B ) Left : quantification of total centrosome numbers in control and EPZ treated sections in 100 µm . Right : number of centrosomes in ventricular zone ( VZ ) , subventricular zone ( SVZ ) , and cortical plate ( CP ) expressed as percentage of total per condition . C ) Left : quantification of total cilia numbers in control and EPZ treated sections in 100 µm . Right : number of cilia in VZ , SVZ , and CP expressed as percentage of total per condition . Data information : T - test , two - tails was performed for all quantifications , * p < 0 . 05 , * * * p < 0 . 001 , error bars represent SD .

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

B) Left: quantification of total centrosome numbers in control and EPZ treated sections in 100 µm. Right: number of centrosomes in ventricular zone (VZ), subventricular zone (SVZ), and cortical plate (CP) expressed as percentage of total per condition. C) Left: quantification of total cilia numbers in control and EPZ treated sections in 100 µm. Right: number of cilia in VZ, SVZ, and CP expressed as percentage of total per condition. Data information: T-test, two-tails was performed for all quantifications, *p<0.05, ***p<0.001, error bars represent SD.


D ) Overview of VZ showing TUBG1 and ARL13B immunofluorescence staining in control ( top ) and EPZ ( bottom ) condition . Squared parentheses indicate the ventricular and abventricular compartment . Dotted lines mark ventricular surface of tissue slide . Data information : Scale bars in all panels : 10 µm .

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

D) Overview of VZ showing TUBG1 and ARL13B immunofluorescence staining in control (top) and EPZ (bottom) condition. Squared parentheses indicate the ventricular and abventricular compartment. Dotted lines mark ventricular surface of tissue slide. Data information: Scale bars in all panels: 10 µm.


E ) Quantification of TUBG1 - and ( F ) ARL13B - positive structures in ventricular and abventricular compartment expressed as percentage of the total number per condition . Data information : T - test , two - tails was performed for all quantifications , * p < 0 . 05 , * * * p < 0 . 001 , error bars represent SD .

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

E) Quantification of TUBG1- and (F) ARL13B-positive structures in ventricular and abventricular compartment expressed as percentage of the total number per condition. Data information: T-test, two-tails was performed for all quantifications, *p<0.05, ***p<0.001, error bars represent SD.


A ) Immunostaining of control and EPZ treated sections showing SOX2 and EOMES expression and EdU labelling . Scale bar : 20 µm . Dotted lines mark ventricular surface of the tissue . B ) Quantification of SOX2 - , EOMES - , EdU - positive cells , and co - labelled cells normalised to 100 μm of apical surface . T - test was performed , * p < 0 . 05 , * * p < 0 . 01 , error bars represent SEM . Total n of independent experiments for EdU labelling : 9 ( n = 4 control samples , n = 5 EPZ - treated samples ) . Total n of independent experiments for SOX2 , EOMES , and double stainings : 6 ( n = 3 control samples , n = 3 EPZ - treated samples ) .

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

A) Immunostaining of control and EPZ treated sections showing SOX2 and EOMES expression and EdU labelling. Scale bar: 20 µm. Dotted lines mark ventricular surface of the tissue. B) Quantification of SOX2-, EOMES-, EdU- positive cells, and co-labelled cells normalised to 100 μm of apical surface. T-test was performed, *p<0.05, **p<0.01, error bars represent SEM. Total n of independent experiments for EdU labelling: 9 (n=4 control samples, n=5 EPZ-treated samples). Total n of independent experiments for SOX2, EOMES, and double stainings: 6 (n=3 control samples, n=3 EPZ-treated samples).


E ) Immunofluorescence staining showing overview of CTIP2 - positive cells in control and EPZ treated sections . Scale bars : 20 µm . Dotted lines mark either ventricular or pial surface of the tissue . F ) Quantification of total CTIP2 - positive cells , normalised to 100 µm of apical surface . Total n of independent experiments : 6 ( n = 3 control samples , n = 3 EPZ - treated samples ) . Error bars represent SEM .

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

E) Immunofluorescence staining showing overview of CTIP2-positive cells in control and EPZ treated sections. Scale bars: 20 µm. Dotted lines mark either ventricular or pial surface of the tissue. F) Quantification of total CTIP2-positive cells, normalised to 100 µm of apical surface. Total n of independent experiments: 6 (n=3 control samples, n=3 EPZ-treated samples). Error bars represent SEM.


G ) Immunofluorescence staining showing SOX2 - , EOMES - , and CTIP2 - positive cells in control and EPZ treated sections . Scale bars : 10 μm . Dotted lines mark the ventricular surface of the tissue . Magnifications on the right show exemplarily a CTIP2 positive cell ( arrowhead ) that is negative for SOX2 and positive for EOMES in EPZ treated condition . H ) Quantification of CTIP2 - positive cells in the VZ , normalised to 100 µm of apical surface . Total n of independent experiments : 6 ( n = 3 control samples , n = 3 EPZ - treated samples ) . T - test was performed , * * p < 0 . 01 , error bars represent SEM .

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

G) Immunofluorescence staining showing SOX2-, EOMES-, and CTIP2-positive cells in control and EPZ treated sections. Scale bars: 10 μm. Dotted lines mark the ventricular surface of the tissue. Magnifications on the right show exemplarily a CTIP2 positive cell (arrowhead) that is negative for SOX2 and positive for EOMES in EPZ treated condition. H) Quantification of CTIP2-positive cells in the VZ, normalised to 100 µm of apical surface. Total n of independent experiments: 6 (n=3 control samples, n=3 EPZ-treated samples). T-test was performed, **p<0.01, error bars represent SEM.


I ) In utero electroporation of cortical tissue of shRNA against DOT1L and control alongside a GFP expressing plasmid , followed by assessment of expression of CTIP2 and TUBB3 as markers for neuronal differentiation to study cell - autonomous and non - autonomous effects . Scale bars : 20 µm . Dotted lines mark either ventricular or pial surface of tissue slide . J ) Quantification of CTIP2 - , and TUBB3 - positive cells in VZ , SVZ , IZ , and CP in control and shDot1l sections . Total n of independent experiments : 6 ( n = 3 control shRNA , n = 3 shDot1l ) . T - test was performed , * p < 0 . 05 , error bars represent SEM .

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

I) In utero electroporation of cortical tissue of shRNA against DOT1L and control alongside a GFP expressing plasmid, followed by assessment of expression of CTIP2 and TUBB3 as markers for neuronal differentiation to study cell-autonomous and non-autonomous effects. Scale bars: 20 µm. Dotted lines mark either ventricular or pial surface of tissue slide. J) Quantification of CTIP2-, and TUBB3-positive cells in VZ, SVZ, IZ, and CP in control and shDot1l sections. Total n of independent experiments: 6 (n=3 control shRNA, n=3 shDot1l ). T-test was performed, *p<0.05, error bars represent SEM.


A ) Microinjected cells in the VZ ( magenta ) in sections treated with DMSO ( Con ) ( upper panel ) or EPZ ( lower panel ) showing Dx - A555 labelled cells after 24 h in slice culture . White arrow indicates the apical process contacting the ventricular surface . White arrowheads indicate the apical - directed process of delaminated cells . Scale bars : 10 µm . B ) Immunofluorescence staining of microinjected sections showing the progeny of microinjected cells ( a daughter cell pair ) and expression of EOMES and TUBB3 . White arrows indicate the apical - directed processes . Note that in the top panel the apical - directed process contacts the ventricular surface ( dotted white line ) ( EOMES - , TUBB3 - negative ) , while in the bottom panel it does not contact the ventricular surface , indicating that the cell has delaminated ( EOMES - , TUBB3 - positive ) . Scale bars MIP : 10 µm ; insets : 5 µm . Data information : VZ - Ventricular Zone , SVZ - Subventricular Zone , CP - Cortical Plate . Scale bars in all main panels : 20 µm , and all insets : 5 µm .

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

A) Microinjected cells in the VZ (magenta) in sections treated with DMSO (Con) (upper panel) or EPZ (lower panel) showing Dx-A555 labelled cells after 24 h in slice culture. White arrow indicates the apical process contacting the ventricular surface. White arrowheads indicate the apical-directed process of delaminated cells. Scale bars: 10 µm. B) Immunofluorescence staining of microinjected sections showing the progeny of microinjected cells (a daughter cell pair) and expression of EOMES and TUBB3. White arrows indicate the apical-directed processes. Note that in the top panel the apical-directed process contacts the ventricular surface (dotted white line) (EOMES-, TUBB3-negative), while in the bottom panel it does not contact the ventricular surface, indicating that the cell has delaminated (EOMES-, TUBB3-positive). Scale bars MIP: 10 µm; insets: 5 µm. Data information: VZ - Ventricular Zone, SVZ - Subventricular Zone, CP - Cortical Plate. Scale bars in all main panels: 20 µm, and all insets: 5 µm.


C ) Quantification of microinjected cells found as single - cell or two - cells clusters , expressed as percentage of total Dx - A555 - positive cells per condition . D ) Quantification of microinjected cells with or without contact to the apical surface of the organotypic slice expressed as percentage of total Dx - A555 - positive cells per condition . Total n of independent microinjection experiments : 6 ( n = 3 wild type embryos , n = 3 in Tis21 - GFP embryos ) . Total number of cells scored : Con , 156 cells ; EPZ , 287 cells . E ) EOMES - positive and EOMES - negative cells plotted as a percentage of total cells scored per condition . F ) TUBB3 - positive and TUBB3 - negative cells in control and EPZ conditions plotted as a percentage of total cells scored per condition . Data information : Cell scoring based on TUBB3 or EOMES expression are from three independent experiments ( n = 3 ; Con : 70 cells , EPZ : 113 cells ) . Fisher ' s exact test was performed for all quantifications , * * * p < 0 . 001 , error bars represent SEM . MIP - maximum intensity projection .

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

C) Quantification of microinjected cells found as single-cell or two-cells clusters, expressed as percentage of total Dx-A555-positive cells per condition. D) Quantification of microinjected cells with or without contact to the apical surface of the organotypic slice expressed as percentage of total Dx-A555-positive cells per condition. Total n of independent microinjection experiments: 6 (n=3 wild type embryos, n=3 in Tis21-GFP embryos). Total number of cells scored: Con, 156 cells; EPZ, 287 cells. E) EOMES-positive and EOMES-negative cells plotted as a percentage of total cells scored per condition. F) TUBB3-positive and TUBB3-negative cells in control and EPZ conditions plotted as a percentage of total cells scored per condition. Data information: Cell scoring based on TUBB3 or EOMES expression are from three independent experiments (n=3; Con: 70 cells, EPZ: 113 cells). Fisher's exact test was performed for all quantifications, ***p<0.001, error bars represent SEM. MIP - maximum intensity projection.


A ) Microinjected cells in the VZ ( magenta ) of control ( Con ) and EPZ treated slices 24 h after microinjection and slice culture . Left : daughter cell pair in control section with asymmetric GFP expression , with cell 1 ( basal location ) being mild GFP - positive , and cell 2 ( apical location ) GFP - negative . Right : EPZ treated section showing a daughter cell pair with symmetric mild GFP - positive expression . Scale bars : 5 µm . Dotted shapes indicate the contour of Dx - A555 cell soma . B ) Graph showing proportion of labelled cells expressing GFP - positive , - mild positive , or - negative , expressed as percentage of total cells per condition . Data information : Quantifications are based on three independent experiments ( n = 3 , biological replicates ) and Chi - square test was performed in all cases . Error bars represent SEM , * * * p < 0 . 001 , * * * * p < 0 . 0001 , alpha = 0 . 05 . VZ - ventricular zone . The following number of cells were scored : B ) TIS21 - GFP - 267 total cells were quantified ( Con : 86 cells , EPZ : 181 cells ) .

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

A) Microinjected cells in the VZ (magenta) of control (Con) and EPZ treated slices 24 h after microinjection and slice culture. Left: daughter cell pair in control section with asymmetric GFP expression, with cell 1 (basal location) being mild GFP-positive, and cell 2 (apical location) GFP-negative. Right: EPZ treated section showing a daughter cell pair with symmetric mild GFP-positive expression. Scale bars: 5 µm. Dotted shapes indicate the contour of Dx-A555 cell soma. B) Graph showing proportion of labelled cells expressing GFP-positive, -mild positive, or -negative, expressed as percentage of total cells per condition. Data information: Quantifications are based on three independent experiments (n=3, biological replicates) and Chi-square test was performed in all cases. Error bars represent SEM, ***p<0.001, ****p<0.0001, alpha = 0.05. VZ - ventricular zone. The following number of cells were scored: B) TIS21-GFP - 267 total cells were quantified (Con: 86 cells, EPZ: 181 cells).


C ) Symmetry or asymmetry in daughter cells ' apical contact ( yes / yes : both cells have apical contact ; yes / no : one cell has apical contact and the other not ; no / no : both cells lack apical contact ) . D ) Symmetry ( pos / pos , neg / neg ) or asymmetry ( pos / neg ) in GFP expression in daughter cells expressed as percentage of total cells per condition . E ) Symmetry or asymmetry in TUBB3 expression in daughter cells plotted ( pos / pos : both cells express TUBB3 ; pos / neg : one cell expresses TUBB3 and the other not ; neg / neg : none of the cells expresses TUBB3 ) . Data information : Quantifications are based on three independent experiments ( n = 3 , biological replicates ) and Chi - square test was performed in all cases . Error bars represent SEM , * * * p < 0 . 001 , * * * * p < 0 . 0001 , alpha = 0 . 05 . The following number of cells were scored : C ) Apical contact - 85 total daughter cell pairs ( Con : 31 pairs , EPZ : 54 pairs ) . D ) TIS21 - GFP - 58 total daughter cell pairs ( Con : 21 pairs , EPZ : 37 pairs ) . E ) TUBB3 - 50 total daughter cell pairs ( Con : 17 pairs , EPZ : 33 pairs ) .

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

C) Symmetry or asymmetry in daughter cells' apical contact (yes/yes: both cells have apical contact; yes/no: one cell has apical contact and the other not; no/no: both cells lack apical contact). D) Symmetry (pos/pos, neg/neg) or asymmetry (pos/neg) in GFP expression in daughter cells expressed as percentage of total cells per condition. E) Symmetry or asymmetry in TUBB3 expression in daughter cells plotted (pos/pos: both cells express TUBB3; pos/neg: one cell expresses TUBB3 and the other not; neg/neg: none of the cells expresses TUBB3). Data information: Quantifications are based on three independent experiments (n=3, biological replicates) and Chi-square test was performed in all cases. Error bars represent SEM, ***p<0.001, ****p<0.0001, alpha = 0.05. The following number of cells were scored: C) Apical contact - 85 total daughter cell pairs (Con: 31 pairs, EPZ: 54 pairs). D) TIS21-GFP - 58 total daughter cell pairs (Con: 21 pairs, EPZ: 37 pairs). E) TUBB3 - 50 total daughter cell pairs (Con: 17 pairs, EPZ: 33 pairs).


F ) Phalloidin staining of control and EPZ treated sections showing cells during mitosis . Scale bar : 5 µm . G ) Pie chart showing the proportion of cells with different angles of the cleavage plane in respect to the ventricular surface , divided in groups with increments of 30 ° . H ) Percentage of cells undergoing symmetric or asymmetric cell division , defined by the cleavage plane bypassing or bysecting the apical plasma membrane , in control and EPZ treated samples . Data information : Quantifications are based on three independent experiments ( n = 3 , biological replicates ) and Chi - square test was performed in all cases . Error bars represent SEM , * * * p < 0 . 001 , * * * * p < 0 . 0001 , alpha = 0 . 05 . VZ - ventricular zone . The following number of cells were scored : G ) For control condition 41 cells from four independent samples , 5 sections and 15 different images were assessed . EPZ treated quantifications were performed on a total of 26 cells from 3 independent samples , 5 sections and 16 images . H ) For control condition a total of 37 cells from four independent samples , 5 sections and 15 different images were assessed . EPZ treated quantifications were performed on a total of 23 cells from 3 independent samples , 5 sections and 16 images .

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

F) Phalloidin staining of control and EPZ treated sections showing cells during mitosis. Scale bar: 5 µm. G) Pie chart showing the proportion of cells with different angles of the cleavage plane in respect to the ventricular surface, divided in groups with increments of 30° . H) Percentage of cells undergoing symmetric or asymmetric cell division, defined by the cleavage plane bypassing or bysecting the apical plasma membrane, in control and EPZ treated samples. Data information: Quantifications are based on three independent experiments (n=3, biological replicates) and Chi-square test was performed in all cases. Error bars represent SEM, ***p<0.001, ****p<0.0001, alpha = 0.05. VZ - ventricular zone. The following number of cells were scored: G) For control condition 41 cells from four independent samples, 5 sections and 15 different images were assessed. EPZ treated quantifications were performed on a total of 26 cells from 3 independent samples, 5 sections and 16 images. H) For control condition a total of 37 cells from four independent samples, 5 sections and 15 different images were assessed. EPZ treated quantifications were performed on a total of 23 cells from 3 independent samples, 5 sections and 16 images.


C ) Immunostaining showing ASNS - positive cells in the VZ , SVZ and CP of control and EPZ treated sections ; and signal intensity for each condition . Scale bars : 50 µm . Dotted lines mark either ventricular or pial surface of the tissue .

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

C) Immunostaining showing ASNS-positive cells in the VZ, SVZ and CP of control and EPZ treated sections; and signal intensity for each condition. Scale bars: 50 µm. Dotted lines mark either ventricular or pial surface of the tissue .


D ) ChIP - seq and ATAC - seq tracks showing distribution of peaks for selected chromatin marks on the Asns locus for NPCs and cerebral cortex of E12 . 5 and E14 . 5 mice . Tracks for control and EPZ conditions are compared in NPCs . Indicated is the TSS of Asns .

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

D) ChIP-seq and ATAC-seq tracks showing distribution of peaks for selected chromatin marks on the Asns locus for NPCs and cerebral cortex of E12.5 and E14.5 mice. Tracks for control and EPZ conditions are compared in NPCs. Indicated is the TSS of Asns.


F ) ChIP - qPCR analysis for DOT1L and IgG ( n = 7 ; biological replicates ) at the TSS on Asns genomic locus in in vitro - derived NPCs . One - sided paired Wilcoxon statistical test was performed , error bars represent SEM . * * p < 0 . 01 . G ) ChIP - qPCR analyses for EZH2 ( n = 5 ; biological replicates ) and H3K27me3 ( n = 9 ; biological replicates ) at the TSS on Asns genomic locus in control and EPZ treated condition of in vitro - derived NPCs . One - sided paired Wilcoxon statistical test was performed , error bars represent SEM . * p < 0 . 04 .

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

F) ChIP-qPCR analysis for DOT1L and IgG (n=7; biological replicates) at the TSS on Asns genomic locus in in vitro-derived NPCs. One-sided paired Wilcoxon statistical test was performed, error bars represent SEM. **p<0.01. G) ChIP-qPCR analyses for EZH2 (n=5; biological replicates) and H3K27me3 (n=9; biological replicates) at the TSS on Asns genomic locus in control and EPZ treated condition of in vitro-derived NPCs. One-sided paired Wilcoxon statistical test was performed, error bars represent SEM. *p<0.04.


C ) Ex utero electroporated cells expressing GFP ( green ) or TUBB3 ( magenta ) in control ( Con ; pCMV - GFP ) or in ASNS overexpression ( ASNS OE ; pCMV - ASNS - GFP ) condition 24 h after slice culture . Scale bars : 20 µm . Dotted lines mark ventricular surface of the tissue . Magnifications on the right show exemplarily a GFP and TUBB3 double positive cell upon ASNS overexpression . Dotted shapes indicate the border of cell soma based on GFP expression . D ) Fraction of TUBB3 - positive and - negative cells expressed as percentage of total cells in Con and upon ASNS OE . Quantifications were made from independent experiments ( n = 3 ) . Student ' s t - test , * * p < 0 . 01 , error bars represent SEM .

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

C) Ex utero electroporated cells expressing GFP (green) or TUBB3 (magenta) in control (Con; pCMV-GFP) or in ASNS overexpression (ASNS OE; pCMV-ASNS-GFP) condition 24 h after slice culture. Scale bars: 20 µm. Dotted lines mark ventricular surface of the tissue. Magnifications on the right show exemplarily a GFP and TUBB3 double positive cell upon ASNS overexpression. Dotted shapes indicate the border of cell soma based on GFP expression. D) Fraction of TUBB3-positive and -negative cells expressed as percentage of total cells in Con and upon ASNS OE. Quantifications were made from independent experiments (n=3). Student's t-test, **p<0.01, error bars represent SEM.


E ) Representative images of mCherry ( red ) or TBR1 - expressing ( green ) cells in control ( Con ; pCAG - mCherry ) or in ASNS overexpression ( ASNS OE ; pCAG - ASNS ) condition , 48 h after in utero electroporation . Scale bars : 20 µm . Dotted lines mark ventricular surface of the tissue . Magnifications on the right show exemplarily mCherry and TBR1 double or single positive cells upon ASNS overexpression . F ) Fraction of TBR1 - positive and - negative cells expressed as percentage of total cells in Con and upon ASNS OE . Quantifications were made from 1141 cells in control group and 606 cells in ASNS OE group . Fisher ' s exact test , * * * * p < 0 . 0001 .

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

E) Representative images of mCherry (red) or TBR1-expressing (green) cells in control (Con; pCAG-mCherry) or in ASNS overexpression (ASNS OE; pCAG-ASNS) condition, 48 h after in utero electroporation. Scale bars: 20 µm. Dotted lines mark ventricular surface of the tissue. Magnifications on the right show exemplarily mCherry and TBR1 double or single positive cells upon ASNS overexpression. F) Fraction of TBR1-positive and -negative cells expressed as percentage of total cells in Con and upon ASNS OE. Quantifications were made from 1141 cells in control group and 606 cells in ASNS OE group. Fisher's exact test, ****p<0.0001.


Widefield images of RPA - mCherry - ssDNA molecules ( magenta ) in the presence of GFP - Rdh54 ( green ; 1 , 3 , 10 and 30 nM ) .

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

Widefield images of RPA-mCherry-ssDNA molecules (magenta) in the presence of GFP-Rdh54 (green; 1, 3, 10 and 30 nM).


Widefield images of Rad51 - ssDNA molecules ( unlabeled ) in the presence of GFP - Rdh54 ( green ; 1 , 3 , 10 and 30 nM ) .

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

Widefield images of Rad51-ssDNA molecules (unlabeled) in the presence of GFP-Rdh54 (green; 1, 3, 10 and 30 nM).


Kymograph illustrating the dissociation of GFP - Rdh54 from Rad51 - ssDNA ; arrowhead highlights the time point at which free GFP - Rdh54 was flushed from the sample chamber . Graph depicting the dissociation rate of GFP - Rdh54 from Rad51 - ssDNA . Error bars represent s . d . of individual ssDNA molecules ( N = 25 ) .

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

Kymograph illustrating the dissociation of GFP-Rdh54 from Rad51-ssDNA; arrowhead highlights the time point at which free GFP-Rdh54 was flushed from the sample chamber.   Graph depicting the dissociation rate of GFP-Rdh54 from Rad51-ssDNA. Error bars represent s.d. of individual ssDNA molecules (N=25).


Kymograph showing the association of GFP - Rdh54 with a Rad51 - ssDNA molecule ; arrowhead highlights the injection time point for GFP - Rdh54 . Association rates of GFP - Rdh54 on individual Rad51 PSCs ( N = 44 ) . Red lines represent the mean and s . d .

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

Kymograph showing the association of GFP-Rdh54 with a Rad51-ssDNA molecule; arrowhead highlights the injection time point for GFP-Rdh54.   Association rates of GFP-Rdh54 on individual Rad51 PSCs (N=44). Red lines represent the mean and s.d.


Images of Rad51 - ssDNA molecules bound by both mCherry - Rdh54 ( 30 nM ; magenta ) and GFP - Rad54 ( green ; 30 nM ) .

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

Images of Rad51-ssDNA molecules bound by both mCherry-Rdh54 (30 nM; magenta) and GFP-Rad54 (green; 30 nM).


Quantification of bound GFP - Rad54 ( 10 nM ) signal intensity for Rad51 - ssDNA molecules with and without bound mCherry - Rdh54 ( 10 nM ) . Error bars represent s . d . of individual ssDNA molecules ( N = 21 ) .

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

Quantification of bound GFP-Rad54 (10 nM) signal intensity for Rad51-ssDNA molecules with and without bound mCherry-Rdh54 (10 nM). Error bars represent s.d. of individual ssDNA molecules (N = 21).


Images showing increasing concentrations of GFP - Rad54 ( green ) only or mCherry - Rdh54 ( magenta ) only bound separately to Rad51 - ssDNA molecules .

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

Images showing increasing concentrations of GFP-Rad54 (green) only or mCherry-Rdh54 (magenta) only bound separately to Rad51-ssDNA molecules.


Images of GFP - Rad54 ( green ) and mCherry - Rdh54 ( magenta ) co - bound to the same Rad51 - ssDNA molecules .

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

Images of GFP-Rad54 (green) and mCherry-Rdh54 (magenta) co-bound to the same Rad51-ssDNA molecules.


Quantification of GFP - Rad54 binding in the absence and presence of mCherry - Rdh54 . The data was fit by non - linear regression and error bars represent s . d . of individual molecules ( N = 43 ) .

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

Quantification of GFP-Rad54 binding in the absence and presence of mCherry-Rdh54. The data was fit by non-linear regression and error bars represent s.d. of individual molecules (N = 43).


Quantification of mCherry - Rdh54 binding in the absence or presence of GFP - Rad54 . The data was fit by non - linear regression and error bars represent s . d . of individual molecules ( N = 43 ) .

Quantification of mCherry-Rdh54 binding in the absence or presence of GFP-Rad54. The data was fit by non-linear regression and error bars represent s.d. of individual molecules (N = 43).


Images of GFP - tagged Rad54 , Rdh54NRad54 , Rdh54 and Rad54NRdh54 ( 1 , 3 , 10 and 30 nM each ) bound to Rad51 - ssDNA molecules .

Images of GFP-tagged Rad54, Rdh54NRad54, Rdh54 and Rad54NRdh54 (1, 3, 10 and 30 nM each) bound to Rad51-ssDNA molecules.


Images of GFP - tagged Rad54 , Rdh54NRad54 , Rdh54 and Rad54NRdh54 ( 1 , 3 , 10 and 30 nM each ) bound to Rad51 - ssDNA molecules in the presence of 30 nM Hed1 .

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

Images of GFP-tagged Rad54, Rdh54NRad54, Rdh54 and Rad54NRdh54 (1, 3, 10 and 30 nM each) bound to Rad51-ssDNA molecules in the presence of 30 nM Hed1.


Quantification of the integrated GFP signal intensity for GFP - tagged Rad54 ( N = 75 ) , Rdh54NRad54 ( N = 46 ) , Rdh54 ( N = 50 ) and Rad54NRdh54 ( N = 35 ; 1 , 3 , 10 and 30 nM each ) bound to Rad51 - ssDNA molecules . The data was fit by non - linear regression and error bars represent s . d . for individual DNA molecules .

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

Quantification of the integrated GFP signal intensity for GFP-tagged Rad54 (N = 75), Rdh54NRad54 (N = 46), Rdh54 (N = 50) and Rad54NRdh54 (N = 35; 1, 3, 10 and 30 nM each) bound to Rad51-ssDNA molecules. The data was fit by non-linear regression and error bars represent s.d. for individual DNA molecules.


Comparison of fold binding inhibition for GFP - tagged Rad54 , Rdh54NRad54 , Rdh54 and Rad54NRdh54 in reactions conducted in the presence of 30 nM Hed1 . The dot represents the mean and the error bars represent the range for two independent experiments .

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

Comparison of fold binding inhibition for GFP-tagged Rad54, Rdh54NRad54, Rdh54 and Rad54NRdh54 in reactions conducted in the presence of 30 nM Hed1. The dot represents the mean and the error bars represent the range for two independent experiments.


Widefield images of Atto565 - labeled PSCs ( magenta ) bound to dsDNA curtains stained with YOYO1 ( green ) . The PSCs were prepared with the indicated combinations of Rad51 , Rdh54 and GFP - Rad54 . Number of Atto565 - labeled PSC binding events per dsDNA molecule for PCS prepared with Rad54 and Rdh54 only ( minus Rad51 ; N = 372 ) , Rad51 plus Rad54 only ( N = 117 ) , Rad51 plus Rdh54 only ( N = 258 ) , Rad51 plus Rdh54 and Rad54 ( N = 303 ) . The error bars represent the s . d . for three independent experiments .

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

Widefield images of Atto565-labeled PSCs (magenta) bound to dsDNA curtains stained with YOYO1 (green). The PSCs were prepared with the indicated combinations of Rad51, Rdh54 and GFP-Rad54.   Number of Atto565-labeled PSC binding events per dsDNA molecule for PCS prepared with Rad54 and Rdh54 only (minus Rad51 ; N = 372), Rad51 plus Rad54 only (N= 117), Rad51 plus Rdh54 only (N = 258), Rad51 plus Rdh54 and Rad54 (N = 303). The error bars represent the s.d. for three independent experiments.


Kymographs of Rad51 - tailed duplex PSCs ( magenta ) containing the indicated combinations of Rad54 and Rdh54 proteins .

Kymographs of Rad51-tailed duplex PSCs (magenta) containing the indicated combinations of Rad54 and Rdh54 proteins.


Translocation velocities for tailed duplex PSCs prepared with Rad54 only ( lane 1 ; N = 86 ) , Rad54 plus Rdh54 ( lane 2 ; N = 144 ) , Rad54 - K341R plus Rdh54 ( lane 3 ; N = 47 ) , Rad54 plus Rdh54 - K318R ( lane 4 ; N = 112 ) , or Rad54 - K341R plus Rdh54 - K318R ( lane 5 ; N = 64 ) . The error bars represent s . d . Translocation distances for tailed duplex PSCs prepared with Rad54 only ( lane 1 ; N = 86 ) , Rad54 plus Rdh54 ( lane 2 ; N = 144 ) , Rad54 - K341R plus Rdh54 ( lane 3 ; N = 47 ) , Rad54 plus Rdh54 - K318R ( lane 4 ; N = 112 ) , or Rad54 - K341R plus Rdh54 - K318R ( lane 5 ; N = 64 ) . Error bars were generated by bootstrapping . First passage recognition efficiency for tailed duplex PSCs prepared with Rad54 only ( lane 1 ; N = 145 ) , Rad54 plus Rdh54 ( lane 2 ; N = 115 ) , Rad54 - K341R plus Rdh54 ( lane 3 ; N = 45 ) , Rad54 plus Rdh54 - K318R ( lane 4 ; N = 39 ) , or Rad54 - K341R plus Rdh54 - K318R ( lane 5 ; no translocation observed ) . Error bars ( s . d . ) were generated from three independent experiments .

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

Translocation velocities for tailed duplex PSCs prepared with Rad54 only (lane 1; N = 86), Rad54 plus Rdh54 (lane 2; N = 144), Rad54-K341R plus Rdh54 (lane 3; N = 47), Rad54 plus Rdh54-K318R (lane 4; N = 112), or Rad54-K341R plus Rdh54-K318R (lane 5; N = 64). The error bars represent s.d.   Translocation distances for tailed duplex PSCs prepared with Rad54 only (lane 1; N = 86), Rad54 plus Rdh54 (lane 2; N = 144), Rad54-K341R plus Rdh54 (lane 3; N = 47), Rad54 plus Rdh54-K318R (lane 4; N = 112), or Rad54-K341R plus Rdh54-K318R (lane 5; N = 64). Error bars were generated by bootstrapping.   First passage recognition efficiency for tailed duplex PSCs prepared with Rad54 only (lane 1; N = 145), Rad54 plus Rdh54 (lane 2; N = 115), Rad54-K341R plus Rdh54 (lane 3; N = 45), Rad54 plus Rdh54-K318R (lane 4; N = 39), or Rad54-K341R plus Rdh54-K318R (lane 5; no translocation observed). Error bars (s.d.) were generated from three independent experiments.


PSC binding distributions following 10 min reactions for PSCs prepared with Rad54 plus Rdh54 ( N = 181 ) ; Rad54 plus Rdh54 - K318R ( N = 115 ) ; Rad54 - K341R plus Rdh54 ( N = 90 ) ; or Rad54 - K341R plus Rdh54 - K318R ( N = 54 ) , as indicated . Error bars were generated by bootstrapping . The asterisks denote the targeted region of homology on the donor dsDNA molecules .

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

PSC binding distributions following 10 min reactions for PSCs prepared with Rad54 plus Rdh54 (N= 181); Rad54 plus Rdh54-K318R (N= 115); Rad54-K341R plus Rdh54 (N = 90); or Rad54-K341R plus Rdh54-K318R (N = 54), as indicated. Error bars were generated by bootstrapping. The asterisks denote the targeted region of homology on the donor dsDNA molecules.


D - loop stimulation by Rad54 or Rdh54NRad54 in reactions with Rad51 ( 300 nM ) , tailed duplex substrate ( 15 nM ) and a pUC19 plasmid dsDNA ( 0 . 3 nM ) . Quantification of D - loop formation for Rad54 and Rdh54NRad54 . Error bars represent s . d . of three independent experiments .

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

D-loop stimulation by Rad54 or Rdh54NRad54 in reactions with Rad51 (300 nM), tailed duplex substrate (15 nM) and a pUC19 plasmid dsDNA (0.3 nM).   Quantification of D-loop formation for Rad54 and Rdh54NRad54. Error bars represent s.d. of three independent experiments.


D - loop stimulation by Rdh54 and Rad54NRdh54 . Quantification of D - loop formation for Rad54 and Rdh54NRad54 . Error bars represent s . d . of three independent experiments .

D-loop stimulation by Rdh54 and Rad54NRdh54.   Quantification of D-loop formation for Rad54 and Rdh54NRad54. Error bars represent s.d. of three independent experiments.


ATPase stimulation for GFP - tagged Rad54 , Rdh54NRad54 , Rdh54 or Rad54NRdh54 in the presence of Rad51 - ssDNA . Error bars represent s . d . of three independent experiments .

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

ATPase stimulation for GFP-tagged Rad54, Rdh54NRad54, Rdh54 or Rad54NRdh54 in the presence of Rad51-ssDNA. Error bars represent s.d. of three independent experiments.


Quantification of Rad51 Atto565 - tailed duplex PSCs bound per dsDNA molecule in dsDNA curtain homology search assays with Rad54 ( N = 117 ) , Rdh54NRad54 ( N = 150 ) or Rad54NRdh54 ( N = 175 ) . The error bars represent the s . d . for three independent experiments .

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

Quantification of Rad51 Atto565-tailed duplex PSCs bound per dsDNA molecule in dsDNA curtain homology search assays with Rad54 (N = 117), Rdh54NRad54 (N = 150) or Rad54NRdh54 (N=175). The error bars represent the s.d. for three independent experiments.


Fraction translocating Rad51 Atto565 - tailed duplex PSCs in reactions with Rad54 ( N = 117 ) , Rdh54NRad54 ( N = 150 ) , Rad54NRdh54 ( N = 0 ) and Rdh54 ( N = 0 ) .

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

Fraction translocating Rad51 Atto565-tailed duplex PSCs in reactions with Rad54 (N = 117), Rdh54NRad54 (N = 150), Rad54NRdh54 (N = 0) and Rdh54 (N = 0).


Translocation velocities of Rad51 Atto565 - tailed duplex PSCs in reactions with Rad54 ( N = 86 ) and Rdh54NRad54 ( N = 18 ) . The error bars represent the s . d . of the data .

Translocation velocities of Rad51 Atto565-tailed duplex PSCs in reactions with Rad54 (N = 86) and Rdh54NRad54 (N = 18). The error bars represent the s.d. of the data.


Yeast spot growth assays to monitor the ability of Rad54 , Rdh54 , Rad54NRdh54 , and Rdh54NRad54 to complement MMS sensitivity in rad54 ∆ yeast strains . Yeast spot growth assays to monitor the ability of Rad54 , Rdh54 , Rad54NRdh54 , and Rdh54NRad54 to complement MMS sensitivity in rdh54 ∆ yeast strains .

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

Yeast spot growth assays to monitor the ability of Rad54, Rdh54, Rad54NRdh54, and Rdh54NRad54 to complement MMS sensitivity in rad54∆ yeast strains.   Yeast spot growth assays to monitor the ability of Rad54, Rdh54, Rad54NRdh54, and Rdh54NRad54 to complement MMS sensitivity in rdh54∆ yeast strains.


H & amp ; amp ; E staining of adult head sections of ( A ) repo > GFP . nls and ( B ) repo > Shits1 at 29 ° C for 3 days . Lamina degeneration was identified as vacuoles in ( B ) . Cryosection of adult ( C ) repo > H2B - RFP exhibited the expression of the nuclear red fluorescent protein ( RFP ) in the glia ( epithelialglialnucleus : arrowhead ) and ( D ) repo > Shits1 at 28 ° C for 12 days . Vacuoles were identified in the lamina neuropile ( D ) . Epithelial ( arrowhead ) ( E ) , marginal ( arrow ) and distal satellite ( arrowhead ) glianuclei ( F ) are labeled by HisCl - GAL4 and NP2109 - GAL4 , respectively . Note that HisCl - GAL4 is not expressed in all epithelialglia . ( G , H ) Weak lamina vacuolization was identified in HisCl > Shits1 ( G , 0 . 92 % ) and NP2109 > Shits1 ( H , 1 . 46 % ) at 29 ° C for 14 days . ( I , J ) A single MARCMglia clone ( GFP , green ) expressed Shits1 at 21 ° C ( I ) and 29 ° C ( J ) . ( J ) A vacuole occurred within a glial clone . DAPI ( white ) stains the nuclei ( C , D , and G - J ) . ( K ) The percentage of the vacuole area in the lamina of the repo > Shits1flies at 28 ° C progressively increased . n indicated in each column . P - values were calculated using one - way ANOVA with Bonferroni ' s post - test . ( L ) When repo > Shits1flies were shifted to 28 ° C for 12 days and then shifted to 17 ° C for 9 additional days , the vacuolization was not alleviated . P - values were calculated using one - way ANOVA with Tukey ' s post - test . n indicated in each column .

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

H&amp;E staining of adult head sections of (A) repo>GFP.nls and (B) repo>Shits1 at 29°C for 3 days. Lamina degeneration was identified as vacuoles in (B). Cryosection of adult (C) repo>H2B-RFP exhibited the expression of the nuclear red fluorescent protein (RFP) in the glia (epithelialglialnucleus: arrowhead) and (D) repo>Shits1 at 28°C for 12 days. Vacuoles were identified in the lamina neuropile (D). Epithelial (arrowhead) (E), marginal (arrow) and distal satellite (arrowhead) glianuclei (F) are labeled by HisCl-GAL4 and NP2109-GAL4, respectively. Note that HisCl-GAL4 is not expressed in all epithelialglia. (G, H) Weak lamina vacuolization was identified in HisCl>Shits1 (G, 0.92%) and NP2109>Shits1 (H, 1.46%) at 29°C for 14 days. (I, J) A single MARCMglia clone (GFP, green) expressed Shits1 at 21°C (I) and 29°C (J). (J) A vacuole occurred within a glial clone. DAPI (white) stains the nuclei (C, D, and G-J). (K) The percentage of the vacuole area in the lamina of the repo>Shits1flies at 28°C progressively increased. n indicated in each column. P-values were calculated using one-way ANOVA with Bonferroni's post-test. (L) When repo>Shits1flies were shifted to 28°C for 12 days and then shifted to 17°C for 9 additional days, the vacuolization was not alleviated. P-values were calculated using one-way ANOVA with Tukey's post-test. n indicated in each column.


( A - E ) Horizontal sections of the adult head lamina cartridge . ( A ) In repo > GFP . nls flies , two lamina neurons L1 , L2 axons ( L ) and R1 - 6 axons ( blue area ) were surrounded by the electron - dense cytoplasm of the epithelial glia ( G ) . The section was examined at three different depths , and the size of a single cartridge is not significantly different at different depths . ( B - D ) repo > Shits1 adults maintained at 29 ° C for 4 days . Axons were enlarged but intact . A large number of small and medium vacuoles were identified in the epithelial glia . ( C ) Small vacuoles in the glia that contained a double - membrane structure ( arrowhead ) . ( C ' ) Higher magnification of the boxed area in ( C ) . ( D ) A double - membrane autophagosome - like structure ( arrowhead ) in the glialcytoplasm . ( E ) In repots > DERDN adults maintained at 29 ° C for 2 days , large vacuoles were identified in between the lamina cartridges . N : glialnuclei . ( E ' ) Higher magnification of the boxed area in ( E ) , which shows autophagosome - like vesicles ( arrowhead ) in the gliacytoplasm .

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

(A-E) Horizontal sections of the adult head lamina cartridge. (A) In repo>GFP.nls flies, two lamina neurons L1, L2 axons (L) and R1-6 axons (blue area) were surrounded by the electron-dense cytoplasm of the epithelial glia (G). The section was examined at three different depths, and the size of a single cartridge is not significantly different at different depths. (B-D) repo>Shits1 adults maintained at 29°C for 4 days. Axons were enlarged but intact. A large number of small and medium vacuoles were identified in the epithelial glia. (C) Small vacuoles in the glia that contained a double-membrane structure (arrowhead). (C') Higher magnification of the boxed area in (C). (D) A double-membrane autophagosome-like structure (arrowhead) in the glialcytoplasm. (E) In repots>DERDN adults maintained at 29°C for 2 days, large vacuoles were identified in between the lamina cartridges. N: glialnuclei. (E') Higher magnification of the boxed area in (E), which shows autophagosome-like vesicles (arrowhead) in the gliacytoplasm.


( A ) Rh1 > lacZ + H2B - RFP exhibited nuclearRFP expression in the R1 - 6 photoreceptors ( red ) . ( B ) Rh1 > lacZ + Shits1 maintained at 29 ° C for 14 days resulted in lamina vacuolization . ( C ) Pan - Rh7 > H2B - RFP exhibited expression in R7 ( arrow ) and R8 ( arrowhead ) ( red ) . ( D ) Pan - Rh7 > Shits1 incubated at 29 ° C for 14 days did not exhibit lamina degeneration . ( E ) Lamina monopolar ( L ) neurons ( red ) were selectively labeled by the Ln - GAL4 driven H2B - RFP with repo - GAL80 . ( F ) Ln > Shits1 , repoGAL80 shifted to 29 ° C for 14 days did not exhibit lamina degeneration . ( G ) R1 - 6 photoreceptors were killed in Rh1ts > lacZ + Hid shifted to 29 ° C for 14 days . Rh1ts indicates tubGAL80ts ; Rh1 - GAL4 . Degeneration was induced in the lamina in addition to the retina . ( H ) rdgC306 / + mutant exposed to constant light for 14 days exhibited degeneration in the retina , as well as the lamina . DAPI : nuclei ( white in A - H ) . ( I , K ) Rh1 > lacZ + Shits1 at 29 ° C for 4 days exhibited vacuoles in the electron dense cytoplasm of epithelialglia or near the glianucleus . Arrow : vacuole with internal debris . Arrowhead : large vacuole . ( J ) Lamina cartridge of Rh1 > lacZ + H2B - RFP . N indicates glialnuclei . ( L ) The percentage of the vacuole area in the lamina at 29 ° C for 14 days was examined . All P - values were calculated using one - way ANOVA with Tukey ' s post - test . Scale bar : 20 μm .

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

(A) Rh1>lacZ+H2B-RFP exhibited nuclearRFP expression in the R1-6 photoreceptors (red). (B) Rh1>lacZ+Shits1 maintained at 29°C for 14 days resulted in lamina vacuolization. (C) Pan-Rh7>H2B-RFP exhibited expression in R7 (arrow) and R8 (arrowhead) (red). (D) Pan-Rh7>Shits1 incubated at 29°C for 14 days did not exhibit lamina degeneration. (E) Lamina monopolar (L) neurons (red) were selectively labeled by the Ln-GAL4 driven H2B-RFP with repo-GAL80. (F) Ln>Shits1, repoGAL80 shifted to 29°C for 14 days did not exhibit lamina degeneration. (G) R1-6 photoreceptors were killed in Rh1ts>lacZ+Hid shifted to 29°C for 14 days. Rh1ts indicates tubGAL80ts; Rh1-GAL4. Degeneration was induced in the lamina in addition to the retina. (H) rdgC306/+ mutant exposed to constant light for 14 days exhibited degeneration in the retina, as well as the lamina. DAPI: nuclei (white in A-H). (I, K) Rh1>lacZ+Shits1 at 29°C for 4 days exhibited vacuoles in the electron dense cytoplasm of epithelialglia or near the glianucleus. Arrow: vacuole with internal debris. Arrowhead: large vacuole. (J) Lamina cartridge of Rh1>lacZ+H2B-RFP. N indicates glialnuclei. (L) The percentage of the vacuole area in the lamina at 29°C for 14 days was examined. All P-values were calculated using one-way ANOVA with Tukey's post-test. Scale bar: 20 μm.


( A ) repots > Shits1 + H2B - RFP , ( B ) repots > Shits1 + Egfrλtop and ( C ) repots > Shits1 + RlSem incubated for 12 days at 28 ° C . ( D ) repots > DERDN for 7 days at 28 ° C . ( E ) Epithelial glia MARCM clone and ( F ) Egfrco mutant glia MARCM clone ( labeled with Tomato , green ) . The penetrance is indicated as the number of samples with vacuoles over the total number of samples . DAPI : nuclei ( white in A - F ) . ( G ) Percentage of vacuole area in the lamina neuropile of ( A - D ) . The P - values were calculated using one - way ANOVA with Dunnett ' s post - test . ( H ) Percentage of vacuole area in the lamina of repots > Shits1 flies in the indicated genetic background . The adults were shifted to 28 ° C for 12 days . The P - values were calculated using one - way ANOVA with Tukey ' s post - test . Scale bar : 20 μm .

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

(A) repots>Shits1+H2B-RFP, (B) repots>Shits1+Egfrλtop and (C) repots>Shits1+RlSem incubated for 12 days at 28°C. (D) repots>DERDN for 7 days at 28°C. (E) Epithelial glia MARCM clone and (F) Egfrco mutant glia MARCM clone (labeled with Tomato, green). The penetrance is indicated as the number of samples with vacuoles over the total number of samples. DAPI: nuclei (white in A-F). (G) Percentage of vacuole area in the lamina neuropile of (A-D). The P-values were calculated using one-way ANOVA with Dunnett's post-test. (H) Percentage of vacuole area in the lamina of repots>Shits1 flies in the indicated genetic background. The adults were shifted to 28°C for 12 days. The P-values were calculated using one-way ANOVA with Tukey's post-test. Scale bar: 20 μm.


( A , A ' ) Anti - Spitz ( red ) immunostaining of w1118 adult head . Spitz can be detected in the retina and lamina . ( B , B ' ) The full - length transmembrane form of Spitz - GFP ( mSpi , green ) expressed in the retina in GMR > mSpiGFP flies was predominantly identified in the photoreceptor soma and terminally localized in the lamina cartridge . mSpitz - GFP requires processing by Rho and Star to become a secreted form . Overexpressed mSpitzGFP has been demonstrated to be retained in the perinuclear ER even in the presence of endogenous Rho / Star [ 56 ] ( C ) Knockdown of both EGFR ligands Spi and Krn in R1 - 6 photoreceptors in Rh1 > Dcr2 + Spi - RNAi + Krn - RNAi and ( D ) blockade of Spi processing in Rh1 > lacZ + iRhom exhibited lamina degeneration after shifting to 29 ° C for 14 days . ( E - E ' ) Lamina degeneration in whole eye rho7M43ru1 double mutant clones at 28 ° C for 12 days . The clone is labeled by DsRed ( red ) . ( F ) Spi secretion is inhibited in Rh1 > lacZ + Rab11S25N . ( G ) The percentages of the vacuole areas of ( C , D , F ) in lamina at 29 ° C for 14 days were examined . All P - values were calculated using one - way ANOVA with Tukey ' s post - test . DAPI : nuclei ( white in A ' , B ' , C - F ) . Scale bar : 20 μm .

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

(A, A') Anti-Spitz (red) immunostaining of w1118 adult head. Spitz can be detected in the retina and lamina. (B, B') The full-length transmembrane form of Spitz-GFP (mSpi, green) expressed in the retina in GMR>mSpiGFP flies was predominantly identified in the photoreceptor soma and terminally localized in the lamina cartridge. mSpitz-GFP requires processing by Rho and Star to become a secreted form. Overexpressed mSpitzGFP has been demonstrated to be retained in the perinuclear ER even in the presence of endogenous Rho/Star [56] (C) Knockdown of both EGFR ligands Spi and Krn in R1-6 photoreceptors in Rh1>Dcr2+Spi-RNAi+Krn-RNAi and (D) blockade of Spi processing in Rh1>lacZ+iRhom exhibited lamina degeneration after shifting to 29°C for 14 days. (E-E') Lamina degeneration in whole eye rho7M43ru1 double mutant clones at 28°C for 12 days. The clone is labeled by DsRed (red). (F) Spi secretion is inhibited in Rh1>lacZ+Rab11S25N. (G) The percentages of the vacuole areas of (C, D, F) in lamina at 29°C for 14 days were examined. All P-values were calculated using one-way ANOVA with Tukey's post-test. DAPI: nuclei (white in A', B', C-F). Scale bar: 20 μm.


( A , A ' ) Anti - Spitz detected Spitz ( magenta ) colocalizing ( arrowhead ) with epithelialgliacytoplasm marked by anti - Black ( green ) . Scale bar : 5 μm . ( B - B ' ) EGFR reporter pointed - lacZ ( pnt - lacZ ) exhibited expression in the epithelial ( arrow ) and marginal glia ( arrowhead ) . ( C , C ' ) Dominant - negative form of EGFR ( DERDN ) expressed in glia at 28 ° C for 3 days inhibited pnt - lacZ expression . ( D , D ' ) pnt - laZ expression was lost in Shits - expressing glia at 28 ° C for 3 days . ( E , E ' ) The knockdown of Spi in R1 - 6 photoreceptors at 28 ° C for 12 days also inhibited pnt - lacZ expression in the glia . The penetrances of ( B - E ) are shown in the upper right corner of each panel . DAPI : nuclei ( white in B ' - E ' ) . Scale bar : 20 μm .

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

(A, A') Anti-Spitz detected Spitz (magenta) colocalizing (arrowhead) with epithelialgliacytoplasm marked by anti-Black (green). Scale bar: 5 μm. (B-B') EGFR reporter pointed-lacZ (pnt-lacZ) exhibited expression in the epithelial (arrow) and marginal glia (arrowhead). (C, C') Dominant-negative form of EGFR (DERDN) expressed in glia at 28°C for 3 days inhibited pnt-lacZ expression. (D, D') pnt-laZ expression was lost in Shits-expressing glia at 28°C for 3 days. (E, E') The knockdown of Spi in R1-6 photoreceptors at 28°C for 12 days also inhibited pnt-lacZ expression in the glia. The penetrances of (B-E) are shown in the upper right corner of each panel. DAPI: nuclei (white in B'-E'). Scale bar: 20 μm.


( A - C ) Immunostaining of TUNEL assay ( red ) , active Caspase - 3 ( magenta , A ' - C ' ) and Repo ( green , A " - C " ) . ( A ) GMR - wIR ; repots > lacZ . ( B ) GMR - wIR ; repots > Shits1 . ( C ) GMR - wIR ; repots > DERDN . GMR - wIR was used to reduce the autofluorescence of the eye pigments . Scale bar : 20 μm . ( D - F ) The in vivo fluorescent sensor of caspase activity ( Apoliner ) indicated there was no active caspase activity in the glia . ( D ) repots > Apoliner + lacZ . ( E ) repots > Apoliner + Shits1 . ( F ) repots > Apoliner + DERDN . All adults were shifted to 28 ° C for 5 days . nls - GFP ( green , D - F ) ; merge of GFP ( green ) and mRFP ( red ) in ( D ' - F ' ) ; merge of nls - GFP and DAPI ( magenta ) in ( D " - F " ) . Scale bar : 10 μm . ( G ) Percentage of vacuole area in the lamina in repots > Shits1flies that coexpressed the anti - apoptotic factors P35 and Diap1 . The adults were shifted to 28 ° C for 21 days . The P - values were calculated using one - way ANOVA with Tukey ' s post - test . ( H ) The cell number of the epithelialglia was not reduced in the vacuolated lamina . The numbers of epithelial glia in the lamina of repo > H2B - RFP and repo > H2B - RFP + Shits1 female adults incubated at 28 ° C for 14 days were examined by counting the nuclear RFP at the epithelial layer from the entire Z - stacks of confocal images . P - values were calculated via Mann - Whitney tests .

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

(A-C) Immunostaining of TUNEL assay (red), active Caspase-3 (magenta, A'-C') and Repo (green, A"-C"). (A) GMR-wIR; repots>lacZ. (B) GMR-wIR; repots>Shits1. (C) GMR-wIR; repots>DERDN. GMR-wIR was used to reduce the autofluorescence of the eye pigments. Scale bar: 20 μm. (D-F) The in vivo fluorescent sensor of caspase activity (Apoliner) indicated there was no active caspase activity in the glia. (D) repots>Apoliner+lacZ. (E) repots>Apoliner+Shits1. (F) repots>Apoliner+DERDN. All adults were shifted to 28°C for 5 days. nls-GFP (green, D-F); merge of GFP (green) and mRFP (red) in (D'-F'); merge of nls-GFP and DAPI (magenta) in (D"-F"). Scale bar: 10 μm. (G) Percentage of vacuole area in the lamina in repots>Shits1flies that coexpressed the anti-apoptotic factors P35 and Diap1. The adults were shifted to 28°C for 21 days. The P-values were calculated using one-way ANOVA with Tukey's post-test. (H) The cell number of the epithelialglia was not reduced in the vacuolated lamina. The numbers of epithelial glia in the lamina of repo>H2B-RFP and repo>H2B-RFP+Shits1 female adults incubated at 28°C for 14 days were examined by counting the nuclear RFP at the epithelial layer from the entire Z-stacks of confocal images. P-values were calculated via Mann-Whitney tests.


In repots > GFP - LC3 + DERDN shifted to 28 ° C , the GFP signal ( green ) was weak on day 1 ( A - A " ) and progressively increased on days 2 ( B - B " ) and 3 ( C - C " ) and colocalized with Ref ( 2 ) P ( magenta ) . ( D ) Ref ( 2 ) P ( magenta ) accumulated within the Egfrco mutant MARCM clone ( green ) . The double - tagged GFP - mCherry - Atg8a is used to distinguish the autophagosomes ( GFP and mCherry , yellow ) and autolysosomes ( mCherry , red ) in autophagic flux . ( E ) In repots > GFP - mCherry - Atg8a + dAtg1 , autophagosomes ( arrowhead ) and autolysosomes ( arrow ) were induced in the glia with normal autophagic flux . ( F ) repots > GFP - mCherry - Atg8a . ( G ) repots > GFP - mCherry - Atg8a + Shits1 . Epithelialglial nuclei are indicated ( arrow ) . ( H ) repots > GFP - mCherry - Atg8a + DERDN . The adults were shifted to 28 ° C for 3 days . GFP : green ( E - H ) ; mCherry : red ( E ' - H ' ) ; merge ( E " - H " ) . Scale bar : 10 μm .

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

In repots>GFP-LC3+DERDN shifted to 28°C, the GFP signal (green) was weak on day 1 (A-A") and progressively increased on days 2 (B-B") and 3 (C-C") and colocalized with Ref(2)P (magenta). (D) Ref(2)P (magenta) accumulated within the Egfrco mutant MARCM clone (green). The double-tagged GFP-mCherry-Atg8a is used to distinguish the autophagosomes (GFP and mCherry, yellow) and autolysosomes (mCherry, red) in autophagic flux. (E) In repots>GFP-mCherry-Atg8a+dAtg1, autophagosomes (arrowhead) and autolysosomes (arrow) were induced in the glia with normal autophagic flux. (F) repots>GFP-mCherry-Atg8a. (G) repots>GFP-mCherry-Atg8a+Shits1. Epithelialglial nuclei are indicated (arrow). (H) repots>GFP-mCherry-Atg8a+DERDN. The adults were shifted to 28°C for 3 days. GFP: green (E-H); mCherry: red (E'-H'); merge (E"-H"). Scale bar: 10 μm.


( A - C ) repots > GFP - LAMP1 . ( D - F ) repots > GFP - LAMP1 + Shits1 . ( G - I ) repots > GFP - LAMP1 + DERDN . Adults were incubated at 28 ° C for 12 h ( A , D , G ) , 24 h ( B , E , H ) , and 48 h ( C , F , I ) , respectively . The GFP - LAMP1 signal ( green ) was induced at 12 h and progressively enhanced in ( D - F ) and ( G - I ) . ( J ) repots > Rab7 - mCherry + H2B - RFP . ( K ) repots > Rab7 - mCherry + Shits1 . ( L ) repots > Rab7 - mCherry + DERDN . The adults were shifted to 28 ° C for 2 days . Rab7 - mCherry puncta ( red ) were increased in ( K , L ) . DAPI : nuclei ( white in J ' - L ' ) . Scale bar : 20 μm .

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

(A-C) repots>GFP-LAMP1. (D-F) repots>GFP-LAMP1+Shits1. (G-I) repots>GFP-LAMP1+DERDN. Adults were incubated at 28°C for 12 h (A, D, G), 24 h (B, E, H), and 48 h (C, F, I), respectively. The GFP-LAMP1 signal (green) was induced at 12 h and progressively enhanced in (D-F) and (G-I). (J) repots>Rab7-mCherry+H2B-RFP. (K) repots>Rab7-mCherry+Shits1. (L) repots>Rab7-mCherry+DERDN. The adults were shifted to 28°C for 2 days. Rab7-mCherry puncta (red) were increased in (K, L). DAPI: nuclei (white in J'-L'). Scale bar: 20 μm.


( A ) repots > Rab5S43N . ( B ) repots > Rab7T22N . ( C ) repots > Rab11S25N . ( D - H ) MARCM clones labeled by RFP or GFP ( green ) of control ( D ) , Rab52 ( E ) , α - Ada3 ( F ) , HrsD28 ( G ) , Rab7KO ( H ) , dor8 ( I ) and carΔ146 ( J ) . The penetrance ( number of samples with vacuole over the number of samples examined ) is indicated in each panel . Adults of all genotypes were incubated at 28 ° C for 14 days . DAPI : nuclei ( white in A - J ) . Scale bar : 20 μm . ( K ) The percentage of the vacuole area in ( A - C ) was summarized . Adults of these genotypes were incubated at 28 ° C for 12 days . P - values were calculated using Kruskal - Wallis with Dunn ' s post - tests .

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

(A) repots>Rab5S43N. (B) repots>Rab7T22N. (C) repots>Rab11S25N. (D-H) MARCM clones labeled by RFP or GFP (green) of control (D), Rab52 (E), α-Ada3 (F), HrsD28 (G), Rab7KO (H), dor8 (I) and carΔ146 (J). The penetrance (number of samples with vacuole over the number of samples examined) is indicated in each panel. Adults of all genotypes were incubated at 28°C for 14 days. DAPI: nuclei (white in A-J). Scale bar: 20 μm. (K) The percentage of the vacuole area in (A-C) was summarized. Adults of these genotypes were incubated at 28°C for 12 days. P-values were calculated using Kruskal-Wallis with Dunn's post-tests.


repots > DERDN was coexpressed with ( A ) H2B - RFP , ( B ) Dorwt , ( C ) Dor - RNAi , ( D ) H2B - RFP , ( E ) Car , ( F ) Car - RNAi , ( G ) lacZ , ( H ) Car - RNAi + Dor - RNAi and ( I ) Car + Dorwt . The autophagy reporter Ref ( 2 ) P ( green ) was stained ( G ' - H ' ) . Scale bar : 20 μm . ( J , K , L ) The percentages of the vacuole areas in the lamina of ( A - C , D - F , G - I ) were summarized , respectively . The adults were shifted to 28 ° C for 7 days . DAPI : nuclei ( white in A - I ) . All P - values were calculated using Kruskal - Wallis tests with Dunn ' s post - tests .

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

repots>DERDN was coexpressed with (A) H2B-RFP, (B) Dorwt, (C) Dor-RNAi, (D) H2B-RFP, (E) Car, (F) Car-RNAi, (G) lacZ, (H) Car-RNAi+Dor-RNAi and (I) Car+Dorwt. The autophagy reporter Ref(2)P (green) was stained (G'-H'). Scale bar: 20 μm. (J, K, L) The percentages of the vacuole areas in the lamina of (A-C, D-F, G-I) were summarized, respectively. The adults were shifted to 28°C for 7 days. DAPI: nuclei (white in A-I). All P-values were calculated using Kruskal-Wallis tests with Dunn's post-tests.


B , Multiplexed activation of Ascl1 , Lmx1a , NeuroD1 and of Ascl1 , Lmx1a , Nr4a2 in primary astrocytic cultures . n = 3 , representative experiment from 2 - 3 independent experiments , additional data in supplement . Activation levels are depicted as fold change between cells transfected with and without sgRNAs . All levels were normalized to β - Actin . Error bars represent mean ± SD between technical replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Multiplexed activation of Ascl1, Lmx1a, NeuroD1 and of Ascl1, Lmx1a, Nr4a2 in primary astrocytic cultures. n=3, representative experiment from 2-3 independent experiments, additional data in supplement. Activation levels are depicted as fold change between cells transfected with and without sgRNAs. All levels were normalized to β-Actin. Error bars represent mean ± SD between technical replicates.


A , Representative photomicrographs taken from the dorsal striatum 13 weeks after AAV - injection . In mice injected with GFP control virus , virtually all GFP positive cells depict an astrocytic morphology , many GFP positive cells in ALNe - 218 and ALN - treated animals show a neuron - like morphology . Data information : Scale bars indicate 20 µm .

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

A, Representative photomicrographs taken from the dorsal striatum 13 weeks after AAV-injection. In mice injected with GFP control virus, virtually all GFP positive cells depict an astrocytic morphology, many GFP positive cells in ALNe-218 and ALN-treated animals show a neuron-like morphology. Data information: Scale bars indicate 20 µm.


B , Immunohistochemical analysis showing GFP + / GFAP + double positive cells 13 wpi . Arrows indicate double positive GFP + / GFAP + cells , arrowheads indicate GFP + / GFAP - cells . Quantification of GFAP + / GFP + cells shows a significant decrease upon ALNe - 218 and ALN - treatment ( GFP vs . ALNe - 218 P = 0 . 0064 , GFP vs . ALN P = 0 . 0002 and ALN vs . ALNe - 218 P = 0 . 0127 ) . Multiple comparison ANOVA F ( 2 , 7 ) = 32 . 06 . C , Double immunostaining for GFP and the neuronal marker NeuN . Arrowheads indicate double positive GFP + / NeuN + cell 13 wpi . Quantification demonstrate a significant increase in NeuN + / GFP + cells upon ALNe - 218 and ALN - induction ( GFP vs . ALNe - 218 P < 0 . 0001 , GFP vs . ALN P < 0 . 0001 and ALN vs . ALNe - 218 P = 0 . 0012 ) . Multiple comparison ANOVA F ( 2 , 6 ) = 170 . 3 . Data information : Scale bars indicate 20 µm . Tukey ' s multiple comparisons test * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . n = 3 - 4 mice per condition . Error bars represent mean ± SD .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Immunohistochemical analysis showing GFP+/GFAP+ double positive cells 13 wpi. Arrows indicate double positive GFP+/GFAP+ cells, arrowheads indicate GFP+/GFAP- cells. Quantification of GFAP+/GFP+ cells shows a significant decrease upon ALNe-218 and ALN-treatment (GFP vs. ALNe-218 P=0.0064, GFP vs. ALN P=0.0002 and ALN vs. ALNe-218 P=0.0127). Multiple comparison ANOVA F(2,7)=32.06. C, Double immunostaining for GFP and the neuronal marker NeuN. Arrowheads indicate double positive GFP+/NeuN+ cell 13 wpi. Quantification demonstrate a significant increase in NeuN+/GFP+ cells upon ALNe-218 and ALN-induction (GFP vs. ALNe-218 P<0.0001, GFP vs. ALN P<0.0001 and ALN vs. ALNe-218 P=0.0012). Multiple comparison ANOVA F(2,6)=170.3. Data information: Scale bars indicate 20 µm. Tukey's multiple comparisons test * P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. n = 3-4 mice per condition. Error bars represent mean ± SD.


D , Double immunostaining for GFP and TH as a marker for dopaminergic neurons in the dorsal striatum of GFP - control , ALNe - 218 and ALN - treated animals . Arrowheads indicate GFP + / TH - cells with neuronal morphology . No GFP + / TH + cell could be detected in any of the experimental groups . Scale bars indicate 20 µm . Data information : Scale bars indicate 20 µm .

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

D, Double immunostaining for GFP and TH as a marker for dopaminergic neurons in the dorsal striatum of GFP-control, ALNe-218 and ALN-treated animals. Arrowheads indicate GFP+/TH- cells with neuronal morphology. No GFP+/TH+ cell could be detected in any of the experimental groups. Scale bars indicate 20 µm. Data information: Scale bars indicate 20 µm.


B , RT - qPCR analysis of Ascl1 induction comparing the activation capacity of full - length versus split - dCas9 in Neuro2A cells ( data in fold change normalized to non - activated control ; dCas9 6116 ± 847 . 3 , split - dCas9 4415 ± 748 . 8 , n = 3 biological replicates , box - and - whiskers plots indicate median , 25th - 75th percentile and min / max whiskers ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, RT-qPCR analysis of Ascl1 induction comparing the activation capacity of full-length versus split-dCas9 in Neuro2A cells (data in fold change normalized to non-activated control; dCas9 6116 ± 847.3, split-dCas9 4415 ± 748.8, n=3 biological replicates, box-and-whiskers plots indicate median, 25th-75th percentile and min/max whiskers).


C , Immunocytochemistry analysis of reprogrammed primary astrocytes cells 16 days after lentiviral transduction revealed successful in vitro reprogramming into neurons using CRISPRa . Astrocytes are infected with two lentiviruses expressing dCas9 - VPR in a intein - split version similar to the AAV - dCAS system but driven by a Tet - O promoter . Tet - O driven dsRed and Ascl1 cDNA expressing construct were used as negative and positive controls respectively . Arrows indicate single MAP2 positive background neurons , arrowheads indicate double positive induced neurons . Scale bar indicates 20 µm . Data information : Abbreviations : OE -

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

C, Immunocytochemistry analysis of reprogrammed primary astrocytes cells 16 days after lentiviral transduction revealed successful in vitro reprogramming into neurons using CRISPRa. Astrocytes are infected with two lentiviruses expressing dCas9-VPR in a intein-split version similar to the AAV-dCAS system but driven by a Tet-O promoter. Tet-O driven dsRed and Ascl1 cDNA expressing construct were used as negative and positive controls respectively. Arrows indicate single MAP2 positive background neurons, arrowheads indicate double positive induced neurons. Scale bar indicates 20 µm. Data information: Abbreviations: OE - overexpression


E , Multiplexed activation of Ascl1 / Lmx1a / NeuroD1 , Ascl1 / Lmx1a / Nr4a2 and Ascl1 / Lmx1a / Nr4a2 / Pitx3 / FoxA2 in primary astrocytic cells . n = 2 - 3 biological replicates , one representative run is shown , additional data in supplement . Activation levels are depicted as fold change between cells transfected with and without sgRNAs . All levels were normalized to β - Actin . Error bars represent mean ± SD between technical replicates .

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

E, Multiplexed activation of Ascl1/Lmx1a/NeuroD1, Ascl1/Lmx1a/Nr4a2 and Ascl1/ Lmx1a/ Nr4a2/Pitx3/FoxA2 in primary astrocytic cells. n=2-3 biological replicates, one representative run is shown, additional data in supplement. Activation levels are depicted as fold change between cells transfected with and without sgRNAs. All levels were normalized to β-Actin. Error bars represent mean ± SD between technical replicates.


A , Representative photomicrographs taken from the dorsal striatum 13 weeks after AAV - injection . In the GFP control condition , virtually all GFP positive cells depict an astrocytic morphology , many GFP positive cells in ALNe - 218 and ALN - treated animals show a neuron - like morphology . Data information : Scale bars indicate 20 µm .

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

A, Representative photomicrographs taken from the dorsal striatum 13 weeks after AAV-injection. In the GFP control condition, virtually all GFP positive cells depict an astrocytic morphology, many GFP positive cells in ALNe-218 and ALN-treated animals show a neuron-like morphology. Data information: Scale bars indicate 20 µm.


B , Immunohistochemical analysis showing GFP + / GFAP + double positive cells 13 wpi . Arrows indicate double positive GFP + / GFAP + cells , arrowheads indicate GFP + / GFAP - cells . Quantification of GFAP + / GFP + cells shows a significant decrease upon ALNe - 218 and ALN - treatment ( GFP vs . ALNe - 218 P = 0 . 0083 , GFP vs . ALN P < 0 . 0001 and ALN vs . ALNe - 218 P = 0 . 0006 , multiple comparison ANOVA F ( 2 , 6 ) = 79 . 76 ) . C , Photomicrographs showing GFP + / NeuN + neurons 13 wpi . Arrowheads indicating GFP + / NeuN + induced neurons . Quantification demonstrate a significant increase in NeuN + / GFP + cells upon ALNe - 218 and ALN - induction ( GFP vs . ALN P = 0 . 0008 , ALN vs . ALNe - 218 P = 0 . 0092 , multiple comparison ANOVA F ( 2 , 7 ) = 21 . 74 ) . Data information : Scale bars indicate 20 µm . Error bars represent mean ± SD . n = 3 - 4 mice ( b , c ) per condition . Tukey ' s multiple comparisons test * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Immunohistochemical analysis showing GFP+/GFAP+ double positive cells 13 wpi. Arrows indicate double positive GFP+/GFAP+ cells, arrowheads indicate GFP+/GFAP- cells. Quantification of GFAP+/GFP+ cells shows a significant decrease upon ALNe-218 and ALN-treatment (GFP vs. ALNe-218 P=0.0083, GFP vs. ALN P<0.0001 and ALN vs. ALNe-218 P=0.0006, multiple comparison ANOVA F(2,6)=79.76). C, Photomicrographs showing GFP+/NeuN+ neurons 13 wpi. Arrowheads indicating GFP+/NeuN+ induced neurons. Quantification demonstrate a significant increase in NeuN+/GFP+ cells upon ALNe-218 and ALN-induction (GFP vs. ALN P=0.0008, ALN vs. ALNe-218 P=0.0092, multiple comparison ANOVA F(2,7)=21.74). Data information: Scale bars indicate 20 µm. Error bars represent mean ± SD. n = 3-4 mice (b,c) per condition. Tukey's multiple comparisons test * P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.


D , In ALN - induced neurons , confocal analysis is demonstrating a co - localization of GFP and glutamic acid decarboxylase ( Gad65 / 67 ) , a marker specific for GABAergic neurons . E , Quantification of GAD6567 + / GFP + cells with neuronal morphology shows that the majority of ALN - induced neurons are colocalizing with this GABAergic marker both in the AAV - dCAS as well as in the dCAM setting . Data information : Scale bars indicate 20 µm . Error bars represent mean ± SD . n = 6 - 8 mice ( e ) per condition . Tukey ' s multiple comparisons test * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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

D, In ALN-induced neurons, confocal analysis is demonstrating a co-localization of GFP and glutamic acid decarboxylase (Gad65/67), a marker specific for GABAergic neurons. E, Quantification of GAD6567+/GFP+ cells with neuronal morphology shows that the majority of ALN-induced neurons are colocalizing with this GABAergic marker both in the AAV-dCAS as well as in the dCAM setting. Data information: Scale bars indicate 20 µm. Error bars represent mean ± SD. n= 6-8 mice (e) per condition. Tukey's multiple comparisons test * P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.


A , Gait analysis using the CatWalk XT system reveals motor defects in 6 - OHDA lesion model . Both dCAM and AAV - dCAS animals transduce with AAV containing specific gRNAs show a significant improvement in different aspects of voluntary movement like the average speed of tread . Naive vs . 6 - OHDA P = 0 . 0063 . AAV - dCAS : GFP vs . ALN P = 0 . 015 multiple comparison ANOVA F ( 2 , 17 ) = 12 . 81 ) Data information : Statistics : naive vs . 6 - OHDA unpaired t - test ( two - tailed ) * P < 0 . 05 , * * P < 0 . 01 . GFP vs ALN , GFP vs ALNe - 218 and ALN vs ALNe - 218 Tukey ' s multiple comparisons test * P < 0 . 05 , * * P < 0 . 01 . n = 4 - 8 mice per condition .

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

A, Gait analysis using the CatWalk XT system reveals motor defects in 6-OHDA lesion model. Both dCAM and AAV-dCAS animals transduce with AAV containing specific gRNAs show a significant improvement in different aspects of voluntary movement like the average speed of tread. Naive vs. 6-OHDA P=0.0063. AAV-dCAS: GFP vs. ALN P=0.015 multiple comparison ANOVA F(2,17)=12.81) Data information: Statistics: naive vs. 6-OHDA unpaired t-test (two-tailed) * P<0.05, ** P<0.01. GFP vs ALN, GFP vs ALNe-218 and ALN vs ALNe-218 Tukey's multiple comparisons test * P<0.05, ** P<0.01. n = 4-8 mice per condition.


the stride length of front paws : B , Naive vs . 6 - OHDA P = 0 . 0164 . AAV - dCAS : GFP vs . ALN P = 0 . 005 , ALN vs . ALNe - 218 P = 0 . 0042 , multiple comparison ANOVA F ( 4 , 22 ) = 9 . 9 ) Data information : Statistics : naive vs . 6 - OHDA unpaired t - test ( two - tailed ) * P < 0 . 05 , * * P < 0 . 01 . GFP vs ALN , GFP vs ALNe - 218 and ALN vs ALNe - 218 Tukey ' s multiple comparisons test * P < 0 . 05 , * * P < 0 . 01 . n = 4 - 8 mice per condition .

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

the stride length of front paws: B, Naive vs. 6-OHDA P=0.0164. AAV-dCAS: GFP vs. ALN P=0.005, ALN vs. ALNe-218 P=0.0042, multiple comparison ANOVA F(4,22)=9.9) Data information: Statistics: naive vs. 6-OHDA unpaired t-test (two-tailed) * P<0.05, ** P<0.01. GFP vs ALN, GFP vs ALNe-218 and ALN vs ALNe-218 Tukey's multiple comparisons test * P<0.05, ** P<0.01. n = 4-8 mice per condition.


the duty cycle of left front paws . C , Naive vs . 6 - OHDA P = 0 . 0096 . dCAM : GFP vs . ALN P = 0 . 036 and ALN vs . ALNe - 218 P = 0 . 0252 , multiple comparison ANOVA F ( 2 , 20 ) = 5 . 199 ) . Data information : Statistics : naive vs . 6 - OHDA unpaired t - test ( two - tailed ) * P < 0 . 05 , * * P < 0 . 01 . GFP vs ALN , GFP vs ALNe - 218 and ALN vs ALNe - 218 Tukey ' s multiple comparisons test * P < 0 . 05 , * * P < 0 . 01 . n = 4 - 8 mice per condition .

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

the duty cycle of left front paws. C, Naive vs. 6-OHDA P=0.0096. dCAM: GFP vs. ALN P=0.036 and ALN vs. ALNe-218 P=0.0252, multiple comparison ANOVA F(2,20)=5.199). Data information: Statistics: naive vs. 6-OHDA unpaired t-test (two-tailed) * P<0.05, ** P<0.01. GFP vs ALN, GFP vs ALNe-218 and ALN vs ALNe-218 Tukey's multiple comparisons test * P<0.05, ** P<0.01. n = 4-8 mice per condition.


D , In contrast to this , dopamine dependent drug - induced behavior does not show rescue effects : amphetamine - induced rotation analysis : change in rotational behavior in lesioned animals upon treatment with dopamine releaser substance . Net rotation = ipsilateral rotation - contralateral rotation ( naïve vs . 6 - OHDA P = 0 . 09 ) . Data information : Statistics : naive vs . 6 - OHDA unpaired t - test ( two - tailed ) * P < 0 . 05 , * * P < 0 . 01 . GFP vs ALN , GFP vs ALNe - 218 and ALN vs ALNe - 218 Tukey ' s multiple comparisons test * P < 0 . 05 , * * P < 0 . 01 . n = 4 - 8 mice per condition . CatWalk error bars represent mean ± SD . Rotation analysis error bars represent mean ± SEM .

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

D, In contrast to this, dopamine dependent drug-induced behavior does not show rescue effects: amphetamine-induced rotation analysis: change in rotational behavior in lesioned animals upon treatment with dopamine releaser substance. Net rotation = ipsilateral rotation-contralateral rotation (naïve vs. 6-OHDA P=0.09). Data information: Statistics: naive vs. 6-OHDA unpaired t-test (two-tailed) * P<0.05, ** P<0.01. GFP vs ALN, GFP vs ALNe-218 and ALN vs ALNe-218 Tukey's multiple comparisons test * P<0.05, ** P<0.01. n = 4-8 mice per condition. CatWalk error bars represent mean ± SD. Rotation analysis error bars represent mean ± SEM.


( E - G ) Primary cortical neurons from E15 cerebral cortices were transfected with the indicated plasmids , incubated for two days in vitro and treated with Tf - 555 for 30 minutes before fixation . Cells were immunostained with the indicated antibodies . The images are obtained with high - resolution microscopy ( Nikon ) . Blue alone channels are shown in black and white images . Lower panels in ( E ) and ( F ) are high magnification images indicated by white or blue rectangles in upper panels . The graph in ( G ) shows the Pearson ' s correlation coefficient of Tf - 555 and Rab11 or APPL1 . Each score represents the mean with the individual points . Control : n = 23 cells , Rab21 - sh115 : n = 18 cells , Rab5 - sh232 ( Tf - Rab11 ) : n = 23 cells , Rab5 - sh232 ( Tf - APPL1 ) : n = 22 cells .

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

(E-G) Primary cortical neurons from E15 cerebral cortices were transfected with the indicated plasmids, incubated for two days in vitro and treated with Tf-555 for 30 minutes before fixation. Cells were immunostained with the indicated antibodies. The images are obtained with high-resolution microscopy (Nikon). Blue alone channels are shown in black and white images. Lower panels in (E) and (F) are high magnification images indicated by white or blue rectangles in upper panels. The graph in (G) shows the Pearson's correlation coefficient of Tf-555 and Rab11 or APPL1. Each score represents the mean with the individual points. Control: n = 23 cells, Rab21-sh115: n = 18 cells, Rab5-sh232 (Tf - Rab11): n = 23 cells, Rab5-sh232 (Tf - APPL1): n = 22 cells.


Cerebral cortices at E17 , electroporated with the indicated plasmids plus pCAG - EGFP at E14 . ( C ) The ratio of locomoting neurons with more than three primary neurites in the IZ . Each score represents the mean with the individual points . Control : n = 4 brains ( 60 cells ) , Rab21 - sh115 : n = 3 brains ( 65 cells ) , Rab5 - sh232 : n = 4 brains ( 111 cells ) .

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

Cerebral cortices at E17, electroporated with the indicated plasmids plus pCAG-EGFP at E14. (C) The ratio of locomoting neurons with more than three primary neurites in the IZ. Each score represents the mean with the individual points. Control: n = 4 brains (60 cells), Rab21-sh115: n = 3 brains (65 cells), Rab5-sh232: n = 4 brains (111 cells).


( E ) Time - lapse imaging of control and Rab21 - sh115 - electroporated cells in cortical slices from E16 cerebral cortices , electroporated with the indicated plasmids at E14 . After formation of the leading process , control neurons rapidly eliminated their immature neurites ( yellow arrows ) , whereas the Rab21 - knockdown neurons retained the immature neurites ( white arrows ) for long periods . Time interval of each frame is 30 min .

(E) Time-lapse imaging of control and Rab21-sh115-electroporated cells in cortical slices from E16 cerebral cortices, electroporated with the indicated plasmids at E14. After formation of the leading process, control neurons rapidly eliminated their immature neurites (yellow arrows), whereas the Rab21-knockdown neurons retained the immature neurites (white arrows) for long periods. Time interval of each frame is 30 min.


Primary cortical neurons from E15 cerebral cortices were transfected with pCAG - PM - mAG1 ( green ) and incubated for two days in vitro . Cells were immunostained with the indicated antibodies ( red and blue / white ) . The transfected PM - mAG1 is a marker for the plasma membrane . The images are obtained with high - resolution microscopy ( Nikon ) and the lower panels are high magnification images near the plasma membrane . Blue alone channels are shown in black and white images . Each score represents the mean of ratios with the individual points . Rab5 : n = 18 cells ( middle and right ) , Rab21 : n = 31 cells ( left and middle ) or 20 cells ( right ) , Caveolin - 1 : n = 43 cells ( left ) .

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

Primary cortical neurons from E15 cerebral cortices were transfected with pCAG-PM-mAG1 (green) and incubated for two days in vitro. Cells were immunostained with the indicated antibodies (red and blue/white). The transfected PM-mAG1 is a marker for the plasma membrane. The images are obtained with high-resolution microscopy (Nikon) and the lower panels are high magnification images near the plasma membrane. Blue alone channels are shown in black and white images. Each score represents the mean of ratios with the individual points. Rab5: n = 18 cells (middle and right), Rab21: n = 31 cells (left and middle) or 20 cells (right), Caveolin-1: n = 43 cells (left).


( H - I ) HeLa cells were immunostained with the indicated antibodies . The images are obtained with high - resolution microscopy ( Nikon ) . The graph in ( I ) shows the Pearson ' s correlation coefficient between caveolin - 1 and Rab5 or Rab21 . Each score represents the mean of ratios with the individual points . Rab5 : n = 80 cells , Rab21 : n = 62 cells .

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

(H-I) HeLa cells were immunostained with the indicated antibodies. The images are obtained with high-resolution microscopy (Nikon). The graph in (I) shows the Pearson's correlation coefficient between caveolin-1 and Rab5 or Rab21. Each score represents the mean of ratios with the individual points. Rab5: n = 80 cells, Rab21: n = 62 cells.


Primary cortical neurons from E15 cerebral cortices were transfected with the indicated plasmids plus pCAG - EGFP , incubated for two days in vitro and treated with BODIPY - LacCer ( LacCer ) ( green ) ( A - B ) for 30 minutes before fixation . White arrows in ( A ) indicate the perinuclear accumulation of LacCer . Blue alone channels are shown in black and white images . The graphs in ( B ) show the ratio of cells with perinuclear accumulation of LacCer ( B ) which was quantified in a blinded counting . Each score represents the mean with the individual points . B : n = 5 biological replicates ( Control : 115 cells , Cav1 - sh490 : 127 cells , Rab21 - sh115 : 136 cells , Rab5 - sh232 : 216 cells , Rab21 - sh115 + wt - Rab21 : 132 cells )

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

Primary cortical neurons from E15 cerebral cortices were transfected with the indicated plasmids plus pCAG-EGFP , incubated for two days in vitro and treated with BODIPY-LacCer (LacCer) (green) (A-B) for 30 minutes before fixation. White arrows in (A) indicate the perinuclear accumulation of LacCer. Blue alone channels are shown in black and white images. The graphs in (B) show the ratio of cells with perinuclear accumulation of LacCer (B) which was quantified in a blinded counting. Each score represents the mean with the individual points. B: n = 5 biological replicates (Control: 115 cells, Cav1-sh490: 127 cells, Rab21-sh115: 136 cells, Rab5-sh232: 216 cells, Rab21-sh115 + wt-Rab21: 132 cells)


Primary cortical neurons from E15 cerebral cortices were transfected with pCAG - PM - mAG1 , incubated for two days in vitro and stained with the indicated antibodies . Blue alone channels are shown in black and white images . Arrowheads indicate accumulation of N - cadherin at the plasma membrane . Lower panels in ( D ) are high magnification images indicated by white or blue rectangles . The images are obtained with high - resolution microscopy ( Nikon ) .

Primary cortical neurons from E15 cerebral cortices were transfected with pCAG-PM-mAG1, incubated for two days in vitro and stained with the indicated antibodies. Blue alone channels are shown in black and white images. Arrowheads indicate accumulation of N-cadherin at the plasma membrane. Lower panels in (D) are high magnification images indicated by white or blue rectangles. The images are obtained with high-resolution microscopy (Nikon).


Immature neurons in the IZ of the cerebral cortices at E17 , electroporated with the indicated plasmids plus pCAG - PM - mAG1 and pCAG - HA - N - cadherin at E14 . Frozen sections were immunostained with anti - mAG1 and anti - HA antibodies . Arrow and arrowheads indicate the accumulation of HA - N - cadherin at the perinuclear regions or the plasma membrane , respectively . Control : n = 77 cells from 3 biological replicates , Rab21 - sh115 : n = 108 cells from 3 biological replicates .

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

Immature neurons in the IZ of the cerebral cortices at E17, electroporated with the indicated plasmids plus pCAG-PM-mAG1 and pCAG-HA-N-cadherin at E14. Frozen sections were immunostained with anti-mAG1 and anti-HA antibodies. Arrow and arrowheads indicate the accumulation of HA-N-cadherin at the perinuclear regions or the plasma membrane, respectively. Control: n = 77 cells from 3 biological replicates, Rab21-sh115: n = 108 cells from 3 biological replicates.