Figure 4Representative confocal z - stack and colocalization images of Gephyrin ( green ) , IBA1 ( blue ) , and CD68 ( red ) in the IC of control and Cdc50a cKO mice . Scale bar , 20 μm . Bar graphs showing the relative colocalization area of Gephyrin and CD68 in the IC of control and Cdc50a cKO mice ( n = 3 for each group , * * P = 0 . 0024 ) . Representative confocal z - stack images of microglia ( IBA1 , green , upper ) and inhibitory synapses ( Gephyrin : green , VGAT : red , lower ) in the IC of control ; DMSO , control ; PLX3397 , Cdc50a cKO ; DMSO and Cdc50a cKO ; PLX3397 mice . The dashed line represents where the high magnified insert was taken from . Scale bar , 20 μm . Bar graphs showing the relative area of IBA1 - positive cells in the IC of control ; DMSO , control ; PLX3397 , Cdc50a cKO ; DMSO and Cdc50a cKO ; PLX3397 mice ( n = 3 for each group , control ; DMSO versus control ; PLX3397 , * P = 0 . 0428 , control ; DMSO versus Cdc50a cKO ; DMSO , * * * P = 0 . 0002 , Cdc50a cKO ; DMSO versus Cdc50a cKO ; PLX3397 , * * * P = 0 . 0005 ) . Bar graphs showing the number of inhibitory ( Gephyrin - and VGAT - positive ) synapses in the IC of control ; DMSO , control ; PLX3397 , Cdc50a cKO ; DMSO and Cdc50a cKO ; PLX3397 mice ( n = 3 for each group , P * * * * < 0 . 0001 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Representative confocal z-stack and colocalization images of Gephyrin (green), IBA1 (blue), and CD68 (red) in the IC of control and Cdc50a cKO mice. Scale bar, 20 μm. Bar graphs showing the relative colocalization area of Gephyrin and CD68 in the IC of control and Cdc50a cKO mice (n = 3 for each group, **P = 0.0024).Representative confocal z-stack images of microglia (IBA1, green, upper) and inhibitory synapses (Gephyrin: green, VGAT: red, lower) in the IC of control;DMSO, control;PLX3397, Cdc50a cKO;DMSO and Cdc50a cKO;PLX3397 mice. The dashed line represents where the high magnified insert was taken from. Scale bar, 20 μm. Bar graphs showing the relative area of IBA1-positive cells in the IC of control;DMSO, control;PLX3397, Cdc50a cKO;DMSO and Cdc50a cKO;PLX3397 mice (n = 3 for each group, control;DMSO versus control;PLX3397, *P = 0.0428, control;DMSO versus Cdc50a cKO;DMSO, ***P = 0.0002, Cdc50a cKO;DMSO versus Cdc50a cKO;PLX3397, ***P = 0.0005). Bar graphs showing the number of inhibitory (Gephyrin- and VGAT-positive) synapses in the IC of control;DMSO, control;PLX3397, Cdc50a cKO;DMSO and Cdc50a cKO;PLX3397 mice (n = 3 for each group, P**** < 0.0001).


Figure 5Representative confocal z - stack images of Gephyrin ( green ) - and VGAT ( red ) - positive inhibitory synapses in the IC ( inferior colliculus ) of control ; C3 + / + , control ; C3 - / - , Cdc50a cKO ; C3 + / + and Cdc50a cKO ; C3 - / - mice . The dashed line represents where the high magnified insert was taken from . Scale bar , 10 μm . Representative confocal z - stack images of Gephyrin ( green ) - and VGAT ( red ) - positive inhibitory synapses in the IC of control ; Mertk + / + , control ; Mertk - / - , Cdc50a cKO ; Mertk + / + and Cdc50a cKO ; Mertk - / - mice . The dashed line represents where the high magnified insert was taken from . Scale bar , 20 μm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Representative confocal z-stack images of Gephyrin (green)- and VGAT (red)-positive inhibitory synapses in the IC (inferior colliculus) of control;C3+/+, control;C3-/-, Cdc50a cKO;C3+/+ and Cdc50a cKO;C3-/- mice. The dashed line represents where the high magnified insert was taken from. Scale bar, 10 μm.Representative confocal z-stack images of Gephyrin (green)- and VGAT (red)-positive inhibitory synapses in the IC of control;Mertk+/+, control;Mertk-/-, Cdc50a cKO;Mertk+/+ and Cdc50a cKO;Mertk-/- mice. The dashed line represents where the high magnified insert was taken from. Scale bar, 20 μm.


Figure 6Schematic diagram of the experiment in which PS exposure was measured in the brains of 1 - month - and 3 - month - old wild - type mice by injecting AAV9 - GFAP - secA5 - mCherry in the neonatal stage ( P0 ) . Representative confocal z - stack colocalization images of Gephyrin ( green ) , VGAT ( blue ) , and secA5 ( red ) in the HP ( hippocampus ) of 1 - month - and 3 - month - old wild - type mice . The white circles indicate secA5 - positive inhibitory synapses . Scale bar , 10 μm . Bar graphs showing the expression of secA5 ( B ) and IBA1 ( C ) in inhibitory post - synapses ( Gephyrin - positive , green ) and pre - synapses ( VGAT - positive , blue ) in the HP of 1 - month - and 3 - month - old wild - type mice ( n = 4 for each group , * * * P = 0 . 0009 , * P = 0 . 0248 , n . s . , not significant ) . Schematic diagram of the experiment in which Annexin V was injected intravenously ( every two days ) to block PS in the brains of 1 - month - and 3 - month - old wild - type mice . Representative confocal z - stack images of Gephyrin ( green ) - and VGAT ( red ) - positive inhibitory synapses in the HP of 1 - month - old wild - type mice . The white circles indicate inhibitory synapses . Scale bar , 10 μm . Bar graphs showing the number of inhibitory ( Gephyrin - and VGAT - positive ) synapses in the HP of 1 - month - old mice after PBS or Annexin V injection ( n = 4 for each group , * * * P = 0 . 0013 , * * P = 0 . 0090 ) . Schematic diagram of the experiment in which microglial Mertk KO ( CX3CR1 - CreERT2 ; Mertkfl / fl ) and control ( Mertkfl / fl ) mice were generated . Both 1 - month - old microglial Mertk KO mice and 1 - month - old control mice were injected with 4 - OHT for 5 consecutive days . Representative confocal z - stack images of Gephyrin ( green ) - and VGAT ( magenta ) - positive inhibitory synapses in the HP of Mertkfl / fl and CX3CR1 - CreERT2 ; Mertkfl / fl mice . The white circles indicate inhibitory synapses . Scale bar , 10 μm . Bar graphs showing the number of inhibitory ( Gephyrin - and VGAT - positive ) synapses in the HP of Mertkfl / fl and CX3CR1 - CreERT2 ; Mertkfl / fl mice ( n = 4 for each group , * * P = 0 . 0027 , * P = 0 . 0062 ) . Representative confocal z - stack colocalization images of Gephyrin ( green ) , VGAT ( blue ) and secA5 ( red ) in the HP of Mertkfl / fl and CX3CR1 - CreERT2 ; Mertkfl / fl mice . The white circles indicate secA5 - positive inhibitory synapses . Scale bar , 10 μm . Bar graphs showing the relative expression of secA5 in inhibitory synapses in the HP of Mertkfl / fl and CX3CR1 - CreERT2 ; Mertkfl / fl mice ( n = 4 for each group , * P = 0 . 0015 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6Schematic diagram of the experiment in which PS exposure was measured in the brains of 1-month- and 3-month-old wild-type mice by injecting AAV9-GFAP-secA5-mCherry in the neonatal stage (P0). Representative confocal z-stack colocalization images of Gephyrin (green), VGAT (blue), and secA5 (red) in the HP (hippocampus) of 1-month- and 3-month-old wild-type mice. The white circles indicate secA5-positive inhibitory synapses. Scale bar, 10 μm. Bar graphs showing the expression of secA5 (B) and IBA1 (C) in inhibitory post-synapses (Gephyrin-positive, green) and pre-synapses (VGAT-positive, blue) in the HP of 1-month- and 3-month-old wild-type mice (n = 4 for each group, ***P = 0.0009, *P = 0.0248, n.s., not significant).Schematic diagram of the experiment in which Annexin V was injected intravenously (every two days) to block PS in the brains of 1-month- and 3-month-old wild-type mice. Representative confocal z-stack images of Gephyrin (green)- and VGAT (red)-positive inhibitory synapses in the HP of 1-month-old wild-type mice. The white circles indicate inhibitory synapses. Scale bar, 10 μm. Bar graphs showing the number of inhibitory (Gephyrin- and VGAT-positive) synapses in the HP of 1-month-old mice after PBS or Annexin V injection (n = 4 for each group, ***P = 0.0013, **P = 0.0090).Schematic diagram of the experiment in which microglial Mertk KO (CX3CR1-CreERT2;Mertkfl/fl) and control (Mertkfl/fl) mice were generated. Both 1-month-old microglial Mertk KO mice and 1-month-old control mice were injected with 4-OHT for 5 consecutive days. Representative confocal z-stack images of Gephyrin (green)- and VGAT (magenta)-positive inhibitory synapses in the HP of Mertkfl/fl and CX3CR1-CreERT2;Mertkfl/fl mice. The white circles indicate inhibitory synapses. Scale bar, 10 μm. Bar graphs showing the number of inhibitory (Gephyrin- and VGAT-positive) synapses in the HP of Mertkfl/fl and CX3CR1-CreERT2;Mertkfl/fl mice (n = 4 for each group, **P = 0.0027, *P = 0.0062). Representative confocal z-stack colocalization images of Gephyrin (green), VGAT (blue) and secA5 (red) in the HP of Mertkfl/fl and CX3CR1-CreERT2;Mertkfl/fl mice. The white circles indicate secA5-positive inhibitory synapses. Scale bar, 10 μm. Bar graphs showing the relative expression of secA5 in inhibitory synapses in the HP of Mertkfl/fl and CX3CR1-CreERT2;Mertkfl/fl mice (n = 4 for each group, *P = 0.0015).


Figure 1B Sequencing chromatograms depicting the identified 22 nucleotide deletion ( c . 346 _ * 7del ) identified in Subject 1 ( S1 ) , the heterozygous state of the deletion in the father , and the wild - type NDUFC2 sequence in Control DNA . C Sequencing chromatograms depicting the identified missense variant ( c . 173A > T ; p . ( His58Leu ) ) in Subject 3 ( S3 ) and the wild - type NDUFC2 sequence in Control DNA .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1B Sequencing chromatograms depicting the identified 22 nucleotide deletion (c.346_*7del) identified in Subject 1 (S1), the heterozygous state of the deletion in the father, and the wild-type NDUFC2 sequence in Control DNA.C Sequencing chromatograms depicting the identified missense variant (c.173A>T; p.(His58Leu)) in Subject 3 (S3) and the wild-type NDUFC2 sequence in Control DNA.


Figure 2A NDUFC2 mRNA transcript levels in fibroblast cell lines of controls ( white ) , Subject 1 ( grey ) and Subject 3 ( black ) . Quantification of NDUFC2 mRNA transcript levels ( normalised to ACTB ) showed diminished transcript levels in Subject 1 while transcript levels were unaffected in Subject 3 . Bars correspond to standard deviations of two independent experiments , performed in duplicate and analysed by Student ' s t - test ; * p < 0 . 05 . B Assessment of respiratory chain enzyme activities , normalised to citrate synthase ( CS ) , in fibroblast cell lines from controls ( white ) , Subject 1 ( grey ) and Subject 3 ( black ) . Mean residual enzyme activities of control fibroblasts ( n = 8 ) are set at 100 % and error bars represent 1 standard deviation of control activities . Asterisks ( * ) denote enzyme activity below control range . C Assessment of respiratory chain enzyme activities , normalised to citrate synthase ( CS ) , in skeletal muscle from controls ( white ) and Subject 3 ( black ) . Mean residual enzyme activities of control skeletal muscle ( n > 100 samples ) are set at 100 % and bars are 1 standard deviation of control activities . Asterisks ( * ) denote enzyme activity below control range . D Respiration of Subject 3 and Subject 1 derived fibroblasts ( black and grey , respectively ) compared to control fibroblasts ( white ) using high - resolution respirometry . ROX - corrected analysis of basal respiration , Oligomycin inhibited LEAK respiration and maximum uncoupled respiration ( ETS ) . Both subject fibroblasts exhibited a severe mitochondrial respiration defect as measured by oxygen consumption . Data are mean ± SD from at least 3 experiments and analysed by Student ' s t - test . * * p < 0 . 01 , * * * p < 0 . 001 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A NDUFC2 mRNA transcript levels in fibroblast cell lines of controls (white), Subject 1 (grey) and Subject 3 (black). Quantification of NDUFC2 mRNA transcript levels (normalised to ACTB) showed diminished transcript levels in Subject 1 while transcript levels were unaffected in Subject 3. Bars correspond to standard deviations of two independent experiments, performed in duplicate and analysed by Student's t-test; *p<0.05.B Assessment of respiratory chain enzyme activities, normalised to citrate synthase (CS), in fibroblast cell lines from controls (white), Subject 1 (grey) and Subject 3 (black). Mean residual enzyme activities of control fibroblasts (n = 8) are set at 100% and error bars represent 1 standard deviation of control activities. Asterisks (*) denote enzyme activity below control range.C Assessment of respiratory chain enzyme activities, normalised to citrate synthase (CS), in skeletal muscle from controls (white) and Subject 3 (black). Mean residual enzyme activities of control skeletal muscle (n>100 samples) are set at 100% and bars are 1 standard deviation of control activities. Asterisks (*) denote enzyme activity below control range.D Respiration of Subject 3 and Subject 1 derived fibroblasts (black and grey, respectively) compared to control fibroblasts (white) using high-resolution respirometry. ROX-corrected analysis of basal respiration, Oligomycin inhibited LEAK respiration and maximum uncoupled respiration (ETS). Both subject fibroblasts exhibited a severe mitochondrial respiration defect as measured by oxygen consumption. Data are mean ± SD from at least 3 experiments and analysed by Student's t-test. **p<0.01, ***p<0.001.


Figure 3A Western blot analysis of various structural subunits of complex I in fibroblasts from Subjects 1 and 3 ( S1 and S3 respectively ) with three age - matched controls ( C1 , C2 , and C3 ) . B BN - PAGE analysis of OXPHOS complex assembly in enriched mitochondria from subject and control fibroblasts solubilised using DDM . Immunoblotting was performed using antibodies to structural subunits from each OXPHOS complex ( CI [ NDUFB8 ] , CII [ SDHA ] , CIII [ UQCRC2 ] , CIV [ MT - CO1 ] , and CV [ ATP5A ] ) . C Western blot analysis of whole cell lysates from controls ( C1 and C2 ) and subject cell lines ( S1 and S3 ) that were either untransduced ( - ) or transduced with the lentiviral vector ( pLVX ) containing wild - type NDUFC2 ( + ) . Transduced cell lines were either uninduced or induced with 100 or 200 ng / ml doxycycline ( for 48 and 96 hours respectively ) where indicated . D BN - PAGE analysis of OXPHOS complex assembly in enriched mitochondria isolated from controls ( C1 and C2 ) and subject cell lines that were either untransduced ( - ) or transduced with the lentiviral vector ( pLVX ) containing wild - type NDUFC2 ( + ) . Transduced cell lines were either uninduced or induced with 200 ng / ml doxycycline for 72 hours .

O B-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A Western blot analysis of various structural subunits of complex I in fibroblasts from Subjects 1 and 3 (S1 and S3 respectively) with three age-matched controls (C1, C2, and C3).B BN-PAGE analysis of OXPHOS complex assembly in enriched mitochondria from subject and control fibroblasts solubilised using DDM. Immunoblotting was performed using antibodies to structural subunits from each OXPHOS complex (CI [NDUFB8], CII [SDHA], CIII [UQCRC2], CIV [MT-CO1], and CV [ATP5A]).C Western blot analysis of whole cell lysates from controls (C1 and C2) and subject cell lines (S1 and S3) that were either untransduced (-) or transduced with the lentiviral vector (pLVX) containing wild-type NDUFC2 (+). Transduced cell lines were either uninduced or induced with 100 or 200 ng/ml doxycycline (for 48 and 96 hours respectively) where indicated.D BN-PAGE analysis of OXPHOS complex assembly in enriched mitochondria isolated from controls (C1 and C2) and subject cell lines that were either untransduced (-) or transduced with the lentiviral vector (pLVX) containing wild-type NDUFC2 (+). Transduced cell lines were either uninduced or induced with 200 ng/ml doxycycline for 72 hours.


Figure 4A Complexome profiling identified assembly intermediates in subject cells with NDUFC2 variants . Complexes from mitochondrial membranes of Control , Subject 1 and Subject 3 fibroblasts were separated by BN - PAGE ( Fig EV2 ) and analysed by complexome profiling . Sample preparation , mass spectrometry , data processing and raw data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository ( Vizcaíno et al . , 2013 ) with the dataset identifier < PXD014936 > . Assignment of complexes : III , complex III ; IV , complex IV ; III / IV , supercomplex containing dimer of complex III and 1 - 2 copies of complex IV ; S , supercomplex containing complex I , dimer of complex III and 1 - 4 copies of complex IV . Assembly intermediates are highlighted in dashed boxes indicating stalled complex I assembly . In subject complexomes , various stalled complex I intermediates are assembled : ND4 module ( blue dashed box ) ; Q - TIMMDC1 - NDUFA13 complex I intermediate ( orange dashed box ) ; and Q - TIMMDC1 - NDUFA13 - MCIA ( green dashed box ) . In control complexome , the Q module ( orange dashed box ) is assembled without TIMMDC1 or NDUFA13 . Complex I modules are denoted according to Stroud and colleagues ( Stroud et al . , 2016 ) . B Abundance data for all the subunits of complex I were averaged to plot an abundance profile of complex I . Heatmap ( upper panel ) and profile plot ( lower panel ) compare complex I between control ( black ) , Subject 1 ( red ) and Subject 3 ( blue ) . C Profile plot of complex III ( average of all subunits ) . Black arrows indicate shifted complex III to the position of the individual complex and small supercomplex ( III / IV ) in Subjects 1 and 3 . D Profile plot of complex IV ( average of all subunits ) indicate less complex IV at the position of the supercomplexes for Subject 1 and Subject 3 , but higher amounts of individual complex IV and small supercomplex ( III / IV , black arrow ) . E Details of Q module - containing assembly intermediates of Subject 3 . Intensity based absolute quantification ( iBAQ ) values of assembly factors , single subunit and averaged Q - module subunits were plotted to show abundance profiles . The cartoon ( lower panel ) indicate assembled intermediate complexes relative to the plot . F Details about ND4 module assembly intermediate of Subject 3 . Abundance profile of averaged iBAQ values of ND4 subunits and assembly factors show accumulation of a complex containing the ND4 module , FOXRED1 and TMEM70 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A Complexome profiling identified assembly intermediates in subject cells with NDUFC2 variants. Complexes from mitochondrial membranes of Control, Subject 1 and Subject 3 fibroblasts were separated by BN-PAGE (Fig EV2) and analysed by complexome profiling. Sample preparation, mass spectrometry, data processing and raw data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (Vizcaíno et al., 2013) with the dataset identifier <PXD014936>. Assignment of complexes: III, complex III; IV, complex IV; III/IV, supercomplex containing dimer of complex III and 1-2 copies of complex IV; S, supercomplex containing complex I, dimer of complex III and 1-4 copies of complex IV. Assembly intermediates are highlighted in dashed boxes indicating stalled complex I assembly. In subject complexomes, various stalled complex I intermediates are assembled: ND4 module (blue dashed box); Q-TIMMDC1-NDUFA13 complex I intermediate (orange dashed box); and Q-TIMMDC1-NDUFA13-MCIA (green dashed box). In control complexome, the Q module (orange dashed box) is assembled without TIMMDC1 or NDUFA13. Complex I modules are denoted according to Stroud and colleagues (Stroud et al., 2016).B Abundance data for all the subunits of complex I were averaged to plot an abundance profile of complex I. Heatmap (upper panel) and profile plot (lower panel) compare complex I between control (black), Subject 1 (red) and Subject 3 (blue). C Profile plot of complex III (average of all subunits). Black arrows indicate shifted complex III to the position of the individual complex and small supercomplex (III/IV) in Subjects 1 and 3. D Profile plot of complex IV (average of all subunits) indicate less complex IV at the position of the supercomplexes for Subject 1 and Subject 3, but higher amounts of individual complex IV and small supercomplex (III/IV, black arrow). E Details of Q module-containing assembly intermediates of Subject 3. Intensity based absolute quantification (iBAQ) values of assembly factors, single subunit and averaged Q-module subunits were plotted to show abundance profiles. The cartoon (lower panel) indicate assembled intermediate complexes relative to the plot.F Details about ND4 module assembly intermediate of Subject 3. Abundance profile of averaged iBAQ values of ND4 subunits and assembly factors show accumulation of a complex containing the ND4 module, FOXRED1 and TMEM70.


Figure 6The intestine - specific RNAi against irx - 1 reduces the Smurf phenotype at day 8 of adulthood . Mean ± SD . Scale bar : 100 μm . Unpaired t - test , two - tailed . More than 30 worms analysed in each of the three biological replicates . , C The intestine - specific RNAi against irx - 1 reduces the Smurf phenotype at day 8 of adulthood . Mean ± SD . Scale bar : 100 μm . Unpaired t - test , two - tailed . More than 30 worms analysed in each of the three biological replicates .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6The intestine-specific RNAi against irx-1 reduces the Smurf phenotype at day 8 of adulthood. Mean ± SD. Scale bar: 100 μm. Unpaired t-test, two-tailed. More than 30 worms analysed in each of the three biological replicates., C The intestine-specific RNAi against irx-1 reduces the Smurf phenotype at day 8 of adulthood. Mean ± SD. Scale bar: 100 μm. Unpaired t-test, two-tailed. More than 30 worms analysed in each of the three biological replicates.


Figure 7B , C The BWM - specific RNAi against myrf - 2 improves sarcomere in the worms at day 8 of adulthood . Arrowheads and arrows respectively denote the gaps of myofilaments and the disorganised myofilaments with GFP aggregations . Scale Bar : 10 μm . Mean ± SD . Unpaired t - test , two - tailed . n > = 50 . D , E Suppressing myrf - 2 in BWM increases worms ' motility at day 5 of adulthood . The tracks were from worms ' movement in 30 sec ( D ) . The speed of worm movement was measured by the ratio of track length versus body length ( E ) . Mean ± SD . n > = 120 . Unpaired t - test , two - tailed .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7B, C The BWM-specific RNAi against myrf-2 improves sarcomere in the worms at day 8 of adulthood. Arrowheads and arrows respectively denote the gaps of myofilaments and the disorganised myofilaments with GFP aggregations. Scale Bar: 10 μm. Mean ± SD. Unpaired t-test, two-tailed. n>=50.D, E Suppressing myrf-2 in BWM increases worms' motility at day 5 of adulthood. The tracks were from worms' movement in 30 sec (D). The speed of worm movement was measured by the ratio of track length versus body length (E). Mean ± SD. n>=120. Unpaired t-test, two-tailed.


Figure 1B . Relative quantification of all four histone proteins in each tissue replicate ( order of appearance per replicate : H2A , H2B , H3 . 3 and H4 ) . C . Immunohistochemistry images from the Human Protein Atlas ( www . proteinatlas . org ) for protein ANXA1 with nuclear staining ( blue ) for three selected tissues . D . Calibration curves for two of the four histone peptides , with decreasing amount of QPrEST standard spiked into a U2OS cell lysate .

O B-PANEL_START O O O O O O 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-PANEL_START O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1B. Relative quantification of all four histone proteins in each tissue replicate (order of appearance per replicate: H2A, H2B, H3.3 and H4).C. Immunohistochemistry images from the Human Protein Atlas (www.proteinatlas.org) for protein ANXA1 with nuclear staining (blue) for three selected tissues.D. Calibration curves for two of the four histone peptides, with decreasing amount of QPrEST standard spiked into a U2OS cell lysate.


Figure 2A . Absolute copy number of protein in kidney tissue and human embryonal kidney cells ( HEK293 ) , liver tissue and liver cancer cell line ( HepG2 ) , lung tissue and lung cancer cell line ( A549 ) , and breast tissue and breast cancer cell line ( MCF7 ) . The order of proteins are the same in the tissue and corresponding cell line and the proteins have been order according to the abundance in the respective tissue . B . The direct correlation between RNA ( TPM ) and protein abundances ( copy number ) for all quantified genes in the same tissues and cell lines . Spearman ( ρ ) and Pearson ( r ) correlation between the two values across the quantified genes are shown . The other seven tissues and five cell lines are shown in Figure EV4 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A. Absolute copy number of protein in kidney tissue and human embryonal kidney cells (HEK293), liver tissue and liver cancer cell line (HepG2), lung tissue and lung cancer cell line (A549), and breast tissue and breast cancer cell line (MCF7). The order of proteins are the same in the tissue and corresponding cell line and the proteins have been order according to the abundance in the respective tissue.B. The direct correlation between RNA (TPM) and protein abundances (copy number) for all quantified genes in the same tissues and cell lines. Spearman (ρ) and Pearson (r) correlation between the two values across the quantified genes are shown. The other seven tissues and five cell lines are shown in Figure EV4.


Figure 3Subcellular localization by immunofluorescence staining and immunohistochemistry staining in tissue sections by three different antibodies ( SELENBP1 , HPA011731 ; STOM , HPA010961 ; ASS1 , HPA020896 ) . Microtubule - and nuclear probes are visualized in red and blue respectively . Antibody staining is shown in green . RNA to protein ratio across nine cell lines and 11 tissues with Spearman rho , Pearson r and R2 for each gene . All other genes can be found in Figure EV1 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Subcellular localization by immunofluorescence staining and immunohistochemistry staining in tissue sections by three different antibodies (SELENBP1, HPA011731; STOM, HPA010961; ASS1, HPA020896). Microtubule- and nuclear probes are visualized in red and blue respectively. Antibody staining is shown in green. RNA to protein ratio across nine cell lines and 11 tissues with Spearman rho, Pearson r and R2 for each gene. All other genes can be found in Figure EV1.


Figure 2Four to six - week - old C57BL / 6 mice were injected i . p . with 104 - 107 PFU of WNV - poly ( A ) , 104 PFU of WT WNV or PBS ( negative control ) in a volume of 200 μL . Survival ( n = 5 ) in infected mice were monitored daily Data information : Log - rank test was usedFour to six - week - old C57BL / 6 mice were injected i . p . with 104 - 107 PFU of WNV - poly ( A ) , 104 PFU of WT WNV or PBS ( negative control ) in a volume of 200 μL . Histological analysis of mouse brain . Scale bars represent 100 μm and 50 μm for left and right panels , respectively . Severe vacuolization ( black arrows ) and inflammation ( yellow arrow ) in brain small vessels as well as degenerative changes in neurons with cytoplasmic rarefaction ( blue arrow ) and necrosis with nuclear compaction ( red arrow ) were indicated . Scale bars represent 100 μm and 50 μm for left and right panels , respectivelyE Viral load and replication kinetics in different tissues of C57BL / 6 mice . Six - week - old female C57BL / 6 mice were inoculated subcutaneously with 106 PFU of WT WNV or WNV - poly ( A ) in the left rear footpad , and tissues were harvested from two mice at the indicated times post - inoculation . Viral loads in brain , spleen , left lymph node , right lymph node and liver were measured by plaque assay and expressed as PFU per gram of tissue . n = 3 , values are mean ± sd , the dotted line corresponds to the limit of detection ( LOD ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Four to six-week-old C57BL/6 mice were injected i.p. with 104-107 PFU of WNV-poly(A), 104 PFU of WT WNV or PBS (negative control) in a volume of 200 μL. Survival (n=5) in infected mice were monitored daily Data information: Log-rank test was usedFour to six-week-old C57BL/6 mice were injected i.p. with 104-107 PFU of WNV-poly(A), 104 PFU of WT WNV or PBS (negative control) in a volume of 200 μL. Histological analysis of mouse brain. Scale bars represent 100 μm and 50 μm for left and right panels, respectively. Severe vacuolization (black arrows) and inflammation (yellow arrow) in brain small vessels as well as degenerative changes in neurons with cytoplasmic rarefaction (blue arrow) and necrosis with nuclear compaction (red arrow) were indicated. Scale bars represent 100 μm and 50 μm for left and right panels, respectivelyE Viral load and replication kinetics in different tissues of C57BL/6 mice. Six-week-old female C57BL/6 mice were inoculated subcutaneously with 106 PFU of WT WNV or WNV-poly(A) in the left rear footpad, and tissues were harvested from two mice at the indicated times post-inoculation. Viral loads in brain, spleen, left lymph node, right lymph node and liver were measured by plaque assay and expressed as PFU per gram of tissue. n=3, values are mean ± sd, the dotted line corresponds to the limit of detection (LOD).


Figure 3B - C Total anti - WNV IgG antibody ( B ) and neutralizing antibody titers ( C ) in mouse serum at the indicated days post immunization . ( For B and C , the values represent mean ± sd in each group and the horizontal dotted line represents the limit of detection ) Data information : For statistical analysis , one - way ANOVA analysis were used * p < 0 . 5 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , ns represents not significantD - E Survival ( D ) and body - weight changes were monitored daily after challenge . ( The values represent mean ± sd in each group ) ( E ) . Data information : For statistical analysis , two - way ANOVA analysis were used Log - rank test was performed * p < 0 . 5 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , ns represents not significantG - H T - cell responses in vaccinated mice were measured through ELISpot assay . Two groups of C57BL / 6 mice ( n = 4 / group ) were i . p . inoculated with 107 of WNV - poly ( A ) or equal volume of PBS . ELISpot assay specific for IFN - γ in splenocytes was performed at 7 days post - immunization . Representative images were shown in ( G ) and the numbers of antibody secreting cells ( ASCs ) were counted ( The values represent mean ± sd in each group , each symbol represents a mouse ) ( H ) . Data information : Mann - Whitney test was used for H . * p < 0 . 5 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , ns represents not

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3B-C Total anti-WNV IgG antibody (B) and neutralizing antibody titers (C) in mouse serum at the indicated days post immunization. (For B and C, the values represent mean ± sd in each group and the horizontal dotted line represents the limit of detection) Data information: For statistical analysis, one-way ANOVA analysis were used *p<0.5, **p<0.01, *** p<0.001,****p< 0.0001, ns represents not significantD-E Survival (D) and body-weight changes were monitored daily after challenge. (The values represent mean ± sd in each group) (E). Data information: For statistical analysis, two-way ANOVA analysis were used Log-rank test was performed *p<0.5, **p<0.01, *** p<0.001,****p< 0.0001, ns represents not significantG-H T-cell responses in vaccinated mice were measured through ELISpot assay. Two groups of C57BL/6 mice (n = 4 / group) were i.p. inoculated with 107 of WNV-poly(A) or equal volume of PBS. ELISpot assay specific for IFN-γ in splenocytes was performed at 7 days post-immunization. Representative images were shown in (G) and the numbers of antibody secreting cells (ASCs) were counted (The values represent mean ± sd in each group, each symbol represents a mouse) (H). Data information: Mann-Whitney test was used for H. *p<0.5, **p<0.01, *** p<0.001,****p< 0.0001, ns represents not significant


Figure 4C Comparison of growth kinetics between WT WNV and WNV - poly ( A ) viruses in MEF cells . MEF cells were infected with either WT WNV or WNV - poly ( A ) virus at an MOI of 0 . 01 . Values indicate mean ± sd from two replicates of one representative experiment . Data information : Three independent assays were performed for above experiments with similar results and data of one representative data was shown . Scale bars represent 50 μm . D Survival rate of IFNAR - / - mice infected with different dosages of WNV - poly ( A ) ( n = 5 ) . Data information : Three independent assays were performed for above experiments with similar results and data of one representative data was shown . Scale bars represent 50 μm . E Cell morphology changes of MEF cells infected with WT WNV and WNV - poly ( A ) . Data information : Three independent assays were performed for above experiments with similar results and data of one representative data was shown . Scale bars represent 50 μm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4C Comparison of growth kinetics between WT WNV and WNV-poly(A) viruses in MEF cells. MEF cells were infected with either WT WNV or WNV-poly(A) virus at an MOI of 0.01. Values indicate mean ± sd from two replicates of one representative experiment. Data information: Three independent assays were performed for above experiments with similar results and data of one representative data was shown. Scale bars represent 50 μm.D Survival rate of IFNAR-/- mice infected with different dosages of WNV-poly(A) (n=5). Data information: Three independent assays were performed for above experiments with similar results and data of one representative data was shown. Scale bars represent 50 μm.E Cell morphology changes of MEF cells infected with WT WNV and WNV-poly(A). Data information: Three independent assays were performed for above experiments with similar results and data of one representative data was shown. Scale bars represent 50 μm.


Figure 5Virulence and efficacy of the passaged WNV - poly ( A ) ( WNV - poly ( A ) - P50 ) in mice ( n = 5 ) . ( E ) Survival of mice infected with 107 PFU of WNV - poly ( A ) - P50 . Data information : For statistical analysis , one - way ANOVA analysis and Log - rank test were used * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , ns represents not significant . Virulence and efficacy of the passaged WNV - poly ( A ) ( WNV - poly ( A ) - P50 ) in mice ( n = 5 ) . ( F ) Antibody titers of mice infected with WNV - poly ( A ) - P50 at different days post immunization . ( For the values represent mean ± sd in each group ( n = 5 ) and the horizontal dotted line represents the limit of detection ) . Data information : For statistical analysis , Mann - Whitney test were used * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , ns represents not significant . ( I ) Survival of mice after WT WNV challenge . Data information : For statistical analysis , one - way ANOVA analysis and Log - rank test were used * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , ns represents not significant .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Virulence and efficacy of the passaged WNV-poly(A) (WNV-poly(A)-P50) in mice (n=5). (E) Survival of mice infected with 107 PFU of WNV-poly(A)-P50. Data information: For statistical analysis, one-way ANOVA analysis and Log-rank test were used **p<0.01, *** p<0.001,****p< 0.0001, ns represents not significant.Virulence and efficacy of the passaged WNV-poly(A) (WNV-poly(A)-P50) in mice (n=5). (F) Antibody titers of mice infected with WNV-poly(A)-P50 at different days post immunization. (For the values represent mean ± sd in each group (n=5) and the horizontal dotted line represents the limit of detection). Data information: For statistical analysis, Mann-Whitney test were used **p<0.01, *** p<0.001,****p< 0.0001, ns represents not significant.(I) Survival of mice after WT WNV challenge. Data information: For statistical analysis, one-way ANOVA analysis and Log-rank test were used **p<0.01, *** p<0.001,****p< 0.0001, ns represents not significant.


Figure 1A A549 and U937 cells were infected with each IAV at MOI 1 or 5 for 24 h , and lysates were analyzed by Western blotting using polyclonal anti - PB1 - F2 antibody . B A549 cells were transfected with a plasmid encoding Flag - tagged PB1 - F2 derived from the PR8 or 1918 strain and treated with the proteasome inhibitor MG132 ( 25 μM ) or vehicle for 6 h before harvesting . PB1 - F2 transcripts were analyzed by semi - quantitative RT - PCR ( top panel ) , and The PB1 - F2 protein was analyzed by Western blotting with monoclonal anti - Flag antibody . C Representative immunofluorescence images showing expression of PB1 - F2 ( red ) . A549 cells were transfected with plasmids encoding indicated PB1 - F2 . At 18 h after transfection , cells were treated with MG132 for 6 h and subjected to immunofluorescence analysis . Magnification , × 100 ; scale bar , 100 μm . D Each HA - tagged PB1 - F2 was transfected into 293T cells with or without Flag - ubiquitin . At 18 h after transfection , cells were treated with MG132 for 6 h . The lysates were immunoprecipitated with anti - HA antibody and analyzed by Western blotting with anti - Flag antibody . PR8 , A / Puerto Rico / 8 / 34 ( H1N1 ) ; 1918 , A / Brevig Mission / 1 / 1918 ( H1N1 ) .

O B-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A A549 and U937 cells were infected with each IAV at MOI 1 or 5 for 24 h, and lysates were analyzed by Western blotting using polyclonal anti-PB1-F2 antibody.B A549 cells were transfected with a plasmid encoding Flag-tagged PB1-F2 derived from the PR8 or 1918 strain and treated with the proteasome inhibitor MG132 (25 μM) or vehicle for 6 h before harvesting. PB1-F2 transcripts were analyzed by semi-quantitative RT-PCR (top panel), and The PB1-F2 protein was analyzed by Western blotting with monoclonal anti-Flag antibody.C Representative immunofluorescence images showing expression of PB1-F2 (red). A549 cells were transfected with plasmids encoding indicated PB1-F2. At 18 h after transfection, cells were treated with MG132 for 6 h and subjected to immunofluorescence analysis. Magnification, ×100; scale bar, 100 μm.D Each HA-tagged PB1-F2 was transfected into 293T cells with or without Flag-ubiquitin. At 18 h after transfection, cells were treated with MG132 for 6 h. The lysates were immunoprecipitated with anti-HA antibody and analyzed by Western blotting with anti-Flag antibody. PR8, A/Puerto Rico/8/34 (H1N1); 1918, A/Brevig Mission/1/1918 (H1N1).


Figure 2B - D A549 cells were transfected with the indicated plasmids encoding Flag - tagged PB1 - F2 derived from the PR8 or 1918 strain and chimeric PB1 - F2 mutants . At 18 h after transfection , cells were treated with or without MG132 for 6 h . The expression of PB1 - F2 and control genes was detected by semi - quantitative RT - PCR ( upper panel ) or Western blotting ( lower panel ) . Results shown are representative of three independent experiments . E Representative immunofluorescence images of PB1 - F2 expression in A549 cells . Cells transfected with the Flag - tagged PB1 - F2 plasmid were stained with anti - Flag ( red ) antibody and analyzed by immunofluorescence assay . Magnification , × 400 ; scale bar , 50 μm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2B-D A549 cells were transfected with the indicated plasmids encoding Flag-tagged PB1-F2 derived from the PR8 or 1918 strain and chimeric PB1-F2 mutants. At 18 h after transfection, cells were treated with or without MG132 for 6 h. The expression of PB1-F2 and control genes was detected by semi-quantitative RT-PCR (upper panel) or Western blotting (lower panel). Results shown are representative of three independent experiments.E Representative immunofluorescence images of PB1-F2 expression in A549 cells. Cells transfected with the Flag-tagged PB1-F2 plasmid were stained with anti-Flag (red) antibody and analyzed by immunofluorescence assay. Magnification, ×400; scale bar, 50 μm.


Figure 3A Relative activity of NF - kB luciferase reporter was measured in A549 cells transfected with plasmids encoding PB1 - F2 . The mRNA levels of pro - inflammatory cytokines were determined by semi - quantitative RT - PCR . All data are shown as mean ( ± SEM ) from at least three independent experiments ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ) . Cells were transfected with PB1 - F2 - expressing plasmids . Relative IFNβ mRNA levels were determined by semi - quantitative RT - PCR ( top ) and qPCR ( bottom ) . All data are shown as mean ( ± SEM ) from at least three independent experiments ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ) . C U937 cells were co - transfected with the IFNβ luciferase reporter plasmid and the PB1 - F2 or NS1 expressing plasmid and treated with polyI : C for 12 h before harvesting . IFNβ promoter activity was determined by luciferase reporter assay . All data are shown as mean ( ± SEM ) from at least three independent experiments ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ) . Cells were infected for 24 h with each virus at 1 MOI . The mRNA level of IFNβ was determined by semi - quantitative RT - PCR , and PB1 - F2 protein expression was assessed by Western blotting . All data are shown as mean ( ± SEM ) from at least three independent experiments ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ) . E Cells were transfected with each plasmid encoding PB1 - F2 . 18 h after transfection , cells were treated with MG132 for 6 h and the level of IFNβ mRNA was determined by semi - quantitative RT - PCR ( left ) and qPCR ( right ) . PB1 - F2 protein expression was assessed by Western blotting . All data are shown as mean ( ± SEM ) from at least three independent experiments ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ) . F A549 cells were transfected with PB1 - F2 clones mutated at positions 68 and 69 ; 24 h after transfection , the IFNβ mRNA level and PB1 - F2 protein expression were determined by semi - quantitative RT - PCR and Western blotting , respectively ( left ) . The relative expression level of IFNβ mRNA was also analyzed by qPCR ( right ) . All data are shown as mean ( ± SEM ) from at least three independent experiments ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A Relative activity of NF-kB luciferase reporter was measured in A549 cells transfected with plasmids encoding PB1-F2. The mRNA levels of pro-inflammatory cytokines were determined by semi-quantitative RT-PCR. All data are shown as mean (±SEM) from at least three independent experiments (*p < 0.05, **p <0.01, ***p <0.001).Cells were transfected with PB1-F2-expressing plasmids. Relative IFNβ mRNA levels were determined by semi-quantitative RT-PCR (top) and qPCR (bottom). All data are shown as mean (±SEM) from at least three independent experiments (*p < 0.05, **p <0.01, ***p <0.001).C U937 cells were co-transfected with the IFNβ luciferase reporter plasmid and the PB1-F2 or NS1 expressing plasmid and treated with polyI:C for 12 h before harvesting. IFNβ promoter activity was determined by luciferase reporter assay. All data are shown as mean (±SEM) from at least three independent experiments (*p < 0.05, **p <0.01, ***p <0.001).Cells were infected for 24 h with each virus at 1 MOI. The mRNA level of IFNβ was determined by semi-quantitative RT-PCR, and PB1-F2 protein expression was assessed by Western blotting. All data are shown as mean (±SEM) from at least three independent experiments (*p < 0.05, **p <0.01, ***p <0.001).E Cells were transfected with each plasmid encoding PB1-F2. 18 h after transfection, cells were treated with MG132 for 6 h and the level of IFNβ mRNA was determined by semi-quantitative RT-PCR (left) and qPCR (right). PB1-F2 protein expression was assessed by Western blotting. All data are shown as mean (±SEM) from at least three independent experiments (*p < 0.05, **p <0.01, ***p <0.001).F A549 cells were transfected with PB1-F2 clones mutated at positions 68 and 69; 24 h after transfection, the IFNβ mRNA level and PB1-F2 protein expression were determined by semi-quantitative RT-PCR and Western blotting, respectively (left). The relative expression level of IFNβ mRNA was also analyzed by qPCR (right). All data are shown as mean (±SEM) from at least three independent experiments (*p < 0.05, **p <0.01, ***p <0.001).


Figure 4A Mice ( n = 5 or 6 per group ) were infected with IAV with indicated pfu . Survival was monitored daily for 2 weeks . B Mice ( n = 6 - 9 per group ) were infected with each IAV as indicated . Body weight was recorded daily after infection for 2 weeks . C Mice ( n = 5 or 6 per group ) were infected with viruses ( 1000 pfu ) carrying PR8 PB1 - F2 with the I68T , L69P , or I68T + L69P mutation . Body weight changes and survival rate were monitored daily . Detailed analyses on the virulence necessitated the monitoring of body weight below the standard level ( 80 % ) of mortality . Three cohorts of mice ( 5 or 6 per group ) were infected with 500 pfu of one of the three IAVs . Viral protein expression in mouse lung was determined by Western blotting 2 days after infection . E Mice ( 6 per group ) were infected with 500 pfu of one of the three IAVs . Viral titration by plaque assay was performed at 2 days post - infection using lung homogenates . F Mice ( 5 - 9 per group ) were infected with 500 pfu of each virus . At 2 days post - infection , IFNβ mRNA levels were determined by qPCR . G Mice ( 5 - 9 per group ) were infected with 1 × 105 pfu of each virus . At 2 days post - infection , IFNβ protein levels in lung homogenates were measured by ELISA .

O B-PANEL_START O O 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-PANEL_START O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A Mice (n = 5 or 6 per group) were infected with IAV with indicated pfu. Survival was monitored daily for 2 weeks.B Mice (n = 6-9 per group) were infected with each IAV as indicated. Body weight was recorded daily after infection for 2 weeks.C Mice (n = 5 or 6 per group) were infected with viruses (1000 pfu) carrying PR8 PB1-F2 with the I68T, L69P, or I68T+L69P mutation. Body weight changes and survival rate were monitored daily. Detailed analyses on the virulence necessitated the monitoring of body weight below the standard level (80%) of mortality.Three cohorts of mice (5 or 6 per group) were infected with 500 pfu of one of the three IAVs. Viral protein expression in mouse lung was determined by Western blotting 2 days after infection.E Mice (6 per group) were infected with 500 pfu of one of the three IAVs. Viral titration by plaque assay was performed at 2 days post-infection using lung homogenates.F Mice (5-9 per group) were infected with 500 pfu of each virus. At 2 days post-infection, IFNβ mRNA levels were determined by qPCR.G Mice (5-9 per group) were infected with 1 × 105 pfu of each virus. At 2 days post-infection, IFNβ protein levels in lung homogenates were measured by ELISA.


Figure 5A SDS - PAGE analysis of proteins immunoprecipitated from A549 cell lysates using HA - antibody . Bands were stained by Coomassie Blue . Results were confirmed in three independent experiments , and representative data are shown . E Analysis of interaction between DDX3 and 1918 PB1 - F2 . Cells were transfected with indicated plasmids ; 18 h later , cells were treated with MG132 for 6 h . Cell lysates were immunoprecipitated with anti - HA antibody . Proteins were detected by immunoblotting with the indicated antibodies ( left panel ) . IAV - infected cell lysates were immunoprecipitated with anti - PB1 - F2 antibody and immunoblotted with the indicated antibodies ( middle panel ) . Purified recombinant 1918 PB1 - F2 protein was incubated with recombinant LysRS or LysRS - DDX3 , and the interaction was analyzed by immunoblotting ( right panel ) . Results were confirmed in three independent experiments , and representative data are shown . F Analysis of interaction between DDX3 and PB1 - F2 mutants . A549 cells were transfected with the indicated clones ; 18 h after transfection , cells were treated with MG132 for 6 h and analyzed by immunoprecipitation . Results were confirmed in three independent experiments , and representative data are shown . G A549 cells were treated with IFNβ for the indicated time periods and subjected to Western blotting ( top ) . A549 cells were infected with indicated IAV . At 24 h post - infection , cell lysates were analyzed by Western blotting ( bottom ) . Results were confirmed in three independent experiments , and representative data are shown . H Representative fluorescence images showing expression level of GFP - DDX3 ( green ) . A549 cells were co - transfected with plasmids encoding the GEP - DDX3 and Flag - tagged PR8 or 1918 PB1 - F2 . At 18 h after transfection , cells were treated with MG132 for 6 h and subjected to fluorescence analysis . Results were confirmed in three independent experiments , and representative data are shown . I Mice were infected with 1 × 105 pfu of each virus , and lung homogenates were examined for endogenous Ddx3 expression . Results were confirmed in three independent experiments , and representative data are shown . J A549 cells were transfected with the indicated plasmids or treated with poly I : C . Cytoplasmic and nuclear fractions were separated and subjected to Western blotting . Results were confirmed in three independent experiments , and representative data are shown . K A549 cells were transfected with the indicated plasmids , and IFNβ mRNA levels were determined by semi - quantitative RT - PCR ( top ) and qPCR ( middle ) . IFNβ promoter activity was determined by luciferase reporter assay ( bottom ) . Results were confirmed in three independent experiments , and representative data are shown .

O B-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A SDS-PAGE analysis of proteins immunoprecipitated from A549 cell lysates using HA-antibody. Bands were stained by Coomassie Blue. Results were confirmed in three independent experiments, and representative data are shown.E Analysis of interaction between DDX3 and 1918 PB1-F2. Cells were transfected with indicated plasmids; 18 h later, cells were treated with MG132 for 6 h. Cell lysates were immunoprecipitated with anti-HA antibody. Proteins were detected by immunoblotting with the indicated antibodies (left panel). IAV-infected cell lysates were immunoprecipitated with anti-PB1-F2 antibody and immunoblotted with the indicated antibodies (middle panel). Purified recombinant 1918 PB1-F2 protein was incubated with recombinant LysRS or LysRS-DDX3, and the interaction was analyzed by immunoblotting (right panel). Results were confirmed in three independent experiments, and representative data are shown.F Analysis of interaction between DDX3 and PB1-F2 mutants. A549 cells were transfected with the indicated clones; 18 h after transfection, cells were treated with MG132 for 6 h and analyzed by immunoprecipitation. Results were confirmed in three independent experiments, and representative data are shown.G A549 cells were treated with IFNβ for the indicated time periods and subjected to Western blotting (top). A549 cells were infected with indicated IAV. At 24 h post-infection, cell lysates were analyzed by Western blotting (bottom). Results were confirmed in three independent experiments, and representative data are shown.H Representative fluorescence images showing expression level of GFP-DDX3 (green). A549 cells were co-transfected with plasmids encoding the GEP-DDX3 and Flag-tagged PR8 or 1918 PB1-F2. At 18 h after transfection, cells were treated with MG132 for 6 h and subjected to fluorescence analysis. Results were confirmed in three independent experiments, and representative data are shown.I Mice were infected with 1×105 pfu of each virus, and lung homogenates were examined for endogenous Ddx3 expression. Results were confirmed in three independent experiments, and representative data are shown.J A549 cells were transfected with the indicated plasmids or treated with poly I:C. Cytoplasmic and nuclear fractions were separated and subjected to Western blotting. Results were confirmed in three independent experiments, and representative data are shown.K A549 cells were transfected with the indicated plasmids, and IFNβ mRNA levels were determined by semi-quantitative RT-PCR (top) and qPCR (middle). IFNβ promoter activity was determined by luciferase reporter assay (bottom). Results were confirmed in three independent experiments, and representative data are shown.


Figure 6Groups of five mice were intranasally infected with IAV ( 1918 ) , and recombinant DDX3 protein was administered intranasally . Survival was monitored daily for 2 weeks . Groups of five mice were intranasally infected with IAV ( 1918 ) , and recombinant DDX3 protein was administered intranasally . Two days post - infection , virus titers in lung homogenates were determined by plaque assay . A p value of < 0 . 05 was considered significant ( * * p < 0 . 01 ) . Groups of five mice were intranasally infected with IAV ( 1918 ) , and recombinant DDX3 protein was administered intranasally . Two days post - infection , the expression level of IFNβ in the lungs was analyzed by semi - quantitative RT - PCR ( left panel ) and qPCR ( right panel ) . A p value of < 0 . 05 was considered significant ( * * p < 0 . 01 ) .

O B-PANEL_START O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6Groups of five mice were intranasally infected with IAV (1918), and recombinant DDX3 protein was administered intranasally. Survival was monitored daily for 2 weeks.Groups of five mice were intranasally infected with IAV (1918), and recombinant DDX3 protein was administered intranasally. Two days post-infection, virus titers in lung homogenates were determined by plaque assay. A p value of <0.05 was considered significant (** p < 0.01).Groups of five mice were intranasally infected with IAV (1918), and recombinant DDX3 protein was administered intranasally. Two days post-infection, the expression level of IFNβ in the lungs was analyzed by semi-quantitative RT-PCR (left panel) and qPCR (right panel). A p value of <0.05 was considered significant (** p < 0.01).


Figure 1 ( A - B ' ' ) Nr2f1 ( red ) and Pax6 ( green , retinal progenitors ) immunofluorescences ( IF ) on sagittal sections of E10 . 5 and E12 . 5 mouse optic vesicles . Note the high - ventral to low - dorsal Nr2f1 gradient in the presumptive neural retina ( pNR ) , and high Nr2f1 expression in both the presumptive dorsal and ventral optic stalk ( pvOS and pdOS ) . Cr : crystal lens ; Di , distal ; Pr , proximal ; presumptive retinal pigmented epithelium ( pRPE ) . Scale bars : 50µm for mouse sections ( C - D " ) IF on E13 . 5 mouse eye sagittal sections with Nr2f1 ( red ) and Sox2 ( green , progenitors in C , C ' ) or Brn3a ( green , RGCs in D , D ' ) showing Nr2f1 expression in NR progenitors and post - mitotic RGCs ( insets in C ' - D " ) . Scale bars : 50µm for mouse sections ( E - F " ) Nr2f1 ( red ) and Brn3a ( green ) IF on E18 . 5 and P7 mouse eyes depicting Nr2f1 expression in virtually all RGCs in the ganglion cell layer ( GCL ) . Scale bars : 50µm for mouse sections ( G , G ' ) Nr2f1 ( red ) and Sox2 ( green ) IF on P7 mouse retina illustrating low Nr2f1 expression in the inner nuclear layer ( INL ) and progenitors ( NPs ) , and high levels in the GCL and ciliary marginal zone ( CMZ ) . No expression in the outer nuclear layer ( ONL ) . Scale bars : 50µm for mouse sections ( H - K " ) IF on cross - sections of E13 . 5 optic stalks ( OS ) , and E18 . 5 and P7 optic nerves ( ONs ) with Nr2f1 ( red ) and Glast ( green , astrocytic progenitors in H - H ' ' ) , NF1A ( green , astrocytes in I - I ' ' ) or Sox10 ( green , oligodendrocyte precursors in J - K ' ' ) showing Nr2f1 expression in both astrocytic and oligodendrocytic lineages . Arrowheads point to double labeled cells . Scale bars : 50µm for mouse sections ( L - L " ) NR2F1 ( red ) and PAX6 ( green ; NR domain ) IF on sagittal sections of human eye primordia at gestational week ( GW ) 11 illustrating high NR2F1 expression in both ON and NR cells . Scale bars : 100µm for human sections ( M - N " ) NR2F1 ( red ) and S100β ( green , astrocytes ) IF on cross - sections of human GW11 and GW14 ONs . Higher magnifications ( M ' - N " ) show that most of S100β + astrocytes co - express NR2F1 . Scale bars : 100µm for human sections , ( O - Q " ' ) NR2F1 ( red ) and SOX2 ( green in O - P ' ) or BRN3a ( green in Q - Q ' ' ' ) IF on sagittal sections of human GW14 eyes indicating high NR2F1 expression in virtually all NR progenitors ( O ' ' ' , P , P ' ) , differentiating RGCs ( Q - Q ' ' ' ) and in the majority of ON astrocytic progenitors ( O ' ) . Nuclei counterstaining ( blue ) was obtained with DAPI . Scale bars 100µm for human

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A-B'') Nr2f1 (red) and Pax6 (green, retinal progenitors) immunofluorescences (IF) on sagittal sections of E10.5 and E12.5 mouse optic vesicles. Note the high-ventral to low-dorsal Nr2f1 gradient in the presumptive neural retina (pNR), and high Nr2f1 expression in both the presumptive dorsal and ventral optic stalk (pvOS and pdOS). Cr: crystal lens; Di, distal; Pr, proximal; presumptive retinal pigmented epithelium (pRPE). Scale bars: 50µm for mouse sections(C-D") IF on E13.5 mouse eye sagittal sections with Nr2f1 (red) and Sox2 (green, progenitors in C,C') or Brn3a (green, RGCs in D,D') showing Nr2f1 expression in NR progenitors and post-mitotic RGCs (insets in C'-D"). Scale bars: 50µm for mouse sections(E-F") Nr2f1 (red) and Brn3a (green) IF on E18.5 and P7 mouse eyes depicting Nr2f1 expression in virtually all RGCs in the ganglion cell layer (GCL). Scale bars: 50µm for mouse sections(G,G') Nr2f1 (red) and Sox2 (green) IF on P7 mouse retina illustrating low Nr2f1 expression in the inner nuclear layer (INL) and progenitors (NPs), and high levels in the GCL and ciliary marginal zone (CMZ). No expression in the outer nuclear layer (ONL). Scale bars: 50µm for mouse sections(H-K") IF on cross-sections of E13.5 optic stalks (OS), and E18.5 and P7 optic nerves (ONs) with Nr2f1 (red) and Glast (green, astrocytic progenitors in H-H''), NF1A (green, astrocytes in I-I'') or Sox10 (green, oligodendrocyte precursors in J-K'') showing Nr2f1 expression in both astrocytic and oligodendrocytic lineages. Arrowheads point to double labeled cells. Scale bars: 50µm for mouse sections(L-L") NR2F1 (red) and PAX6 (green; NR domain) IF on sagittal sections of human eye primordia at gestational week (GW) 11 illustrating high NR2F1 expression in both ON and NR cells. Scale bars: 100µm for human sections(M-N") NR2F1 (red) and S100β (green, astrocytes) IF on cross-sections of human GW11 and GW14 ONs. Higher magnifications (M'-N") show that most of S100β+ astrocytes co-express NR2F1. Scale bars: 100µm for human sections,(O-Q"') NR2F1 (red) and SOX2 (green in O-P') or BRN3a (green in Q-Q''') IF on sagittal sections of human GW14 eyes indicating high NR2F1 expression in virtually all NR progenitors (O''',P,P'), differentiating RGCs (Q-Q''') and in the majority of ON astrocytic progenitors (O'). Nuclei counterstaining (blue) was obtained with DAPI. Scale bars 100µm for human sections


Figure 2 ( 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 ) 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 " ) and Pax6 + ( D - D " ) cells . Arrowheads in ( D ' , D " ) point to ectopic Tuj1 / Pax6 + neurons . ( 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 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 ) 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 ' ) 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 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 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 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 and HET retinae indicating decreased number of Brn3a + neurons ( arrowheads ) in the GCL of HET animals . ( 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 and KO pups ( R , R ' ) Tuj1 + P8 ON fibers in WT and KO pups showing a progressive volume loss ( 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 ' ) 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 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(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) 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") and Pax6+ (D-D") cells. Arrowheads in (D',D") point to ectopic Tuj1/Pax6+ neurons.(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 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) 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') 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 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 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 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 and HET retinae indicating decreased number of Brn3a+ neurons (arrowheads) in the GCL of HET animals.(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 and KO pups(R,R') Tuj1+ P8 ON fibers in WT and KO pups showing a progressive volume loss(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') 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 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.


Figure 3 ( A - F ) Pax2 ( magenta , astrocytic progenitors ) and Tuj1 ( green , RGC axons ) IF on E13 . 5 , E15 . 5 and E18 . 5 OS / ONs cross - sections showing an increase of Pax2 - expressing cells within the optic nerve ( ON ) of KO embryos at all stages . Histograms quantifying the average number of Pax2 + ( G , H ) cells in WT , HET and KO at different stages and axial length of the ON , as indicated . Astrocytic progenitors are significantly increased along the nerve of mutants . data are represented as means ± SEM ; N = 3 - 5 . Statistical significance was obtained by Student t - test ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) . Nuclei ( blue ) were stained with DAPI . Scale bars : 50µmHistograms quantifying the average number of NF1A + ( I ) cells in WT , HET and KO at different stages and axial length of the ON , as indicated . Astrocytic progenitors are significantly increased along the nerve of mutants . data are represented as means ± SEM ; N = 3 - 5 . Statistical significance was obtained by Student t - test ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) . Nuclei ( blue ) were stained with DAPI . Scale bars : 50µm ( J , K ) NF1A ( red , astrocyte precursors ) IF on P7 WT and KO ON cross - sections illustrating a high astrocytic density in KO ON ( delineated by thin dashed lines ) . ( L ) Histogram confirming the increased percentage of NF1A + astrocytes in HET and KO ONs compared to WT . data are represented as means ± SEM ; N = 3 - 5 . Statistical significance was obtained by Student t - test ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) . Nuclei ( blue ) were stained with DAPI . Scale bars : 50µm ( M - O " ) Tuj1 ( green , RGC axons ) and S100β ( red , astrocytes ) IF on P7 WT , HET and KO ONs . Arrowheads point to S100β + astrocyte groups ( N ' , O ' ) and absence of Tuj1 + fibers ( N ' ' , O ' ' ) in HET and KO ONs . ( P . Q ) TEM images of WT and KO P8 ONs showing thick astrocytic processes filled with intermediate filaments ( red dotted lines and arrowheads ) in KO nerves . Scale bars ( 500nm ) ( R - S ' ) S100β ( green , astrocytes ) and Sox2 ( red , reactive / inflamed astrocytes ) IF on P28 WT and HET ONs illustrating strong Sox2 expression in reactive astrocytes . ( T ) Graph quantifying the average number of Sox2 + astrocytes per P28 ON section in KO and WT animals . data are represented as means ± SEM ; N = 3 - 5 . Statistical significance was obtained by Student t - test ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) . Nuclei ( blue ) were stained with DAPI . Scale bars : 50µm ( U , V ) EM sections of P28 WT and HET ONs illustrating the expanded astrocytic processes ( surrounded by red dotted lines ) among myelinated fibers of P28 ONs . Scale bars : ( U , V ) ( 4µm ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A-F) Pax2 (magenta, astrocytic progenitors) and Tuj1 (green, RGC axons) IF on E13.5, E15.5 and E18.5 OS/ONs cross-sections showing an increase of Pax2-expressing cells within the optic nerve (ON) of KO embryos at all stages.Histograms quantifying the average number of Pax2+ (G,H) cells in WT, HET and KO at different stages and axial length of the ON, as indicated. Astrocytic progenitors are significantly increased along the nerve of mutants. data are represented as means ± SEM; N=3-5. Statistical significance was obtained by Student t-test (*P<0.05; **P<0.01; ***P<0.001). Nuclei (blue) were stained with DAPI. Scale bars: 50µmHistograms quantifying the average number of NF1A+ (I) cells in WT, HET and KO at different stages and axial length of the ON, as indicated. Astrocytic progenitors are significantly increased along the nerve of mutants. data are represented as means ± SEM; N=3-5. Statistical significance was obtained by Student t-test (*P<0.05; **P<0.01; ***P<0.001). Nuclei (blue) were stained with DAPI. Scale bars: 50µm(J,K) NF1A (red, astrocyte precursors) IF on P7 WT and KO ON cross-sections illustrating a high astrocytic density in KO ON (delineated by thin dashed lines). (L) Histogram confirming the increased percentage of NF1A+ astrocytes in HET and KO ONs compared to WT. data are represented as means ± SEM; N=3-5. Statistical significance was obtained by Student t-test (*P<0.05; **P<0.01; ***P<0.001). Nuclei (blue) were stained with DAPI. Scale bars: 50µm(M-O") Tuj1 (green, RGC axons) and S100β (red, astrocytes) IF on P7 WT, HET and KO ONs. Arrowheads point to S100β+ astrocyte groups (N',O') and absence of Tuj1+ fibers (N'',O'') in HET and KO ONs.(P.Q) TEM images of WT and KO P8 ONs showing thick astrocytic processes filled with intermediate filaments (red dotted lines and arrowheads) in KO nerves. Scale bars (500nm)(R-S') S100β (green, astrocytes) and Sox2 (red, reactive/inflamed astrocytes) IF on P28 WT and HET ONs illustrating strong Sox2 expression in reactive astrocytes.(T) Graph quantifying the average number of Sox2+ astrocytes per P28 ON section in KO and WT animals. data are represented as means ± SEM; N=3-5. Statistical significance was obtained by Student t-test (*P<0.05; **P<0.01; ***P<0.001). Nuclei (blue) were stained with DAPI. Scale bars: 50µm(U,V) EM sections of P28 WT and HET ONs illustrating the expanded astrocytic processes (surrounded by red dotted lines) among myelinated fibers of P28 ONs. Scale bars: (U,V) (4µm).


Figure 4 ( A - C ) NF1A ( red , astrocytes ) and Sox10 ( green , oligodendrocytes ) IF on cross - sections of WT and KO P8 ONs showing altered ratio of astrocytes and oligodendrocytes in mutants , particularly in KOs , as quantified in ( C ) . data are represented as means ± SEM . N = 4 - 5 Statistical significance was obtained by Student t - test or by two - way ANOVA when comparing 2 or multiple conditions , respectively ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 Scale bars : 50µm ( D - J ) MBP ( yellow , fully differentiated oligodendrocytes ) IF on cross - sections of WT , HET and KO P7 optic nerves ( ONs ) at the chiasm ( D - F ) and between the chiasm and the eye ( G - I ) . Note the dramatic decrease of MBP + oligodendrocytes at the chiasm in HET and KO , quantified in ( J ) . The ratio of MBP + cells is calculated over the total DAPI + cell number per chiasmal / ON section . data are represented as means ± SEM . N = 4 - 5 Statistical significance was obtained by Student t - test or by two - way ANOVA when comparing 2 or multiple conditions , respectively ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) . Nuclei ( blue ) were stained with DAPI . Scale bars : 50µm ( K - N ) EM thin sections of P8 WT , HET and KO ONs depicting myelin as a dense , dark staining around axonal fibers illustrate a significantly lower number of myelinated fibers in HET and KO , as quantified in ( N ) . Higher magnification images ( insets ) showing different degrees of myelination in HET and KO ONs . data are represented as means ± SEM . N = 3 Statistical significance was obtained by Student t - test or by two - way ANOVA when comparing 2 or multiple conditions , respectively ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) . Nuclei ( blue ) were stained with DAPI . Scale bars : 50µm ( 4µm ) TEM images of P28 WT and HET confirming persistent hypomyelination in mutants at P28 Scale bars : ( 4µm ) P28 WT and HET confirming persistent hypomyelination in mutants at P28 , as quantified in ( Q ) . data are represented as means ± SEM . N = 3 Statistical significance was obtained by Student t - test or by two - way ANOVA when comparing 2 or multiple conditions , respectively ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) . Nuclei ( blue ) were stained with DAPI . Scale bars : 50µm ( R , S ) High magnification EM images of P28 WT and HET ONs showing the difference in myelin compaction between WT ( arrowheads in R ) and HET ( arrowheads in S ) . Scale bars : ( 1µm ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A-C) NF1A (red, astrocytes) and Sox10 (green, oligodendrocytes) IF on cross-sections of WT and KO P8 ONs showing altered ratio of astrocytes and oligodendrocytes in mutants, particularly in KOs, as quantified in (C). data are represented as means ± SEM. N=4-5 Statistical significance was obtained by Student t-test or by two-way ANOVA when comparing 2 or multiple conditions, respectively (*P<0.05; **P<0.01; ***P<0.001 Scale bars: 50µm(D-J) MBP (yellow, fully differentiated oligodendrocytes) IF on cross-sections of WT, HET and KO P7 optic nerves (ONs) at the chiasm (D-F) and between the chiasm and the eye (G-I). Note the dramatic decrease of MBP+ oligodendrocytes at the chiasm in HET and KO, quantified in (J). The ratio of MBP+ cells is calculated over the total DAPI+ cell number per chiasmal/ON section. data are represented as means ± SEM. N=4-5 Statistical significance was obtained by Student t-test or by two-way ANOVA when comparing 2 or multiple conditions, respectively (*P<0.05; **P<0.01; ***P<0.001). Nuclei (blue) were stained with DAPI. Scale bars: 50µm(K-N) EM thin sections of P8 WT, HET and KO ONs depicting myelin as a dense, dark staining around axonal fibers illustrate a significantly lower number of myelinated fibers in HET and KO, as quantified in (N). Higher magnification images (insets) showing different degrees of myelination in HET and KO ONs. data are represented as means ± SEM. N=3 Statistical significance was obtained by Student t-test or by two-way ANOVA when comparing 2 or multiple conditions, respectively (*P<0.05; **P<0.01; ***P<0.001). Nuclei (blue) were stained with DAPI. Scale bars: 50µm (4µm)TEM images of P28 WT and HET confirming persistent hypomyelination in mutants at P28 Scale bars: (4µm)P28 WT and HET confirming persistent hypomyelination in mutants at P28, as quantified in (Q). data are represented as means ± SEM. N=3 Statistical significance was obtained by Student t-test or by two-way ANOVA when comparing 2 or multiple conditions, respectively (*P<0.05; **P<0.01; ***P<0.001). Nuclei (blue) were stained with DAPI. Scale bars: 50µm(R,S) High magnification EM images of P28 WT and HET ONs showing the difference in myelin compaction between WT (arrowheads in R) and HET (arrowheads in S). Scale bars: (1µm).