of differentiating RGCs in bending ventral retinae of KO ( N ' ) .

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(M-N') Double Tuj1/Brn3a IF on E13.5 WT and KO optic cup sections revealing decreased numbers of differentiating RGCs in bending ventral retinae of KO embryos (N').


( O , O ' ) Details of P28 WT HET retinae indicating decreased number of Brn3a + neurons ( arrowheads ) in GCL of HET .

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

(O,O') Details of P28 WT and HET retinae indicating decreased number of Brn3a+ neurons (arrowheads) in the GCL of HET animals.


( P ) Graph illustrating dynamics of RGC differentiation from E13 . 5 to P28 , normalized to WT . data are represented as mean ± ; N = 3 - 4 Student t - ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . Scale bars : 50µm .

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(P) Graph illustrating the dynamics of RGC differentiation from E13.5 to P28, normalized to WT animals. data are represented as mean ± SEM; N=3-4 Student t-test (*P<0.05, **P<0.01, ***P<0.001). Scale bars: 50µm.


Q - Q " ) Cross - sections of DiI - labelled P0 ONs in WT KO

Q-Q") Cross-sections of DiI-labelled P0 ONs in WT and KO pups


( R , R ' ) Tuj1 + P8 ON in WT KO pups showing a progressive volume

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(R,R') Tuj1+ P8 ON fibers in WT and KO pups showing a progressive volume loss


( S ) of surface occupied by Tuj1 + or DiI + in different genotypes ages , as indicated . data are represented as mean ± ; N = 4 - 5 Student t - ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . Scale bars : 50µm .

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(S) Quantification of the surface occupied by Tuj1+ or DiI+ fibers in different genotypes and ages, as indicated. data are represented as mean ± SEM; N=4-5 Student t-test (*P<0.05, **P<0.01, ***P<0.001). Scale bars: 50µm.


( T - U ' ) - Caspase - 3 ( green , apoptotic ) Brn3a ( red , RGCs ) IF on transverse sections of E18 . 5 retinae in WT KO fetuses . High magnifications ( T ' - T ' " ) highlight an example of a double - labeled RGC undergoing apoptosis ( arrowheads ) in ganglion layer ( GCL ) . Apoptotic RGCs are increased in KO ( arrowheads in U ' ) , compared to WT ( U ) .

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

(T-U') Cleaved-Caspase-3 (green, apoptotic cells) and Brn3a (red, RGCs) IF on transverse sections of E18.5 retinae in WT and KO fetuses. High magnifications (T'-T'") highlight an example of a double-labeled RGC undergoing apoptosis (arrowheads) in the ganglion cell layer (GCL). Apoptotic RGCs are increased in KO animals (arrowheads in U'), compared to WT (U).


( V ) Histogram quantifying number of RGC apoptotic in WT , HET KO from E18 . 5 to P5 . Note significant increase of dying at E18 . 5 . Nuclei ( blue ) were stained with DAPI . data are represented as mean ± ; N = 3 - 4 Student t - ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . Scale bars : 50µm .

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

(V) Histogram quantifying the number of RGC apoptotic cells in WT, HET and KO from E18.5 to P5. Note the significant increase of dying cells at E18.5. Nuclei (blue) were stained with DAPI. data are represented as mean ± SEM; N=3-4 Student t-test (*P<0.05, **P<0.01, ***P<0.001). Scale bars: 50µm.


( A - F ) Pax2 ( magenta , astrocytic progenitors ) Tuj1 ( green , RGC axons ) IF on E13 . 5 , E15 . 5 E18 . 5 OS / ONs cross - sections showing an increase of Pax2 - expressing within optic nerve ( ON ) of KO at stages .

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

(A-F) Pax2 (magenta, astrocytic progenitors) and Tuj1 (green, RGC axons) IF on E13.5, E15.5 and E18.5 OS/ONs cross-sections showing an increase of Pax2-expressing cells within the optic nerve (ON) of KO embryos at all stages.


Histograms quantifying average number of Pax2 + ( G , H ) in WT , HET KO at different stages axial of ON , as indicated . Astrocytic progenitors are significantly increased along nerve of mutants . data are represented as means ± ; N = 3 - 5 . Statistical significance was obtained by Student t - ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) . Nuclei ( blue ) were stained with DAPI . Scale bars :

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

Histograms quantifying the average number of Pax2+ (G,H) cells in WT, HET and KO at different stages and axial length of the ON, as indicated. Astrocytic progenitors are significantly increased along the nerve of mutants. data are represented as means ± SEM; N=3-5. Statistical significance was obtained by Student t-test (*P<0.05; **P<0.01; ***P<0.001). Nuclei (blue) were stained with DAPI. Scale bars: 50µm


Histograms quantifying average number of NF1A + ( I ) in WT , HET KO at different stages axial of ON , as indicated . Astrocytic progenitors are significantly increased along nerve of mutants . data are represented as means ± ; N = 3 - 5 . Statistical significance was obtained by Student t - ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) . Nuclei ( blue ) were stained with DAPI . Scale bars :

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

Histograms quantifying the average number of NF1A+ (I) cells in WT, HET and KO at different stages and axial length of the ON, as indicated. Astrocytic progenitors are significantly increased along the nerve of mutants. data are represented as means ± SEM; N=3-5. Statistical significance was obtained by Student t-test (*P<0.05; **P<0.01; ***P<0.001). Nuclei (blue) were stained with DAPI. Scale bars: 50µm


( J , K ) NF1A ( red , astrocyte precursors ) IF on P7 WT KO ON cross - sections illustrating a high astrocytic density in KO ON ( delineated by thin dashed lines ) . ( L ) Histogram confirming increased of NF1A + astrocytes in HET KO ONs compared to WT . data are represented as means ± ; N = 3 - 5 . Statistical significance was obtained by Student t - ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) . Nuclei ( blue ) were stained with DAPI . Scale bars :

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

(J,K) NF1A (red, astrocyte precursors) IF on P7 WT and KO ON cross-sections illustrating a high astrocytic density in KO ON (delineated by thin dashed lines). (L) Histogram confirming the increased percentage of NF1A+ astrocytes in HET and KO ONs compared to WT. data are represented as means ± SEM; N=3-5. Statistical significance was obtained by Student t-test (*P<0.05; **P<0.01; ***P<0.001). Nuclei (blue) were stained with DAPI. Scale bars: 50µm


( M - O " ) Tuj1 ( green , RGC axons ) S100β ( red , astrocytes ) IF on P7 WT , HET KO ONs . Arrowheads point to S100β + astrocyte groups ( N ' , O ' ) absence of Tuj1 + ( N ' ' , O ' ' ) in HET KO ONs .

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(M-O") Tuj1 (green, RGC axons) and S100β (red, astrocytes) IF on P7 WT, HET and KO ONs. Arrowheads point to S100β+ astrocyte groups (N',O') and absence of Tuj1+ fibers (N'',O'') in HET and KO ONs.


( P . Q ) of WT KO P8 ONs showing thick astrocytic processes filled with intermediate filaments ( red dotted lines arrowheads ) in KO nerves . Scale bars ( 500nm )

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(P.Q) TEM images of WT and KO P8 ONs showing thick astrocytic processes filled with intermediate filaments (red dotted lines and arrowheads) in KO nerves. Scale bars (500nm)


( R - S ' ) S100β ( green , astrocytes ) Sox2 ( red , reactive / inflamed astrocytes ) IF on P28 WT HET ONs illustrating strong Sox2 expression in reactive astrocytes .

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(R-S') S100β (green, astrocytes) and Sox2 (red, reactive/inflamed astrocytes) IF on P28 WT and HET ONs illustrating strong Sox2 expression in reactive astrocytes.


( T ) Graph quantifying average number of Sox2 + astrocytes per P28 ON section in KO WT . data are represented as means ± ; N = 3 - 5 . Statistical significance was obtained by Student t - ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) . Nuclei ( blue ) were stained with DAPI . Scale bars :

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(T) Graph quantifying the average number of Sox2+ astrocytes per P28 ON section in KO and WT animals. data are represented as means ± SEM; N=3-5. Statistical significance was obtained by Student t-test (*P<0.05; **P<0.01; ***P<0.001). Nuclei (blue) were stained with DAPI. Scale bars: 50µm


( U , V ) EM sections of P28 WT HET ONs illustrating expanded astrocytic processes ( surrounded by red dotted lines ) among myelinated of P28 ONs . Scale bars : ( U , V ) ( 4µm ) .

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(U,V) EM sections of P28 WT and HET ONs illustrating the expanded astrocytic processes (surrounded by red dotted lines) among myelinated fibers of P28 ONs. Scale bars: (U,V) (4µm).


( A - C ) NF1A ( red , astrocytes ) Sox10 ( green , oligodendrocytes ) IF on cross - sections of WT KO P8 ONs showing altered of astrocytes oligodendrocytes in mutants , particularly in KOs , as quantified in ( C ) . data are represented as means ± . N = 4 - 5 Statistical significance was obtained by Student t - or by two - way ANOVA when comparing 2 or multiple conditions , respectively ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 Scale bars :

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(A-C) NF1A (red, astrocytes) and Sox10 (green, oligodendrocytes) IF on cross-sections of WT and KO P8 ONs showing altered ratio of astrocytes and oligodendrocytes in mutants, particularly in KOs, as quantified in (C). data are represented as means ± SEM. N=4-5 Statistical significance was obtained by Student t-test or by two-way ANOVA when comparing 2 or multiple conditions, respectively (*P<0.05; **P<0.01; ***P<0.001 Scale bars: 50µm


( D - J ) MBP ( yellow , fully differentiated oligodendrocytes ) IF on cross - sections of WT , HET KO P7 optic nerves ( ONs ) at chiasm ( D - F ) between chiasm eye ( G - I ) . Note dramatic decrease of MBP + oligodendrocytes at chiasm in HET KO , quantified in ( J ) . of MBP + is calculated over total DAPI + number per chiasmal / ON section . data are represented as means ± . N = 4 - 5 Statistical significance was obtained by Student t - or by two - way ANOVA when comparing 2 or multiple conditions , respectively ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) . Nuclei ( blue ) were stained with DAPI . Scale bars :

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

(D-J) MBP (yellow, fully differentiated oligodendrocytes) IF on cross-sections of WT, HET and KO P7 optic nerves (ONs) at the chiasm (D-F) and between the chiasm and the eye (G-I). Note the dramatic decrease of MBP+ oligodendrocytes at the chiasm in HET and KO, quantified in (J). The ratio of MBP+ cells is calculated over the total DAPI+ cell number per chiasmal/ON section. data are represented as means ± SEM. N=4-5 Statistical significance was obtained by Student t-test or by two-way ANOVA when comparing 2 or multiple conditions, respectively (*P<0.05; **P<0.01; ***P<0.001). Nuclei (blue) were stained with DAPI. Scale bars: 50µm


( K - N ) EM thin sections of P8 WT , HET KO ONs depicting myelin as a dense , around axonal illustrate a significantly lower number of myelinated in HET KO , as quantified in ( N ) . Higher magnification ( insets ) showing different degrees of myelination in HET KO ONs . data are represented as means ± . N = 3 Statistical significance was obtained by Student t - or by two - way ANOVA when comparing 2 or multiple conditions , respectively ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) . Nuclei ( blue ) were stained with DAPI . Scale bars : 50µm ( 4µm )

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

(K-N) EM thin sections of P8 WT, HET and KO ONs depicting myelin as a dense, dark staining around axonal fibers illustrate a significantly lower number of myelinated fibers in HET and KO, as quantified in (N). Higher magnification images (insets) showing different degrees of myelination in HET and KO ONs. data are represented as means ± SEM. N=3 Statistical significance was obtained by Student t-test or by two-way ANOVA when comparing 2 or multiple conditions, respectively (*P<0.05; **P<0.01; ***P<0.001). Nuclei (blue) were stained with DAPI. Scale bars: 50µm (4µm)


of P28 WT HET confirming persistent hypomyelination in mutants at P28 Scale bars : ( 4µm )

TEM images of P28 WT and HET confirming persistent hypomyelination in mutants at P28 Scale bars: (4µm)


P28 WT HET confirming persistent hypomyelination in mutants at P28 , as quantified in ( Q ) . data are represented as means ± . N = 3 Statistical significance was obtained by Student t - or by two - way ANOVA when comparing 2 or multiple conditions , respectively ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) . Nuclei ( blue ) were stained with DAPI . Scale bars :

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P28 WT and HET confirming persistent hypomyelination in mutants at P28, as quantified in (Q). data are represented as means ± SEM. N=3 Statistical significance was obtained by Student t-test or by two-way ANOVA when comparing 2 or multiple conditions, respectively (*P<0.05; **P<0.01; ***P<0.001). Nuclei (blue) were stained with DAPI. Scale bars: 50µm


( R , S ) High magnification EM of P28 WT HET ONs showing difference in myelin compaction between WT ( arrowheads in R ) HET ( arrowheads in S ) . Scale bars : ( 1µm ) .

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(R,S) High magnification EM images of P28 WT and HET ONs showing the difference in myelin compaction between WT (arrowheads in R) and HET (arrowheads in S). Scale bars: (1µm).


( A ) Experimental design to treat HET pups from P2 to P8 with Miconazole . DMSO ( controls ) Miconazole - treated pups were sacrificed at P8 or at P28 to evaluate ON myelination . ( B - E ) Sox10 ( red , oligodendrocyte precursors ) MBP ( green , fully differentiated oligodendrocytes ) IF on cross - sections of P8 HET ONs after 6 days of treatment with DMSO ( D ) or Miconazole ( E ) . Miconazole increased number of Sox10 + compared to HETs treated with DMSO ( quantified in B ) rescued MBP pixel of HET pups to level comparable to those of WT pups ( quantified in C ) . Nuclei ( blue ) were stained with DAPI . , error bars represent of means ; N = 3 Statistical significance was obtained by ANOVA ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) . Scale bars :

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(A) Experimental design to treat HET pups from P2 to P8 with Miconazole. DMSO (controls) and Miconazole-treated pups were sacrificed at P8 or at P28 to evaluate the ON myelination. (B-E) Sox10 (red, oligodendrocyte precursors) and MBP (green, fully differentiated oligodendrocytes) IF on cross-sections of P8 HET ONs after 6 days of treatment with DMSO (D) or Miconazole (E). Miconazole increased the number of Sox10+ cells compared to HETs treated with DMSO (quantified in B) and rescued MBP staining pixel intensity of HET pups to level comparable to those of WT pups (quantified in C). Nuclei (blue) were stained with DAPI. , the error bars represent the SEM of the means; N=3 Statistical significance was obtained by ANOVA (*P<0.05; **P<0.01; ***P<0.001). Scale bars: 50µm


( F - I ) Sox10 ( oligodendrocyte marker , red ) Phospho - ERK ( dually phosphorylated forms of active ERK1 ERK2 , green ) IF in WT HET ONs , treated with DMSO ( F ) or Miconazole ( G ) between P2 P8 . As previously reported , Miconazole strongly activate ERK signaling pathway ( G ) . Signal in whole ON is shown in ( H ) , while specific evaluation of pixel around Sox10 + nuclei is shown in ( I ) . Nuclei ( blue ) were stained with DAPI . error bars represent of means N = 2 Statistical significance was obtained by ANOVA ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) . Scale bars :

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

(F-I) Sox10 (oligodendrocyte marker, red) and Phospho-ERK (dually phosphorylated forms of active ERK1 and ERK2, green) IF in WT and HET ONs, treated with DMSO (F) or Miconazole (G) between P2 and P8. As previously reported, Miconazole strongly activate ERK signaling pathway (G). Signal intensity quantification in whole ON area is shown in (H), while specific evaluation of pixel intensity around Sox10+ nuclei is shown in (I). Nuclei (blue) were stained with DAPI. the error bars represent the SEM of the means N=2 Statistical significance was obtained by ANOVA (*P<0.05; **P<0.01; ***P<0.001). Scale bars: 50µm


( J - M ) EM thin sections of P28 WT HET ONs , treated with DMSO ( J , K ) or Miconazole ( L ) between P2 P8 . Different degrees of myelin compaction can be appreciated at high magnification , with HET ONs showing non compacted sheaths ( arrows in K ) vacuoles ( yellow arrows ) . Miconazole treatment rescues almost normal myelin g - , as quantified in M . Nuclei ( blue ) were stained with DAPI . error bars represent of means N = 2 Statistical significance was obtained by ANOVA ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) . Scale bars : 50µm , except ( J - L ) ( 500nm ) .

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(J-M) EM thin sections of P28 WT and HET ONs, treated with DMSO (J,K) or Miconazole (L) between P2 and P8. Different degrees of myelin compaction can be appreciated at high magnification, with HET ONs showing non compacted sheaths (white arrows in K) and vacuoles (yellow arrows). Miconazole treatment rescues almost normal myelin g-ratio, as quantified in M. Nuclei (blue) were stained with DAPI. the error bars represent the SEM of the means N=2 Statistical significance was obtained by ANOVA (*P<0.05; **P<0.01; ***P<0.001). Scale bars: 50µm, except (J-L) (500nm).


( N - P ' ) S100β ( green , astrocytes ) Sox2 ( red , reactive / proliferative astrocytes ) IF in ONs of WT ( N , N ' ) HET ( O - P ' ) after 6 days of treatment with DMSO ( N - O ' ) or Miconazole ( P , P ' ) , then sacrificed at P28 . Astrocytes undergo remodeling express Sox2 in Nr2f1 HET ( O ' , P ' ) , even after Miconazole treatment .

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(N-P') S100β (green, astrocytes) and Sox2 (red, reactive/proliferative astrocytes) IF in the ONs of WT (N,N') and HET (O-P') animals after 6 days of treatment with DMSO (N-O') or Miconazole (P,P'), then sacrificed at P28. Astrocytes undergo remodeling and express Sox2 in Nr2f1 HET animals (O',P'), even after Miconazole treatment.


Histogram showing average number of S100β + / Sox2 + astrocytes per P28 ON section in WT or HET treated with DMSO or Miconazole as indicated , with Sox2 high ( magenta columns ) low ( pink columns ) . Nuclei ( blue ) were stained with DAPI . error bars represent of means ; ; N = 2 Statistical significance was obtained by ANOVA ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) . Scale bars :

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Histogram showing the average number of S100β+/Sox2+ astrocytes per P28 ON section in WT or HET animals treated with DMSO or Miconazole as indicated, with Sox2 high (magenta columns) and low levels (pink columns). Nuclei (blue) were stained with DAPI. the error bars represent the SEM of the means; ; N=2 Statistical significance was obtained by ANOVA (*P<0.05; **P<0.01; ***P<0.001). Scale bars: 50µm


( B ) Nissl - stained sections illustrating location of recording electrodes ( arrows ) . dhc , dorsal hippocampal commissure ; PAC , periaqueductal grey ; Th , thalamus .

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(B) Nissl-stained sections illustrating the location of the recording electrodes (arrows). dhc, dorsal hippocampal commissure; PAC, periaqueductal grey; Th, thalamus.


( C ) Examples of field potentials ( averaged ≥ 20 times ) evoked by flash stimulation in recording sites for WT HET mice . Dotted lines indicate beginning of evoked field potentials .

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(C) Examples of field potentials (averaged ≥ 20 times) evoked by flash stimulation in the recording sites for WT and HET mice. Dotted lines indicate the beginning of evoked field potentials.


( D ) Latency of evoked field potentials for WT ( N = 13 ) HET ( N = 18 ) mice .

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(D) Latency of evoked field potentials for WT (N=13) and HET (N=18) mice.


( E ) In a first series of experiments , [ WT ( N = 13 ) HET ( N = 18 ) ] were trained to press a lever to obtain a pellet of food in an illuminated Skinner box with a fixed - ( 1 : 1 ) schedule . After ten days of training , they were transferred to a on / off protocol where lever presses were only rewarded when a small bulb located over lever was switched on . Lever presses while bulb was off were punished with a time penalty of ≤ 10 s during which bulb would not turn on . ( F , G ) Mean days to criterion ( F ) mean number of lever presses during last two training sessions ( G ) for fixed - ( 1 : 1 ) paradigm . No significant differences were observed .

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(E) In a first series of experiments, animals [WT (N=13) and HET (N=18)] were trained to press a lever to obtain a pellet of food in an illuminated Skinner box with a fixed-ratio (1:1) schedule. After ten days of training, they were transferred to a light on/light off protocol where lever presses were only rewarded when a small light bulb located over the lever was switched on. Lever presses while the bulb was off were punished with a time penalty of ≤ 10 s during which the bulb would not turn on. (F,G) Mean days to criterion (F) and mean number of lever presses during the last two training sessions (G) for the fixed-ratio (1:1) paradigm. No significant differences were observed.


( H ) Performance of WT ( N = 13 ) HET ( N = 18 ) mice in on / off . Criterion ( dotted ) was that had to press lever more times during small bulb on period than during off one . WT mice performed better than HET reached criterion by 6th session . HET did not reached criterion for duration of . While a linear fit to WT data increased significantly across training , same linear fit to HET performance did not present any significant change .

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(H) Performance of WT (N=13) and HET (N=18) mice in the light on/light off test. Criterion (dotted line) was that the animal had to press the lever more times during the small bulb light on period than during the light off one. WT mice performed better than HET animals and reached the criterion by the 6th session. The HET did not reached criterion for the duration of the test. While a linear fit to WT data increased significantly across training, the same linear fit to HET performance did not present any significant change.


( I ) of lever presses during on off periods for WT HET mice was also different , because WT group pressed lever preferentially during period . Data were averaged from 9th 10th sessions . For statistical significance ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) .

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(I) The percentage of lever presses during the light on and light off periods for WT and HET mice was also different, because the WT group pressed the lever preferentially during the light period. Data were averaged from the 9th and 10th sessions. For statistical significance (*P<0.05; **P<0.01; ***P<0.001).


Human first - trimester trophoblast Sw . 71 ( left panel ) human culture ( right panel ) trophoblast were infected with ZIKV ( MOI = 2 ) for 1 h refreshed with regular media over time . was collected for measuring viral titers by qRT - PCR . Data represent as mean ± ; n = 4 biological replicates . NT , no treatment group ; HPC , human culture . ( A ) ZIKV titers in Sw . 71 HPC trophoblasts . * p < 0 . 05 by One - way ANOVA .

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Human first-trimester trophoblast cell line Sw.71 (left panel) and human primary culture (right panel) trophoblast cells were infected with ZIKV (MOI=2) for 1 h and refreshed with regular media over time. RNA was collected for measuring viral titers by qRT-PCR. Data represent as mean ± SEM; n=4 biological replicates. NT, no treatment group; HPC, human primary culture. (A) ZIKV titers in Sw.71 and HPC trophoblasts. *p < 0.05 by One-way ANOVA.


Human first - trimester trophoblast Sw . 71 ( left panel ) human culture ( right panel ) trophoblast were infected with ZIKV ( MOI = 2 ) for 1 h refreshed with regular media over time . was collected for measuring gene expression by qRT - PCR . Data represent as mean ± ; n = 4 biological replicates . NT , no treatment group ; HPC , human culture . ( B ) IFNα is not highly induced in response to ZIKV infection in trophoblast . IFNα mRNA expression was inhibited in first 24 h . p . i . maintained at low level during ZIKV infection . Difference not significant ( n . s . ) by One - way ANOVA .

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

Human first-trimester trophoblast cell line Sw.71 (left panel) and human primary culture (right panel) trophoblast cells were infected with ZIKV (MOI=2) for 1 h and refreshed with regular media over time. RNA was collected for measuring gene expression by qRT-PCR. Data represent as mean ± SEM; n=4 biological replicates. NT, no treatment group; HPC, human primary culture. (B) IFNα is not highly induced in response to ZIKV infection in trophoblast cells. IFNα mRNA expression was inhibited in the first 24 h.p.i. and maintained at low level during ZIKV infection. Difference not significant (n.s.) by One-way ANOVA.


Human first - trimester trophoblast Sw . 71 ( left panel ) human culture ( right panel ) trophoblast were infected with ZIKV ( MOI = 2 ) for 1 h refreshed with regular media over time . was collected for measuring gene expression by qRT - PCR . Data represent as mean ± ; n = 4 biological replicates . NT , no treatment group ; HPC , human culture . ( C ) ZIKV infection induced a time - dependent increase in IFNβ mRNA expression in Sw . 71 HPC . * p < 0 . 05 , * * p < 0 . 01 by One - way ANOVA .

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

Human first-trimester trophoblast cell line Sw.71 (left panel) and human primary culture (right panel) trophoblast cells were infected with ZIKV (MOI=2) for 1 h and refreshed with regular media over time. RNA was collected for measuring gene expression by qRT-PCR. Data represent as mean ± SEM; n=4 biological replicates. NT, no treatment group; HPC, human primary culture. (C) ZIKV infection induced a time-dependent increase in IFNβ mRNA expression in Sw.71 and HPC. *p < 0.05, **p < 0.01 by One-way ANOVA.


Human first - trimester trophoblast Sw . 71 ( left panel ) human culture ( right panel ) trophoblast were infected with ZIKV ( MOI = 2 ) for 1 h refreshed with regular media over time . ( D ) Expression of secreted IFNβ detected in trophoblast supernatants by ELLA . Supernatants were collected from ZIKV - infected control trophoblast cultures at different times , IFNβ secretion was quantified by ELLA . Note increase of secreted IFNβ in ZIKV - infected supernatant in a time - dependent manner . * * * * p < 0 . 0001 by Student ' s t - against time point NT control .

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

Human first-trimester trophoblast cell line Sw.71 (left panel) and human primary culture (right panel) trophoblast cells were infected with ZIKV (MOI=2) for 1 h and refreshed with regular media over time. (D) Expression levels of secreted IFNβ detected in trophoblast supernatants by ELLA assay. Supernatants were collected from ZIKV-infected and control trophoblast cultures at different times, and IFNβ protein secretion was quantified by ELLA assay. Note the increase of secreted IFNβ in the ZIKV-infected supernatant in a time-dependent manner. ****p < 0.0001 by Student's t-test against individual time point NT control.


Sw . 71 ( left panel ) HPC ( right panel ) were treated with IFNβ to detect ISG20 mRNA expression . Data represent as mean ± ; n = 3 biological replicates . NT , no treatment group ; HPC , human culture . ( A ) Sw . 71 HPC were treated with different doses of IFNβ ( 3 , 30 , 300 ng / ml ) for 8 h were collected for determining ISG20 mRNA expressions by qRT - PCR . Note increase of ISG20 mRNA expression in a dose - dependent manner . * p < 0 . 05 , * * p < 0 . 01 by One - way ANOVA . ( B ) Sw . 71 HPC were treated with 300ng / ml IFNβ over time was collected for determining ISG20 mRNA expressions by qRT - PCR . Note increase of ISG20 mRNA expression in a time - dependent manner . * p < 0 . 05 by One - way ANOVA . ( C )

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

Sw.71 (left panel) and HPC cells (right panel) were treated with IFNβ to detect ISG20 mRNA expression. Data represent as mean ± SEM; n=3 biological replicates. NT, no treatment group; HPC, human primary culture. (A) Sw.71 and HPC cells were treated with different doses of IFNβ (3, 30, 300 ng/ml) for 8 h and RNA were collected for determining ISG20 mRNA expressions by qRT-PCR. Note the increase of ISG20 mRNA expression in a dose-dependent manner. *p < 0.05, **p < 0.01 by One-way ANOVA. (B) Sw.71 and HPC cells were treated with 300ng/ml IFNβ over time and RNA was collected for determining ISG20 mRNA expressions by qRT-PCR. Note the increase of ISG20 mRNA expression in a time-dependent manner. *p < 0.05 by One-way ANOVA. (C)


Sw . 71 ( left panel ) HPC ( right panel ) were treated with IFNβ to detect ISG20 expression . ( D ) Sw . 71 HPC were treated with 300ng / ml IFNβ over time was collected for determining ISG20 expressions by western blot . Note that there was no ISG20 expression in no treatment group in both Sw . 71 HPC , only after IFNβ treatment , ISG20 expression exhibited a time - dependent manner . β - actin served as a loading control .

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

Sw.71 (left panel) and HPC cells (right panel) were treated with IFNβ to detect ISG20 protein expression. (D) Sw.71 and HPC cells were treated with 300ng/ml IFNβ over time and protein was collected for determining ISG20 protein expressions by western blot. Note that there was no ISG20 protein expression in the no treatment group in both Sw.71 and HPC, only after IFNβ treatment, ISG20 protein expression exhibited a time-dependent manner. β-actin served as a loading control.


( B ) No ISG20 expression in ISG20 - / - Sw . 71 in ZIKV infection . Sw . 71 ISG20 - / - Sw . 71 were infected with ZIKV ( MOI = 2 ) for 1 h refreshed with regular media for 48 h , were collected for western blot . β - actin served as a loading control .

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(B) No ISG20 protein expression in ISG20-/- Sw.71 in ZIKV infection. Sw.71 and ISG20-/- Sw.71 cells were infected with ZIKV (MOI=2) for 1 h and refreshed with regular media for 48 h, and proteins were collected for western blot analysis. β-actin served as a loading control.


( C ) Higher ZIKA titer was shown in ISG20 - / - Sw . 71 . Sw . 71 ISG20 - / - Sw . 71 were infected with ZIKV ( MOI = 2 ) for 1 h refreshed with regular media for 48 h , was collected for determining viral titer gene expression by qRT - PCR . Data represent as mean ± ; n = 4 biological replicates ; * p < 0 . 05 by Student ' s t - .

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

(C) Higher ZIKA titer was shown in ISG20-/- Sw.71 cells. Sw.71 and ISG20-/- Sw.71 cells were infected with ZIKV (MOI=2) for 1 h and refreshed with regular media for 48 h, and RNA was collected for determining the viral titer and gene expression by qRT-PCR. Data represent as mean ± SEM; n=4 biological replicates; *p < 0.05 by Student's t-test.