( A ) Flow cytometry analysis of l ( 2 ) mbn cells starved for 2 h ( 2h S ) or 4 h ( 4h S ) showed an increase in LTG fluorescence levels ( x axis ) compared with control cells in full - nutrient medium ( C ) . The gate shown on the histogram represents the LTGhigh population . ( B ) Representative images of GFP - LC3 puncta in control and 2 - h starved l ( 2 ) mbn cells . An increase in GFP - LC3 puncta was observed in the starved cells ( left ) . Bar , 10 μm . ( C ) Flow cytometry analysis of 4 - h starved cells were incubated with 3MA ( 4h S + 3MA ) and Baf ( 4h S + Baf ) . Both autophagy inhibitors reduced the LTG fluorescence levels compared with starved cells ( 4h S ) . Control cells in nutrient - full medium ( C ) are represented by the brown line . ( D ) Both autophagy inhibitors , 3MA and Baf , reduced the LTGhigh population significantly . ( 3MA , P = 0 . 00001 ; and Baf , P = 0 . 00006 ) .

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

(A) Flow cytometry analysis of l(2)mbn cells starved for 2 h (2h S) or 4 h (4h S) showed an increase in LTG fluorescence levels (x axis) compared with control cells in full-nutrient medium (C). The gate shown on the histogram represents the LTGhigh population. (B) Representative images of GFP-LC3 puncta in control and 2-h starved l(2)mbn cells. An increase in GFP-LC3 puncta was observed in the starved cells (left). Bar, 10 μm. (C) Flow cytometry analysis of 4-h starved cells were incubated with 3MA (4h S + 3MA) and Baf (4h S + Baf). Both autophagy inhibitors reduced the LTG fluorescence levels compared with starved cells (4h S). Control cells in nutrient-full medium (C) are represented by the brown line. (D) Both autophagy inhibitors, 3MA and Baf, reduced the LTGhigh population significantly. (3MA, P = 0.00001; and Baf, P = 0.00006).


( E ) RNAi of representative Atg genes decreased the LTR fluorescence levels compared with control . ( Atg1 , P = 0 . 01 ; Atg5 , P = 0 . 01 ; Atg7 , P = 0 . 002 ; Atg8a , P = 0 . 008 ; Atg8b , P = 0 . 006 ; and Atg12 , P = 0 . 02 ) .

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(E) RNAi of representative Atg genes decreased the LTR fluorescence levels compared with control. (Atg1, P = 0.01; Atg5, P = 0.01; Atg7, P = 0.002; Atg8a, P = 0.008; Atg8b, P = 0.006; and Atg12, P = 0.02).


( F ) RNAi of all tested genes in the TOR - PI3K pathways had a statistically significant effect on LTG fluorescence levels . Known negative regulators of autophagy are shown with gray bars ; positive regulators are shown with white bars . ( Pten , P = 0 . 007 ; Tsc1 , P = 0 . 027 ; Tsc2 , P = 0 . 025 ; RheB , P = 0 . 005 ; Tor , P = 0 . 016 ; and S6k , P = 0 . 012 ) . dsRNA corresponding to a human gene ( Hs ) was used as a negative control in E and F . Results represent the mean value ± SD from at least three independent experiments .

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(F) RNAi of all tested genes in the TOR-PI3K pathways had a statistically significant effect on LTG fluorescence levels. Known negative regulators of autophagy are shown with gray bars; positive regulators are shown with white bars. (Pten, P = 0.007; Tsc1, P = 0.027; Tsc2, P = 0.025; RheB, P = 0.005; Tor, P = 0.016; and S6k, P = 0.012). dsRNA corresponding to a human gene (Hs) was used as a negative control in E and F. Results represent the mean value ± SD from at least three independent experiments.


( A ) The percentage of LTGhigh cells was reduced by hid - RNAi ( P = 0 . 006 ) but not by rpr , grim , and skl - RNAi .

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(A) The percentage of LTGhigh cells was reduced by hid-RNAi (P = 0.006) but not by rpr, grim, and skl-RNAi.


( B ) Knockdown of Ras , phl , and rl expression by RNAi resulted in an increase in the percentage of LTGhigh cells . ( Ras , P = 0 . 003 ; phl , P = 0 . 001 ; and rl , P = 0 . 028 ) .

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(B) Knockdown of Ras, phl, and rl expression by RNAi resulted in an increase in the percentage of LTGhigh cells. (Ras, P = 0.003; phl, P = 0.001; and rl, P = 0.028).


( C ) th - RNAi treatment ( 24 h ) had no significant effect on LTG levels ; in contrast , RNAi of Bruce resulted in an increase in LTG fluorescence levels ( P = 0 . 01 ) .

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(C) th-RNAi treatment (24 h) had no significant effect on LTG levels; in contrast, RNAi of Bruce resulted in an increase in LTG fluorescence levels (P = 0.01).


( D ) Reduction of debcl , Buffy , or p53 expression by RNAi resulted in a decrease in LTG fluorescence levels . ( debcl , P = 0 . 018 ; Buffy , P = 0 . 006 ; and p53 , P = 0 . 004 ) .

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(D) Reduction of debcl, Buffy, or p53 expression by RNAi resulted in a decrease in LTG fluorescence levels. (debcl, P = 0.018; Buffy, P = 0.006; and p53, P = 0.004).


( E ) RNAi of effector caspase Dcp - 1 resulted in a significant decrease in the LTGhigh population ( P = 0 . 001 ) .

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(E) RNAi of effector caspase Dcp-1 resulted in a significant decrease in the LTGhigh population (P = 0.001).


( F ) Representative images of GFP - LC3 puncta in cells treated with the indicated RNAi after a 2 - h starvation treatment . Bar , 10 μm .

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(F) Representative images of GFP-LC3 puncta in cells treated with the indicated RNAi after a 2-h starvation treatment. Bar, 10 μm.


( G ) Quantification of cells with GFP - LC3 puncta after RNAi treatment . Cells with more than three GFP - LC3 punctate dots were considered to be GFP - LC3 - positive cells . Cells treated with the RNAi indicated here all showed a significant difference ( P < 0 . 05 ) in the percentage of GFP - LC3 - positive cells compared with the human ( Hs ) RNAi control . ( Pten , P = 0 . 006 ; Tor , P = 0 . 034 ; Buffy , P = 0 . 005 ; debcl , P = 0 . 003 ; Bruce , P = 0 . 003 ; Dcp - 1 , P = 0 . 007 ; hid , P = 0 . 002 ; Ras , P = 0 . 006 ; and phl , P = 0 . 050 ) . Results represent the mean value ± SD from three independent experiments .

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(G) Quantification of cells with GFP-LC3 puncta after RNAi treatment. Cells with more than three GFP-LC3 punctate dots were considered to be GFP-LC3-positive cells. Cells treated with the RNAi indicated here all showed a significant difference (P < 0.05) in the percentage of GFP-LC3-positive cells compared with the human (Hs) RNAi control. (Pten, P = 0.006; Tor, P = 0.034; Buffy, P = 0.005; debcl, P = 0.003; Bruce, P = 0.003; Dcp-1, P = 0.007; hid, P = 0.002; Ras, P = 0.006; and phl, P = 0.050). Results represent the mean value ± SD from three independent experiments.


( A ) GFP - LC3 proteins were expressed in nurse cells ( NC ) but not in follicle cells ( FC ) by using the UASp / nanos Gal4 system . DAPI staining of nuclei is shown in blue .

(A) GFP-LC3 proteins were expressed in nurse cells (NC) but not in follicle cells (FC) by using the UASp/nanos Gal4 system. DAPI staining of nuclei is shown in blue.


( B ) UASp - GFP - LC3 ; nanos GAL4 flies were conditioned on yeast paste and had a diffuse GFP - LC3 pattern . Numerous GFP - LC3 puncta ( green ) at region 2 within germarium were observed in nutrient - deprived flies . Ovaries were stained with LTR in w1118 flies . Germarium of nutrient - deprived w1118 flies had an increase in punctate LTR staining ( red ) compared with well - fed germarium .

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(B) UASp-GFP-LC3; nanos GAL4 flies were conditioned on yeast paste and had a diffuse GFP-LC3 pattern. Numerous GFP-LC3 puncta (green) at region 2 within germarium were observed in nutrient-deprived flies. Ovaries were stained with LTR in w1118 flies. Germarium of nutrient-deprived w1118 flies had an increase in punctate LTR staining (red) compared with well-fed germarium.


( C ) Degenerating stage 8 egg chambers ( arrows ) had numerous GFP - LC3 puncta ( green ) and an increase in LTR - positive dots ( red ) compared with healthy egg chambers ( arrowheads ) . DAPI ( blue ) staining of nuclei is shown in the two panels on the right .

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(C) Degenerating stage 8 egg chambers (arrows) had numerous GFP-LC3 puncta (green) and an increase in LTR-positive dots (red) compared with healthy egg chambers (arrowheads). DAPI (blue) staining of nuclei is shown in the two panels on the right.


( D ) Degenerating stage 8 egg chambers ( arrows ) of nutrient - deprived Atg7 mutants ( Atg7d77 / Atg7d14 ) showed a dramatic decrease in LTR staining . DAPI staining of nuclei is shown in blue .

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(D) Degenerating stage 8 egg chambers (arrows) of nutrient-deprived Atg7 mutants (Atg7d77/Atg7d14) showed a dramatic decrease in LTR staining. DAPI staining of nuclei is shown in blue.


( A ) Germaria of the nutrient - deprived Dcp - 1Prev flies showed a dramatic decrease in LTR staining compared with nutrient - deprived wild - type flies shown in Fig . 3 B . DAPI staining of nuclei is shown in blue . ( B ) Degenerating stage 8 egg chambers ( arrows ) of nutrient - deprived Dcp - 1Prev flies showed a dramatic decrease in LTR staining compared with nutrient - deprived wild - type flies shown in Fig . 3 C .

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(A) Germaria of the nutrient-deprived Dcp-1Prev flies showed a dramatic decrease in LTR staining compared with nutrient-deprived wild-type flies shown in Fig. 3 B. DAPI staining of nuclei is shown in blue. (B) Degenerating stage 8 egg chambers (arrows) of nutrient-deprived Dcp-1Prev flies showed a dramatic decrease in LTR staining compared with nutrient-deprived wild-type flies shown in Fig. 3 C.


( C ) Lack of Dcp - 1 function ( UASp - GFP - LC3 Dcp - 1Prev / Dcp - 1Prev ; nanos - GAL4 / + ) resulted in uniform diffuse staining of GFP - LC3 rather than the punctate pattern observed in wild - type degenerating stage 8 egg chambers shown in Fig 3 C . ( D ) Dying egg chambers ( arrows ) of NGT / + ; nanos - GAL4 / UASp - fl - Dcp - 1 flies that were conditioned on yeast paste showed a significant increase in punctate LTR staining ( red ) compared with healthy egg chambers ( arrowheads ) . ( E ) Expression of activated Dcp - 1 ( a truncated form ) and GFP - LC3 in the germ line ( UASp - GFP - LC3 / + ; nanos - GAL4 / nanos - GAL4 UASp - tDcp - 1 ) resulted in abundant degenerating stage 8 egg chambers ( arrows ) with numerous GFP - LC3 puncta ( green ) . DAPI staining of nuclei is shown in blue .

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(C) Lack of Dcp-1 function (UASp-GFP-LC3 Dcp-1Prev/Dcp-1Prev; nanos-GAL4/+) resulted in uniform diffuse staining of GFP-LC3 rather than the punctate pattern observed in wild-type degenerating stage 8 egg chambers shown in Fig 3 C. (D) Dying egg chambers (arrows) of NGT/+; nanos-GAL4/UASp-fl-Dcp-1 flies that were conditioned on yeast paste showed a significant increase in punctate LTR staining (red) compared with healthy egg chambers (arrowheads). (E) Expression of activated Dcp-1 (a truncated form) and GFP-LC3 in the germ line (UASp-GFP-LC3/+; nanos-GAL4/nanos-GAL4 UASp-tDcp-1) resulted in abundant degenerating stage 8 egg chambers (arrows) with numerous GFP-LC3 puncta (green). DAPI staining of nuclei is shown in blue.


( A ) The germarium in well - fed BruceE81 flies showed an increase in LTR staining ( red ) compared with wild - type well - fed flies shown in Fig . 3 B ( top right ) . DAPI staining ( white ) of nuclei is shown on the right . ( B ) In well - fed wild - type flies , mid - oogenesis nurse cell death is a rare event . Lack of Bruce function resulted in an increase in dying stage 8 egg chambers ( arrows ) in ovaries under well - fed conditions , and these degenerating stage 8 egg chambers had numerous LTR ( red ) punctate dots . DAPI staining ( white ) is shown on the right . Bars : ( A ) 20 μm ; ( B ) 50 μm .

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(A) The germarium in well-fed BruceE81 flies showed an increase in LTR staining (red) compared with wild-type well-fed flies shown in Fig. 3 B (top right). DAPI staining (white) of nuclei is shown on the right. (B) In well-fed wild-type flies, mid-oogenesis nurse cell death is a rare event. Lack of Bruce function resulted in an increase in dying stage 8 egg chambers (arrows) in ovaries under well-fed conditions, and these degenerating stage 8 egg chambers had numerous LTR (red) punctate dots. DAPI staining (white) is shown on the right. Bars: (A) 20 μm; (B) 50 μm.


( A ) Ovaries were stained with TUNEL ( green ) to detect DNA fragmentation . Clusters of cysts with TUNEL staining were observed in region 2 in nutrient - deprived w1118 files . In Dcp - 1Prev flies , fewer TUNEL - positive cysts in region 2 were observed . Under well - fed conditions , numerous TUNEL - positive cysts were observed in BruceE81 flies . DAPI staining of nuclei is shown in white . ( B ) Numerous degenerating stage 8 egg chambers ( arrows ) with Bruce - positive staining ( green ) were observed in well - fed BruceE81 flies . DAPI staining of nuclei ( white ) is shown on the right .

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 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

(A) Ovaries were stained with TUNEL (green) to detect DNA fragmentation. Clusters of cysts with TUNEL staining were observed in region 2 in nutrient-deprived w1118 files. In Dcp-1Prev flies, fewer TUNEL-positive cysts in region 2 were observed. Under well-fed conditions, numerous TUNEL-positive cysts were observed in BruceE81 flies. DAPI staining of nuclei is shown in white. (B) Numerous degenerating stage 8 egg chambers (arrows) with Bruce-positive staining (green) were observed in well-fed BruceE81 flies. DAPI staining of nuclei (white) is shown on the right.


( A ) TUNEL - positive staining was observed in dying stage 8 egg chambers ( arrows ) of starved control flies ( CG5335d30 / Atg7d14 ) . DAPI staining of nuclei ( white ) is shown on the right . ( B ) In nutrient - deprived Atg7 mutants ( Atg7d77 / Atg7d14 ) , degenerating stage 8 egg chambers ( arrows ) showed no or low levels of TUNEL staining . Nuclear DNA condensation , detected by DAPI , was still observed . ( C ) Dying stage 8 egg chambers ( arrows ) from nutrient - deprived control siblings ( Atg1Δ3D / TM3 ) generated from the same cross in D had abundant TUNEL - positive staining . ( D ) In nutrient - deprived Atg1 GLCs , degenerating stage 8 egg chambers ( arrows ) showed no or low levels of TUNEL staining . Nuclear DNA condensation ( DAPI , right ) in degenerating egg chambers appeared to occur as in the controls .

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(A) TUNEL-positive staining was observed in dying stage 8 egg chambers (arrows) of starved control flies (CG5335d30/Atg7d14). DAPI staining of nuclei (white) is shown on the right. (B) In nutrient-deprived Atg7 mutants (Atg7d77/Atg7d14), degenerating stage 8 egg chambers (arrows) showed no or low levels of TUNEL staining. Nuclear DNA condensation, detected by DAPI, was still observed. (C) Dying stage 8 egg chambers (arrows) from nutrient-deprived control siblings (Atg1Δ3D/TM3) generated from the same cross in D had abundant TUNEL-positive staining. (D) In nutrient-deprived Atg1 GLCs, degenerating stage 8 egg chambers (arrows) showed no or low levels of TUNEL staining. Nuclear DNA condensation (DAPI, right) in degenerating egg chambers appeared to occur as in the controls.


CSTB gene expression analysis in hCOs , starting from day ( d ) 16 until d140 . For every time point , at least 3 different samples were analyzed ; each sample was made by a pool of 3 to 4 hCOs . Data are represented as mean ± SEM . Unpaired t - test ( * P < 0 . 05 , * * P < 0 . 01 ) .

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CSTB gene expression analysis in hCOs, starting from day (d) 16 until d140. For every time point, at least 3 different samples were analyzed; each sample was made by a pool of 3 to 4 hCOs. Data are represented as mean ± SEM. Unpaired t-test (*P < 0.05, **P < 0.01).


Micrographs of sections of control hCOs immunostained with CSTB antibody at different time points in culture . The dashed lines represent the apical surface of the cavities / ventricles ( V - like ) . The cortical plate like side is indicated ( CP - like ) . Nuclei ( blue ) are stained with DAPI .

Micrographs of sections of control hCOs immunostained with CSTB antibody at different time points in culture. The dashed lines represent the apical surface of the cavities/ventricles (V-like). The cortical plate like side is indicated (CP-like). Nuclei (blue) are stained with DAPI.


Real time qPCR results of PAX6 , NEUN , NESTIN , CSTB gene expression levels in PAX6 - and NEUN - FACS sorted nuclei from f - CTRL d135 hCOs . Data are represented as mean ± SEM . Statistical significance was based on T - student test ( * P < 0 . 05 , * * P < 0 . 01 ) . For PAX6 , 10 samples were analyzed as biological replicates : 5 for Pax6 + sorted Nuclei and 5 for Neun + sorted Nuclei ; for NEUN , NESTIN and CSTB , 9 samples were analyzed as biological replicates : 5 for Pax6 + sorted Nuclei and 4 for Neun + sorted Nuclei .

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Real time qPCR results of PAX6, NEUN, NESTIN, CSTB gene expression levels in PAX6- and NEUN- FACS sorted nuclei from f-CTRL d135 hCOs. Data are represented as mean ± SEM. Statistical significance was based on T-student test (*P < 0.05, **P < 0.01). For PAX6, 10 samples were analyzed as biological replicates: 5 for Pax6+sorted Nuclei and 5 for Neun+sorted Nuclei; for NEUN, NESTIN and CSTB, 9 samples were analyzed as biological replicates: 5 for Pax6+sorted Nuclei and 4 for Neun+sorted Nuclei.


Western blot analysis and relative quantifications ( arbitrary units , a . u . ) on protein extracts from cortical ( Cx ) and ganglionic eminence ( GE ) tissues for Cstb using Actb as a loading control . Cx and GE tissues from mouse embryos at different time of development as indicated in figure .

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Western blot analysis and relative quantifications (arbitrary units, a.u.) on protein extracts from cortical (Cx) and ganglionic eminence (GE) tissues for Cstb using Actb as a loading control. Cx and GE tissues from mouse embryos at different time of development as indicated in figure.


Micrograph of coronal sections of E16 mouse cerebral cortices immunostained for Cstb and acetylated tubulin ( acTub ) as marker for stabilized microtubules . Ventricle ( V ) and cortical plate ( CP ) are indicated . The dashed lines represent the apical surface of the ventricles . Nuclei ( blue ) are stained with DAPI .

Micrograph of coronal sections of E16 mouse cerebral cortices immunostained for Cstb and acetylated tubulin (acTub) as marker for stabilized microtubules. Ventricle (V) and cortical plate (CP) are indicated. The dashed lines represent the apical surface of the ventricles. Nuclei (blue) are stained with DAPI.


Micrograph sections of d40 hCOs electroporated with GFP - empty vector control or GFP - CSTB and analyzed 5 dpe . Sections were then immunostained for GFP and KI67 . The dashed lines represent the apical surface of the cavities / ventricles ( V - like ) . The cortical plate like side is indicated ( CP - like ) . Quantification of the total number of proliferating KI67 + cells / area ( µm2 ) of V - like structures transfected with GFP - empty vector control or GFP - CSTB in ( A ) .

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Micrograph sections of d40 hCOs electroporated with GFP-empty vector control or GFP-CSTB and analyzed 5 dpe. Sections were then immunostained for GFP and KI67. The dashed lines represent the apical surface of the cavities/ventricles (V-like). The cortical plate like side is indicated (CP-like). Quantification of the total number of proliferating KI67+ cells/area (µm2) of V-like structures transfected with GFP-empty vector control or GFP-CSTB in (A).


Micrograph of coronal sections of E16 mouse cerebral cortices electroporated at E14 with GFP - empty vector or GFP - Cstb and analyzed 2 dpe and immunostained with GFP , Ki67 and BrdU 30 minutes after BrdU injection . Ventricle ( V ) and cortical plate ( CP ) are indicated . The dashed lines represent the apical surface of the ventricles . Quantification of the total number of proliferating Ki67 + cells / area ( µm2 ) ( D ) and Ki67 + Brdu + cells / area ( µm2 ) ( E ) of ventricles transfected with GFP - empty vector or GFP - Cstb in ( C ) .

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Micrograph of coronal sections of E16 mouse cerebral cortices electroporated at E14 with GFP-empty vector or GFP-Cstb and analyzed 2 dpe and immunostained with GFP,Ki67 and BrdU 30 minutes after BrdU injection. Ventricle (V) and cortical plate (CP) are indicated. The dashed lines represent the apical surface of the ventricles. Quantification of the total number of proliferating Ki67+ cells/area(µm2)(D) and Ki67+Brdu+ cells/area(µm2)(E) of ventricles transfected with GFP-empty vector or GFP-Cstb in (C).


Micrograph of coronal sections of E16 mouse cerebral cortices electroporated at E14 with GFP - empty vector or GFP - Cstb , analyzed 2 dpe and immunostained with GFP and Phospho - Histone H3 ( PH3 ) ( F ) and with GFP , Pax6 and Tbr2 ( G ) . Ventricle ( V ) and cortical plate ( CP ) are indicated . The dashed lines represent the apical surface of the ventricles .

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Micrograph of coronal sections of E16 mouse cerebral cortices electroporated at E14 with GFP-empty vector or GFP-Cstb, analyzed 2 dpe and immunostained with GFP and Phospho-Histone H3 (PH3)(F) and with GFP, Pax6 and Tbr2 (G). Ventricle (V) and cortical plate (CP) are indicated. The dashed lines represent the apical surface of the ventricles.


Distribution of proliferating Ki67 + / BrdU + cells ( H ) and mitotic progenitor PH3 + cells ( I ) in the mouse cortex . The cortex was subdivided in 5 equal Bins - Bin1 corresponded to apical side and Bin5 to pial side of the cortex and PH3 + cells or Ki67 + / BrdU + cells calculated in each Bin .

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Distribution of proliferating Ki67+/BrdU+cells (H) and mitotic progenitor PH3+ cells (I) in the mouse cortex. The cortex was subdivided in 5 equal Bins -Bin1 corresponded to apical side and Bin5 to pial side of the cortex and PH3+ cells or Ki67+/BrdU+ cells calculated in each Bin.


Distribution of Tbr2 + intermediate progenitors ( J ) and Pax6 + Tbr2 + cells ( K ) in the mouse cortex . The cortex was subdivided in 5 equal Bins - Bin1 corresponded to apical side and Bin5 to pial side of the cortex and Tbr2 + cells or Pax6 + Tbr2 + cells calculated in each Bin .

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

Distribution of Tbr2+ intermediate progenitors (J) and Pax6+Tbr2+ cells (K) in the mouse cortex. The cortex was subdivided in 5 equal Bins -Bin1 corresponded to apical side and Bin5 to pial side of the cortex and Tbr2+ cells or Pax6+Tbr2+ cells calculated in each Bin.


Western blot analysis for CSTB in the CSF from 2 different E14 mouse embryos ( A ) , in the CM from E14 cortical cells in culture for 4

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

Western blot analysis for CSTB in the CSF from 2 different E14 mouse embryos (A), in the CM from E14 cortical cells in culture for 4 days


Western blot analysis for CSTB in the CM from hCOs after 4 days in culture ( C ) . The CM was collected from hCOs at different time points in culture ( d19 , d38 , d60 , d80 ) . Protein extract ( PE ) from 60 days hCOs was used as a positive control . GAPDH immunostaining was used as a negative control on hCOs CM . Ponceau red staining is shown to indicate the presence of proteins at comparable levels in the CM

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Western blot analysis for CSTB in the CM from hCOs after 4 days in culture (C). The CM was collected from hCOs at different time points in culture (d19, d38, d60, d80). Protein extract (PE) from 60 days hCOs was used as a positive control. GAPDH immunostaining was used as a negative control on hCOs CM. Ponceau red staining is shown to indicate the presence of proteins at comparable levels in the CM samples


Micrograph of coronal sections of E17 mouse cerebral cortices electroporated at E14 co - electroporated with mCherry expressing vector and HA - empty vector or - Cstb , and analyzed 3 dpe . Immunostaining with RFP to identify electroporated cells and GFP to identify migrating interneurons in the GAD65 - GFP transgenic mouse line . Ventricle ( V ) and cortical plate ( CP ) are indicated . The dashed lines represent the apical surface of the ventricles . Distribution of GFP + interneurons in the 5 equal Bins of the mouse developing cortex - Bin1 corresponded to apical side and Bin5 to pial side . Ventricle ( V ) is indicated . The dashed lines represent the apical surface of the ventricles .

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

Micrograph of coronal sections of E17 mouse cerebral cortices electroporated at E14 co-electroporated with mCherry expressing vector and HA-empty vector or -Cstb, and analyzed 3 dpe. Immunostaining with RFP to identify electroporated cells and GFP to identify migrating interneurons in the GAD65-GFP transgenic mouse line. Ventricle (V) and cortical plate (CP) are indicated. The dashed lines represent the apical surface of the ventricles. Distribution of GFP+ interneurons in the 5 equal Bins of the mouse developing cortex-Bin1 corresponded to apical side and Bin5 to pial side. Ventricle (V) is indicated. The dashed lines represent the apical surface of the ventricles.


Micrograph of coronal sections of E17 mouse cerebral cortices electroporated at E14 co - electroporated with mCherry and HA - Cstb expressing vector , analyzed 3 dpe . Immunostaining with RFP to identify electroporated cells and GFP to identify migrating interneurons in the GAD67 - GFP transgenic mouse line . Ventricle ( V ) and cortical plate ( CP ) are indicated . The dashed lines represent the apical surface of the ventricles . Distribution of GFP + interneurons in the 5 equal Bins of the mouse developing cortex comparing electroporated sides with the contralateral ones in the same section to avoid different distribution of interneurons rostro - caudally .

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

Micrograph of coronal sections of E17 mouse cerebral cortices electroporated at E14 co-electroporated with mCherry and HA-Cstb expressing vector, analyzed 3 dpe. Immunostaining with RFP to identify electroporated cells and GFP to identify migrating interneurons in the GAD67-GFP transgenic mouse line. Ventricle (V) and cortical plate (CP) are indicated. The dashed lines represent the apical surface of the ventricles. Distribution of GFP+ interneurons in the 5 equal Bins of the mouse developing cortex comparing electroporated sides with the contralateral ones in the same section to avoid different distribution of interneurons rostro-caudally.


Micrograph of coronal sections of E17 mouse cerebral cortices electroporated at E14 with GFP - miR - neg vector ( CTRL ) and GFP - miRNA - Cstb ( KD ) , analyzed 3 dpe and immunostained with GFP . Ventricle ( V ) and cortical plate ( CP ) are indicated . The dashed lines represent the apical surface of the ventricles . Distribution of electroporated GFP + cells in the mouse cortex . The cortex was subdivided in 5 equal Bins - Bin1 corresponded to apical side and Bin5 to pial side of the cortex and GFP + cells calculated in each Bin . Nuclei ( blue ) are stained with DAPI .

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

Micrograph of coronal sections of E17 mouse cerebral cortices electroporated at E14 with GFP-miR-neg vector (CTRL) and GFP-miRNA-Cstb (KD), analyzed 3 dpe and immunostained with GFP. Ventricle (V) and cortical plate (CP) are indicated. The dashed lines represent the apical surface of the ventricles. Distribution of electroporated GFP+ cells in the mouse cortex. The cortex was subdivided in 5 equal Bins -Bin1 corresponded to apical side and Bin5 to pial side of the cortex and GFP+ cells calculated in each Bin. Nuclei (blue) are stained with DAPI.


Micrograph of coronal sections of E17 mouse cerebral cortices electroporated at E14 with GFP - empty vector control and GFP - miRNA - Cstb , analyzed 3 dpe and immunostained with GFP and Ki67 . Ventricle ( V ) is indicated . The dashed lines represent the apical surface of the ventricles . Quantification of the total number of proliferating Ki67 + cells / area ( µm2 ) of ventricular structures transfected with GFP - miR - neg vector or GFP - miRNA - Cstb in ( C ) . Nuclei ( blue ) are stained with DAPI .

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Micrograph of coronal sections of E17 mouse cerebral cortices electroporated at E14 with GFP-empty vector control and GFP-miRNA-Cstb, analyzed 3 dpe and immunostained with GFP and Ki67. Ventricle (V) is indicated. The dashed lines represent the apical surface of the ventricles. Quantification of the total number of proliferating Ki67+ cells/area (µm2) of ventricular structures transfected with GFP-miR-neg vector or GFP-miRNA- Cstb in (C). Nuclei (blue) are stained with DAPI.


Micrograph sections of d40 hCOs electroporated with GFP - CSTB expressing vector or GFP - R68X mutant and analyzed 5 dpe . Sections were immunostained for GFP and Ki67 . The dashed lines represent the apical surface of the cavities / ventricles ( V - like ) . The cortical plate like side is indicated ( CP - like ) . Quantification of the total number of proliferating KI67 + cells / area ( µm2 ) ( F ) and Ki67 + GFP + cells / area ( µm2 ) ( G ) of ventricles like structures transfected with GFP - empty vector control , GFP - CSTB or GFP - R68X mutant in ( E ) .

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Micrograph sections of d40 hCOs electroporated with GFP-CSTB expressing vector or GFP-R68X mutant and analyzed 5 dpe. Sections were immunostained for GFP and Ki67. The dashed lines represent the apical surface of the cavities/ventricles (V-like). The cortical plate like side is indicated (CP-like). Quantification of the total number of proliferating KI67+ cells/area(µm2)(F)and Ki67+GFP+ cells/area(µm2)(G) of ventricles like structures transfected with GFP-empty vector control, GFP-CSTB or GFP-R68X mutant in (E).


Micrograph of coronal sections of E16 mouse cerebral cortices electroporated at E14 with GFP - empty vector control , GFP - Cstb or GFP - R68X , analyzed 2 dpe and immunostained with GFP and Phospho - Histone H3 ( PH3 ) . Ventricle ( V ) and cortical plate ( CP ) are indicated . The dashed lines represent the apical surface of the ventricles . Distribution of electroporated GFP + cells in the mouse cortex . The cortex was subdivided in 5 equal Bins - Bin1 corresponded to apical side and Bin5 to pial side of the cortex and GFP + cells calculated in each Bin .

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

Micrograph of coronal sections of E16 mouse cerebral cortices electroporated at E14 with GFP-empty vector control, GFP-Cstb or GFP-R68X, analyzed 2 dpe and immunostained with GFP and Phospho-Histone H3 (PH3). Ventricle (V) and cortical plate (CP) are indicated. The dashed lines represent the apical surface of the ventricles. Distribution of electroporated GFP+ cells in the mouse cortex. The cortex was subdivided in 5 equal Bins -Bin1 corresponded to apical side and Bin5 to pial side of the cortex and GFP+ cells calculated in each Bin.


Distribution of mitotic progenitor PH3 + cells in the mouse cortex . The cortex was subdivided in 5 equal Bins - Bin1 corresponded to apical side and Bin5 to pial side of the cortex and PH3 + cells calculated in each Bin .

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Distribution of mitotic progenitor PH3+ cells in the mouse cortex. The cortex was subdivided in 5 equal Bins -Bin1 corresponded to apical side and Bin5 to pial side of the cortex and PH3+ cells calculated in each Bin.


K . Quantification of the total number of proliferating PH3 + cells / area ( µm2 ) of ventricular structures transfected with GFP - empty vector control , GFP - Cstb or GFP - R68X mutant in ( H ) .

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

K.Quantification of the total number of proliferating PH3+ cells/area(µm2)of ventricular structures transfected with GFP-empty vector control, GFP-Cstb or GFP-R68X mutant in (H).


Micrograph of coronal sections of E16 mouse cerebral cortices electroporated at E14 with GFP - empty vector control , GFP - Cstb or GFP - R68X mutant , analyzed 2 dpe and immunostained with GFP and Tbr2 . Ventricle ( V ) is indicated . The dashed lines represent the apical surface of the ventricles . Quantification of the total number of Tbr2 + cells / cortical area ( µm2 ) in ( L ) . Data shown as Z - scores relative to the mean of GFP control vector .

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

Micrograph of coronal sections of E16 mouse cerebral cortices electroporated at E14 with GFP-empty vector control, GFP-Cstb or GFP-R68X mutant, analyzed 2 dpe and immunostained with GFP and Tbr2. Ventricle (V) is indicated. The dashed lines represent the apical surface of the ventricles. Quantification of the total number of Tbr2+ cells/ cortical area (µm2) in (L). Data shown as Z-scores relative to the mean of GFP control vector.


Micrograph of coronal sections of E17 mouse cerebral cortices electroporated at E14 co - electroporated with mCherry expressing vector and HA - Cstb or R68X , and analyzed 3 dpe . Immunostaining with RFP to identify electroporated cells and GFP to identify migrating interneurons in the GAD65 - GFP transgenic mouse line . Ventricle ( V ) is indicated . The dashed lines represent the apical surface of the ventricles . Quantification of the total number of GAD65 - GFP interneurons / cortical area ( µm2 ) in ( N ) . Data shown as Z - scores relative to the mean of GFP control vector . Nuclei ( blue ) are stained with DAPI .

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

Micrograph of coronal sections of E17 mouse cerebral cortices electroporated at E14 co-electroporated with mCherry expressing vector and HA-Cstb or R68X, and analyzed 3 dpe. Immunostaining with RFP to identify electroporated cells and GFP to identify migrating interneurons in the GAD65-GFP transgenic mouse line. Ventricle (V) is indicated. The dashed lines represent the apical surface of the ventricles. Quantification of the total number of GAD65-GFP interneurons/ cortical area (µm2) in (N). Data shown as Z-scores relative to the mean of GFP control vector. Nuclei (blue) are stained with DAPI.


Left panel shows representative images of hCOs from the different iPSC lines at the analyzed time points . Right panel shows the difference of the areas of f - CTRL , b - CTRL , UL1 and UL4 hCOs at different time in culture . Data shown as Z - scores relative to the mean of b - CTRL .

Left panel shows representative images of hCOs from the different iPSC lines at the analyzed time points. Right panel shows the difference of the areas of f-CTRL, b-CTRL, UL1 and UL4 hCOs at different time in culture. Data shown as Z-scores relative to the mean of b-CTRL.


Micrograph sections of d30 b - CTRL , UL1 and UL4 hCOs immunostained for proliferation markers KI67 and PH3 . The dashed lines represent the apical surface of the cavities / ventricles . Quantification of the total number of proliferating Ki67 + cells / area ( µm2 ) of ventricles like structures and of the number of PH3 + cells facing the ventricle / apical surface length ( µm ) . Data shown as Z - scores relative to the mean of b - CTRL . Nuclei ( blue ) are stained with DAPI .

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Micrograph sections of d30 b-CTRL, UL1 and UL4 hCOs immunostained for proliferation markers KI67 and PH3. The dashed lines represent the apical surface of the cavities/ventricles. Quantification of the total number of proliferating Ki67+ cells/area (µm2) of ventricles like structures and of the number of PH3+ cells facing the ventricle/apical surface length (µm). Data shown as Z-scores relative to the mean of b-CTRL. Nuclei (blue) are stained with DAPI.


Micrograph sections of d60 b - CTRL , UL1 and UL4 hCOs immunostained for proliferation markers KI67 and PH3 . The dashed lines represent the apical surface of the cavities / ventricles . Quantification of the total number of proliferating KI67 + cells / area of ventricles like structures and of the number of PH3 + cells facing the ventricle / the apical surface length ( µm ) . Data shown as Z - scores relative to the mean of CTRL . Nuclei ( blue ) are stained with DAPI .

Micrograph sections of d60 b-CTRL, UL1 and UL4 hCOs immunostained for proliferation markers KI67 and PH3. The dashed lines represent the apical surface of the cavities/ventricles. Quantification of the total number of proliferating KI67+ cells/ area of ventricles like structures and of the number of PH3+ cells facing the ventricle/the apical surface length (µm). Data shown as Z-scores relative to the mean of CTRL. Nuclei (blue) are stained with DAPI.


Scheme of the experiment with conditioned medium exchange between b - CTRL and UL4 hCOs and quantification of the Ki67 + cells by FACS analysis on 36 days old dissociated hCOs . Four samples were analyzed as biological replicates .

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Scheme of the experiment with conditioned medium exchange between b-CTRL and UL4 hCOs and quantification of the Ki67+ cells by FACS analysis on 36 days old dissociated hCOs. Four samples were analyzed as biological replicates.


Micrograph sections of d30 b - CTRL , UL1 and UL4 hCOs immunostained for the neuronal marker DCX . The dashed lines represent the apical surface of the cavities / ventricles . Quantification of the DCX + area . The area of DCX + cells was calculated and related to the total area ( µm2 ) of the ventricular structure . Data shown as Z - scores relative to the mean of b - CTRL . Nuclei ( blue ) are stained with DAPI .

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

Micrograph sections of d30 b-CTRL, UL1 and UL4 hCOs immunostained for the neuronal marker DCX. The dashed lines represent the apical surface of the cavities/ventricles. Quantification of the DCX+ area. The area of DCX+ cells was calculated and related to the total area (µm2) of the ventricular structure. Data shown as Z-scores relative to the mean of b-CTRL. Nuclei (blue) are stained with DAPI.


Micrograph sections of d60 b - CTRL , UL1 and UL4 hCOs immunostained for neuronal marker DCX . The dashed lines represent the apical surface of the cavities / ventricles . Quantification of the DCX + area . The area ( µm2 ) of DCX + cells was calculated and related to the total area of the ventricular structure . Data shown as Z - scores relative to the mean of b - CTRL . Quantification of the number of neuronal DCX + cells by FACS sorting on 60 days old dissociated hCOs . Data shown as Z - scores relative to the mean of b - CTRL . Nuclei ( blue ) are stained with DAPI .

Micrograph sections of d60 b-CTRL, UL1 and UL4 hCOs immunostained for neuronal marker DCX. The dashed lines represent the apical surface of the cavities/ventricles. Quantification of the DCX+ area. The area (µm2) of DCX+ cells was calculated and related to the total area of the ventricular structure. Data shown as Z-scores relative to the mean of b-CTRL. Quantification of the number of neuronal DCX+ cells by FACS sorting on 60 days old dissociated hCOs. Data shown as Z-scores relative to the mean of b-CTRL. Nuclei (blue) are stained with DAPI.


Micrograph sections of d30 fused - organoid ; ventral GFP - f - CTRL and dorsal UL1 .

Micrograph sections of d30 fused-organoid; ventral GFP-f-CTRL and dorsal UL1.


Micrographs of sections from d30 fused organoids , showing ventral GFP + cells in the dorsal area . The migrating GFP + cells were counted starting from 100 µm far from the ventral / dorsal border . Quantification of ventrally - derived GFP + cells in dorsal / thickness of the counted area ( µm3 ) . The counted area was always 100 µm far from the border between ventral and dorsal area as indicated in Fig . EV5D . Every dot represents a different hCOs and different areas were counted for each hCO . Nuclei ( blue ) are stained with DAPI .

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Micrographs of sections from d30 fused organoids, showing ventral GFP+ cells in the dorsal area. The migrating GFP+ cells were counted starting from 100 µm far from the ventral/dorsal border. Quantification of ventrally-derived GFP+ cells in dorsal/thickness of the counted area (µm3). The counted area was always 100 µm far from the border between ventral and dorsal area as indicated in Fig.EV5D. Every dot represents a different hCOs and different areas were counted for each hCO. Nuclei (blue) are stained with DAPI.


Volcano plot representing proteins identified by high - resolution nanoLC MS / MS analysis . Up - and down - regulated proteins ( - 0 . 3 ≥ log2FC ≥ 0 . 3 ) in human cerebral organoids ( UL1 vs b - CTRL ) are shown in red and green , respectively .

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Volcano plot representing proteins identified by high-resolution nanoLC MS/MS analysis. Up- and down-regulated proteins (-0.3≥log2FC≥0.3) in human cerebral organoids (UL1 vs b-CTRL) are shown in red and green, respectively.


A ) TUBB1 is expressed in the developing human thyroid at 8 gestational weeks ( GW ) , 10 GW , and 12 GW and in the adult human thyroid : quantitative PCR results normalized to one of three thyroid tissues at 8 GW value ( in black ) and peptidylprolyl isomerase A . Experiments with at least three tissues per stage . Statistical comparisons versus 8 GW ( in black ) using ANOVA test showed no significant

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A) TUBB1 is expressed in the developing human thyroid at 8 gestational weeks (GW), 10 GW, and 12 GW and in the adult human thyroid: quantitative PCR results normalized to one of three thyroid tissues at 8 GW value (in black) and peptidylprolyl isomerase A. Experiments with at least three tissues per stage. Statistical comparisons versus 8 GW (in black) using ANOVA test showed no significant differences


B ) Tubb1 is expressed in the developing mouse thyroid at E13 . 5 , E15 . 5 , and E17 . 5 and in the adult mouse thyroid : quantitative PCR results normalized to one of three thyroid tissues at E13 . 5 value and peptidylprolyl isomerase A . Experiments with at least three tissues per stage . Statistical comparisons versus E13 . 5 ( black ) , * * p < 0 . 01 using ANOVA

B) Tubb1 is expressed in the developing mouse thyroid at E13.5, E15.5, and E17.5 and in the adult mouse thyroid: quantitative PCR results normalized to one of three thyroid tissues at E13.5 value and peptidylprolyl isomerase A. Experiments with at least three tissues per stage. Statistical comparisons versus E13.5 (black), **p<0.01 using ANOVA test


C ) Sorted mouse thyroid cells , Tubb1 expression in endothelial cells ( Pecam - positive cells ) , epithelial cells ( Epcam ) , fibroblasts ( Pdgfra ) , and leucocytes ( CD45 ) at E17 . 5 and adulthood : quantitative PCR results ( normalized to Epcam - positive cells at adult stage and peptidylprolyl isomerase A ) . Mean ± SEM of three independent cell - sorting experiments . Statistical comparisons versus epithelial cells ( in black ) at each stage , * p < 0 . 05 using ANOVA

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C) Sorted mouse thyroid cells, Tubb1 expression in endothelial cells (Pecam-positive cells), epithelial cells (Epcam), fibroblasts (Pdgfra), and leucocytes (CD45) at E17.5 and adulthood: quantitative PCR results (normalized to Epcam-positive cells at adult stage and peptidylprolyl isomerase A). Mean±SEM of three independent cell-sorting experiments. Statistical comparisons versus epithelial cells (in black) at each stage, *p<0.05 using ANOVA test


D ) Immunohistofluorescence of β1 - tubulin ( in red ) , thyroglobulin ( TG , in green ) , and both merged ( from left to right ) at 12 GW in human thyroid tissue . The boxes delineate the enlarged areas shown

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D) Immunohistofluorescence of β1-tubulin (in red), thyroglobulin (TG, in green), and both merged (from left to right) at 12 GW in human thyroid tissue. The boxes delineate the enlarged areas shown below


F ) Localization of transfected Myc - tagged wild - type and mutant ( P160L ) β1 - tubulin in Nthy cells . Immunostaining with anti - Myc antibody ( in green ) and microtubule cytoskeleton stained with α - tubulin antibody ( in red ) . Note that β1 - tubulin - P160L is found throughout the cytoplasm showing puncta appearance , whereas wild - type β1 - tubulin colocalizes with the

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F) Localization of transfected Myc-tagged wild-type and mutant (P160L) β1-tubulin in Nthy cells. Immunostaining with anti-Myc antibody (in green) and microtubule cytoskeleton stained with α-tubulin antibody (in red). Note that β1-tubulin -P160L is found throughout the cytoplasm showing puncta appearance, whereas wild-type β1-tubulin colocalizes with the microtubules


A ) Thyroid morphology was investigated using Nkx2 - 1 staining at E9 . 5 and E11 . 5 ( sagittal sections ) and at E13 . 5 , E15 . 5 , and E17 . 5 ( transverse sections ) in wild - type ( wt ) and Tubb1 - / - littermates . Nkx2 - 1was used as a marker of early thyrocyte progenitors and differentiated thyrocytes . Delays in thyroid migration and in fusion of the median anlage to the ultimobranchial bodies were observed at E11 . 5 and E13 . 5 ( arrow ) , respectively , in Tubb1 - / - mice . At E11 . 5 , stained migrating thyroid cells were visible along the tract only in Tubb1 - / - littermates ( arrow ) . At E17 . 5 , a pyramidal lobe was visible in Tubb1 - / - littermates ( arrow ) . tr : trachea ; ub : ultimobranchial body . Scale bar : 50

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A) Thyroid morphology was investigated using Nkx2-1 staining at E9.5 and E11.5 (sagittal sections) and at E13.5, E15.5, and E17.5 (transverse sections) in wild-type (wt) and Tubb1-/- littermates. Nkx2-1was used as a marker of early thyrocyte progenitors and differentiated thyrocytes. Delays in thyroid migration and in fusion of the median anlage to the ultimobranchial bodies were observed at E11.5 and E13.5 (arrow), respectively, in Tubb1-/- mice. At E11.5, stained migrating thyroid cells were visible along the tract only in Tubb1-/- littermates (arrow). At E17.5, a pyramidal lobe was visible in Tubb1-/- littermates (arrow). tr: trachea; ub: ultimobranchial body. Scale bar: 50 μ


B ) Total thyroid surface area ( μm2 ) was quantified using Nkx2 - 1 staining at each embryonic stage . Top : thyroid surface area normalised for weight of each embryo . Bottom : thyroid surface area in μm2 . Right : proliferation ratio calculated as the proportion of Nkx2 - 1 - positive cells labelled with Ki67 at E9 . 5 in wt and Tubb1 - / - thyroid anlages . ma : median anlage . Note that the thyroid ma is larger and the proliferation ratio higher at E . 9 . 5 in Tubb1 - / - mice , whereas from E13 . 5 onwards the Tubb1 - / - thyroids were hypoplastic . Wt in black , KO , Tubb1 - / - in

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B) Total thyroid surface area (μm2) was quantified using Nkx2-1 staining at each embryonic stage. Top: thyroid surface area normalised for weight of each embryo. Bottom: thyroid surface area in μm2. Right: proliferation ratio calculated as the proportion of Nkx2-1-positive cells labelled with Ki67 at E9.5 in wt and Tubb1-/- thyroid anlages. ma: median anlage. Note that the thyroid ma is larger and the proliferation ratio higher at E.9.5 in Tubb1-/- mice, whereas from E13.5 onwards the Tubb1-/- thyroids were hypoplastic. Wt in black, KO, Tubb1-/- in gre


C ) Thyroid marker expression by quantitative PCR in thyroid tissue at E15 . 5 and E17 . 5 in Tubb1 - / - versus wt mice normalised for peptidylprolyl isomerase A . Note the decreases in thyroid differentiation markers at E15 . 5 and E17 . 5 and also in thyroid transcription factors at E17 .

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C) Thyroid marker expression by quantitative PCR in thyroid tissue at E15.5 and E17.5 in Tubb1-/- versus wt mice normalised for peptidylprolyl isomerase A. Note the decreases in thyroid differentiation markers at E15.5 and E17.5 and also in thyroid transcription factors at E17.5


D ) Staining of Nkx2 - 1 and T4 at E17 . 5 and surface area quantification demonstrating T4 retention in Tubb1 - / - versus wt thyroids . The data are the percentage of T4 or calcitonin ( CT ) surface area versus total thyroid area ( estimated from the Nkx2 - 1 - stained surface area ) . Scale bar : 50 μm . Student " s test , * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 . Experiments were done on at least three tissues per stage for each

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D) Staining of Nkx2-1 and T4 at E17.5 and surface area quantification demonstrating T4 retention in Tubb1-/- versus wt thyroids. The data are the percentage of T4 or calcitonin (CT) surface area versus total thyroid area (estimated from the Nkx2-1-stained surface area). Scale bar: 50 μm. Student"s test, *p<0.05, **p<0.01 and ***p<0.001. Experiments were done on at least three tissues per stage for each gen


A ) Serum TSH and T4 levels in 3 - month - old Tubb1 - / - and wild - type mice . Numbers of animals tested were 10 wild - type males and 14 Tubb1 - / - males for TSH and for 14 wild - type and 22 Tubb1 - / - males for T4 . Tubb1 - / - mice had hypothyroidism with elevated TSH and decreased T4 versus wild - type mice . Student " s test , * * p <

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A) Serum TSH and T4 levels in 3-month-old Tubb1-/- and wild-type mice. Numbers of animals tested were 10 wild-type males and 14 Tubb1-/- males for TSH and for 14 wild-type and 22 Tubb1-/-males for T4. Tubb1-/- mice had hypothyroidism with elevated TSH and decreased T4 versus wild-type mice. Student"s test, ** p<


B ) Nkx2 - 1 ( in brown ) immunostaining of adult thyroid tissue . Note the disorganisation of the thyroid tissue in Tubb1 - / - versus wild - type

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B) Nkx2-1 (in brown) immunostaining of adult thyroid tissue. Note the disorganisation of the thyroid tissue in Tubb1-/- versus wild-type mice


C ) Ultrastructural alterations shown by electron microscopy in wild - type and Tubb1 - / - thyroid tissues . In Tubb1 - / - tissues , note the disorganisation of secretion vesicles ( white asterisks ) and rods of identical density to secretion vesicles ( white arrow ) . The ER is considerably dilated in Tubb1 - / - thyrocytes . Representative views . Scale bars at the bottom left for each view . Co :

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C) Ultrastructural alterations shown by electron microscopy in wild-type and Tubb1-/- thyroid tissues. In Tubb1-/- tissues, note the disorganisation of secretion vesicles (white asterisks) and rods of identical density to secretion vesicles (white arrow). The ER is considerably dilated in Tubb1-/- thyrocytes. Representative views. Scale bars at the bottom left for each view. Co: colloi


D ) ER stress in Tubb1 - / - thyroid tissue . Top : Chop and XBPs expression by quantitative PCR in adults , normalized to peptidylprolyl isomerase A . Experiments with at least three tissues per stage for each genotype ; wt in black and Tubb1 - / - in grey . Bottom : Chop and Actin protein expression by western blotting in three representative wild - type and Tubb1 - / - mice . The Chop band quantification normalised for Actin confirms the increased Chop expression demonstrated by quantitative PCR in Tubb1 - / - versus wild - type thyroids . All lanes are from the same blot , which was cut where indicated . Student " s test , * p < 0 . 05 and * * * p <

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D) ER stress in Tubb1-/- thyroid tissue. Top: Chop and XBPs expression by quantitative PCR in adults, normalized to peptidylprolyl isomerase A. Experiments with at least three tissues per stage for each genotype; wt in black and Tubb1-/- in grey. Bottom: Chop and Actin protein expression by western blotting in three representative wild-type and Tubb1-/- mice. The Chop band quantification normalised for Actin confirms the increased Chop expression demonstrated by quantitative PCR in Tubb1-/- versus wild-type thyroids. All lanes are from the same blot, which was cut where indicated. Student"s test, *p<0.05 and ***p<


E ) Co - immunostaining of endoplasmic reticulum ( ER ) marker ( KDEL , in red ) , thyroglobulin ( Tg in green ) , and both merged ( from left to right ) in adult thyroid tissue . Note the thyrocyte disorganisation in Tubb1 - / -

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E) Co-immunostaining of endoplasmic reticulum (ER) marker (KDEL, in red), thyroglobulin (Tg in green), and both merged (from left to right) in adult thyroid tissue. Note the thyrocyte disorganisation in Tubb1-/- mice


A ) Representative blood smears of patients with TUBB1 mutations ( P1 and P3 homozygous for p . P160L , P4 heterozygous for p . Y106X , and P6 and P7 heterozygous for c . 35delG ) after May - Grünwald Giemsa staining . Variable platelet size in patients was observed with all three mutations with presence of macroplatelets ( arrows )

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A) Representative blood smears of patients with TUBB1 mutations (P1 and P3 homozygous for p.P160L, P4 heterozygous for p.Y106X, and P6 and P7 heterozygous for c.35delG) after May-Grünwald Giemsa staining. Variable platelet size in patients was observed with all three mutations with presence of macroplatelets (arrows)


B ) Transmission electron microscopy ( TEM ) analysis of platelet ultrastructure in the patients . The results confirm the abnormal platelet

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B) Transmission electron microscopy (TEM) analysis of platelet ultrastructure in the patients. The results confirm the abnormal platelet morphology


C ) Representative images of proplatelet formation in P1 and P4 . Cultured control and patient megakaryocytes ( MKs ) after thrombopoietin - induced differentiation were spread over a BSA - coated coverslip on day 10 . On day 13 or 14 , MKs were fixed ( 4 % paraformaldehyde ) and proplatelet structure was observed after β - tubulin staining . Scale bars : 20 μm . Only P1 and P4 could be studied . The graphs show the mean ± SEM of the shaft thickness of proplatelet extensions ( left ) and the diameter of coiled elements ( right ) of the future proplatelets , both of which were significantly increased in the patients , indicating that the TUBB1 mutations ( homozygous p . P160L and heterozygous p . Y106X ) affected proplatelet morphology . Statistical significance was determined by one - way ANOVA , followed by Dunnett " s multiple comparisons test ( * p < 0 . 05 , * * * p < 0 . 001 ; 20 - 60 MKs were analysed /

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C) Representative images of proplatelet formation in P1 and P4. Cultured control and patient megakaryocytes (MKs) after thrombopoietin-induced differentiation were spread over a BSA-coated coverslip on day 10. On day 13 or 14, MKs were fixed (4% paraformaldehyde) and proplatelet structure was observed after β-tubulin staining. Scale bars: 20 μm. Only P1 and P4 could be studied. The graphs show the mean ± SEM of the shaft thickness of proplatelet extensions (left) and the diameter of coiled elements (right) of the future proplatelets, both of which were significantly increased in the patients, indicating that the TUBB1 mutations (homozygous p.P160L and heterozygous p.Y106X) affected proplatelet morphology. Statistical significance was determined by one-way ANOVA, followed by Dunnett"s multiple comparisons test (*p<0.05, ***p<0.001; 20-60 MKs were analysed/pat


A ) Relative expression of α - tubulin and β1 - tubulin in platelets from P1 and P3 ( homozygous p . P160L ) , P4 ( heterozygous p . Y106X ) , and P6 and P7 ( heterozygous c . 35delG ) was quantified by Western blotting using specific antibodies against α - tubulin or β1 - tubulin . With both mutations , α - tubulin expression was normal but β1 - tubulin expression was significantly decreased . The β1 - tubulin antibody recognizes C - terminal

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A) Relative expression of α-tubulin and β1-tubulin in platelets from P1 and P3 (homozygous p.P160L), P4 (heterozygous p.Y106X), and P6 and P7 (heterozygous c.35delG) was quantified by Western blotting using specific antibodies against α-tubulin or β1-tubulin. With both mutations, α-tubulin expression was normal but β1-tubulin expression was significantly decreased. The β1-tubulin antibody recognizes C-terminal domain


B ) αIIbβ3 integrin activation was evaluated by flow cytometry ( % of positive platelets and mean fluorescence intensity ) in whole blood from P1 and P3 ( homozygous p . P160L ) and from P6 and P7 ( heterozygous c . 35delG ) ; P4 could not be studied . While no αIIbβ3 integrin activations were detected in P6 and P7 ( family 3 ) , P1 and P3 ( family 1 ) exhibited a significant increase of αIIbβ3 activation , indicating basal platelet

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B) αIIbβ3 integrin activation was evaluated by flow cytometry (% of positive platelets and mean fluorescence intensity) in whole blood from P1 and P3 (homozygous p.P160L) and from P6 and P7 (heterozygous c.35delG); P4 could not be studied. While no αIIbβ3 integrin activations were detected in P6 and P7 (family 3), P1 and P3 (family 1) exhibited a significant increase of αIIbβ3 activation, indicating basal platelet activation


C ) Aggregation of washed platelets induced by ADP ( 5 or 10 μM ) or collagen ( 0 . 6 , 0 . 8 or 2 μg / mL ) was evaluated in at least one member of each family . Patients from families 1 and 2 had increased platelet aggregation in response to low doses of agonist versus controls , suggesting that TUBB1 affected by homozygous p . P160L and heterozygous p . Y106X mutations affected platelet function . Platelet aggregation to both agonists was normal in both patients from family 3 ( heterozygous c . 35delG ) . Family F1 , n = 3 independent experiments ; Family F2 , n = 1 experiment ; Family F3 , n = 2 independent experiments . Statistical significance was determined by one - way ANOVA , followed by Dunnett " s multiple comparisons test ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , nd : statistical significance not determined

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C) Aggregation of washed platelets induced by ADP (5 or 10 μM) or collagen (0.6, 0.8 or 2 μg/mL) was evaluated in at least one member of each family. Patients from families 1 and 2 had increased platelet aggregation in response to low doses of agonist versus controls, suggesting that TUBB1 affected by homozygous p.P160L and heterozygous p.Y106X mutations affected platelet function. Platelet aggregation to both agonists was normal in both patients from family 3 (heterozygous c.35delG). Family F1, n=3 independent experiments; Family F2, n=1 experiment; Family F3, n=2 independent experiments. Statistical significance was determined by one-way ANOVA, followed by Dunnett"s multiple comparisons test (*p<0.05, **p<0.01, ***p<0.001, nd: statistical significance not determined because


( C - C ' ' ' ) Terminal cell expressing reporters for membrane ( C ) , ER ( C ' ) and Golgi apparatus ( C ' ' ) . Red arrowheads : CD4 : : mIFP vesicles and associated markers at the tip of the cell .

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(C-C''') Terminal cell expressing reporters for membrane (C), ER (C') and Golgi apparatus (C''). Red arrowheads: CD4::mIFP vesicles and associated markers at the tip of the cell.


( D ) Mean and 95 % confidence interval of tube and branch length during the first hour of growth . Numbers represent the rate of growth estimated from the slope of the curve + / - SD , p = 0 . 39 , Mann - Whitney test , n = 6 .

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(D) Mean and 95% confidence interval of tube and branch length during the first hour of growth. Numbers represent the rate of growth estimated from the slope of the curve +/-SD, p=0.39, Mann-Whitney test, n=6.


( E ) Distance of the tip of the tube , and the leading ER and Golgi accumulations from the cell junction that defines the base of the cell . Line and shading represent mean and 95 % confidence interval , n = 4 .

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(E) Distance of the tip of the tube, and the leading ER and Golgi accumulations from the cell junction that defines the base of the cell. Line and shading represent mean and 95% confidence interval, n=4.


( F - I ) Terminal cells expressing the general membrane marker CD4 : : mIFP in combination with different membrane or polarity markers : PH : : mCherry , a PIP2 sensor commonly used as apical plasma membrane marker ( F ' ) , an endogenously GFP - tagged Crb ( G ' ) , polarity proteins Par3 : : YFP ( H ' ) and Par6 : : mCherry ( H ' ' ) . The boxed region in ( H ' ' ' ) is shown at higher magnification in ( I ) over 4 time points . Red arrowheads : CD4 : : mIFP vesicles and associated markers at the tip of the cell .

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(F-I) Terminal cells expressing the general membrane marker CD4::mIFP in combination with different membrane or polarity markers: PH::mCherry, a PIP2 sensor commonly used as apical plasma membrane marker (F'), an endogenously GFP-tagged Crb (G'), polarity proteins Par3::YFP (H') and Par6::mCherry (H''). The boxed region in (H''') is shown at higher magnification in (I) over 4 time points. Red arrowheads: CD4::mIFP vesicles and associated markers at the tip of the cell.


TEM tomograms from high - pressure frozen embryos . ( A - A ' ) Low magnification ( 2 . 5 nm voxels ) tomogram and 3D reconstruction from 10 serial 300 nm sections that cover one terminal cell . ( A ' ' - A ' ' ' ) 3D reconstructions derived from higher resolution tomograms showing different organelles in the cell .

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TEM tomograms from high-pressure frozen embryos. (A-A') Low magnification (2.5 nm voxels) tomogram and 3D reconstruction from 10 serial 300 nm sections that cover one terminal cell. (A''-A''') 3D reconstructions derived from higher resolution tomograms showing different organelles in the cell.


( B ) Example of an individual high - resolution ( 0 . 78nm voxels ) tomograms used to generate 3D reconstructions .

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(B) Example of an individual high-resolution (0.78nm voxels) tomograms used to generate 3D reconstructions.


( C ) Low resolution tomogram of another cell ; the dotted box is magnified in ( D ) . ( D ) High - resolution tomogram . ( D ' ) Model derived from 4 serial 3D reconstructions including the one shown in ( D ) .

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(C) Low resolution tomogram of another cell; the dotted box is magnified in (D). (D) High-resolution tomogram. (D') Model derived from 4 serial 3D reconstructions including the one shown in (D).


( B ) Distribution of three commonly seen classes of vesicles , expressed as their density per area of cytoplasm screened , in the two regions of the cell . Box plots represent median , interquartile range ( IQR ) , and IQR * 1 . 5 below and above the IQR . n = 62 tomograms from two different cells . ( C ) Representative examples for each vesicle class are shown next to the quantification plots .

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(B) Distribution of three commonly seen classes of vesicles, expressed as their density per area of cytoplasm screened, in the two regions of the cell. Box plots represent median, interquartile range (IQR), and IQR*1.5 below and above the IQR. n=62 tomograms from two different cells. (C) Representative examples for each vesicle class are shown next to the quantification plots.


( D ) Distribution of endocytic events at the apical or basal membrane compartment , expressed as density per membrane surface screened . Left shows tomograms corresponding to the cell body and branch , and right shows tomograms corresponding to the tip of the cell . Box plots represent median , interquartile range ( IQR ) , and IQR * 1 . 5 below and above the IQR . n = 48 tomograms from two different cells .

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(D) Distribution of endocytic events at the apical or basal membrane compartment, expressed as density per membrane surface screened. Left shows tomograms corresponding to the cell body and branch, and right shows tomograms corresponding to the tip of the cell. Box plots represent median, interquartile range (IQR), and IQR*1.5 below and above the IQR. n=48 tomograms from two different cells.


Distribution of the plasma membrane reporter PH : : GFP in control cells ( A - A ' ' ) and in cells where dynamin activity had been blocked using a temperature - sensitive allele of dynamin ( shibirets In ( B ) , was inactivated at the onset of tube formation ( C - D ) Distribution of PH : : GFP fluorescence intensity in control and shibirets cells . Data from 1 - 2 minute - interval time lapses was collected in windows of 20 minutes each ( except for t = 0 ) . ( C ) Proportion of signal in the apical and in the basal membrane compartment over time in control cells ( n = 4 ) and also in cells where dynamin was inactivated ( n = 5 ) , * p = 0 . 015 , Mann - Whitney test . ( D ) Total fluorescence intensity of PH : : GFP over time in controls ( n = 4 ) and in cells where dynamin was inactivated ( n = 5 ) , n . s . : p = 0 . 063 , Mann - Whitney test .

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Distribution of the plasma membrane reporter PH::GFP in control cells (A-A'') and in cells where dynamin activity had been blocked using a temperature-sensitive allele of dynamin (shibirets In (B), was inactivated at the onset of tube formation (C-D) Distribution of PH::GFP fluorescence intensity in control and shibirets cells. Data from 1-2 minute-interval time lapses was collected in windows of 20 minutes each (except for t=0). (C) Proportion of signal in the apical and in the basal membrane compartment over time in control cells (n=4) and also in cells where dynamin was inactivated (n=5), *p=0.015, Mann-Whitney test. (D) Total fluorescence intensity of PH::GFP over time in controls (n=4) and in cells where dynamin was inactivated (n=5), n.s.: p=0.063, Mann-Whitney test.


Distribution of the plasma membrane reporter PH : : GFP in cells where dynamin activity had been blocked using a temperature - sensitive allele of dynamin ( shibirets in ( E ) it was inactivated ~ 15 minutes after the tube had begun to form .

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Distribution of the plasma membrane reporter PH::GFP in cells where dynamin activity had been blocked using a temperature-sensitive allele of dynamin (shibirets in (E) it was inactivated ~15 minutes after the tube had begun to form.


( F - G ) Distribution of the luminal reporter Gasp : : GFP in control cells ( F ) and in cells where dynamin was inactivated ( G ) . Arrowheads point to protrusions sprouting from the lumen of the terminal cell .

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(F-G) Distribution of the luminal reporter Gasp::GFP in control cells (F) and in cells where dynamin was inactivated (G). Arrowheads point to protrusions sprouting from the lumen of the terminal cell.


( H - I ) Distribution of the general plasma membrane marker CD4 : : mIFP in combination with the Clathrin light chain fused to GFP ( CLC : : GFP , H ) and with Dynamin : : YFP ( I ) .

(H-I) Distribution of the general plasma membrane marker CD4::mIFP in combination with the Clathrin light chain fused to GFP (CLC::GFP, H) and with Dynamin::YFP (I).


( A - F ) TEM tomograms and 3D reconstructions of terminal cells in shibirets embryos . ( A ) Control . ( B - F ) Kept at 34ºC for 1 ( B , E ) or for 2 hours ( C , D , F ) . Boxed regions in ( A ) and ( B ) are magnified in ( A ' ' ) and ( B ' ' ) . ( A ' , B ' ) Reconstructed 3D models . The tube membrane ( cyan ) is characterized by the electron - dense apical extracellular matrix ( aECM ) . Invaginations with connections that can be traced to the tube membrane are red , invaginations from the basal plasma membrane are green . The basal membrane is light pink . ( C - D ' ) shibirets terminal cell after 2 hours at 34ºC . ( C ' ) 3D reconstruction , color coding for basal and tube membrane is the same as above . Membrane that is continuous with both the apical and the basal membrane is shown in green , purple and dark blue . ( D - D ' ) higher magnification of a region in the same cell at a level where membrane sheets ( purple , dark blue ) bridge the apical and basal plasma membrane domains . ( E - E ' ' ) Two focal planes and model of a tomogram from the cell shown in ( B ) where a basal ( pink arrowhead ) and an apical invagination ( blue ) are seen in close proximity ( closest distance is marked by cyan and blue arrowheads ) . ( F - F ' ) apical membrane overgrowth regions of the cell shown in ( C ) . The genotype of the embryos was shibirets ; btl > KDEL : : RFP , Par3 : : YFP . The cell shown in ( C - D ) was found and acquired without the CLEM approach .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F) TEM tomograms and 3D reconstructions of terminal cells in shibirets embryos. (A) Control. (B-F) Kept at 34ºC for 1 (B, E) or for 2 hours (C, D, F). Boxed regions in (A) and (B) are magnified in (A'') and (B''). (A', B') Reconstructed 3D models. The tube membrane (cyan) is characterized by the electron-dense apical extracellular matrix (aECM). Invaginations with connections that can be traced to the tube membrane are red, invaginations from the basal plasma membrane are green. The basal membrane is light pink. (C-D') shibirets terminal cell after 2 hours at 34ºC. (C') 3D reconstruction, color coding for basal and tube membrane is the same as above. Membrane that is continuous with both the apical and the basal membrane is shown in green, purple and dark blue. (D-D') higher magnification of a region in the same cell at a level where membrane sheets (purple, dark blue) bridge the apical and basal plasma membrane domains. (E-E'') Two focal planes and model of a tomogram from the cell shown in (B) where a basal (pink arrowhead) and an apical invagination (blue) are seen in close proximity (closest distance is marked by cyan and blue arrowheads). (F-F') apical membrane overgrowth regions of the cell shown in (C). The genotype of the embryos was shibirets; btl>KDEL::RFP, Par3::YFP. The cell shown in (C-D) was found and acquired without the CLEM approach.


( A - B ) Terminal cells expressing the membrane marker CD4 : : mIFP under btl - gal4 and FGFR : : GFP under its own promoter ( from the fTRG library ) . ( A ) Time lapse imaging of a control cell . Blue arrowheads point to filopodia and basal plasma membrane and red ones point to puncta containing CD4 : : mIFP and FGFR : : GFP . ( B ) shibirets cell imaged before dynamin inactivation , after 23 and 45 minutes of inactivation , and after 30 minutes of recovery . White arrowheads point to FGFR : : GFP accumulation at the apical compartment .

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

(A-B) Terminal cells expressing the membrane marker CD4::mIFP under btl-gal4 and FGFR::GFP under its own promoter (from the fTRG library). (A) Time lapse imaging of a control cell. Blue arrowheads point to filopodia and basal plasma membrane and red ones point to puncta containing CD4::mIFP and FGFR::GFP. (B) shibirets cell imaged before dynamin inactivation, after 23 and 45 minutes of inactivation, and after 30 minutes of recovery. White arrowheads point to FGFR::GFP accumulation at the apical compartment.


( C - E ) Single confocal planes of terminal cells expressing PH : : mCherry and stained for βPS - Integrin . The outline of the cells was traced using the PH : : mCherry signal and is shown as a blue dashed line . ( C ) Control . Blue arrowheads : βPS - Integrin signal in filopodia . ( D ) 1 hour at 34ºC , ( E ) 2 hours at 34ºC . ( C ' ' - E ' ' ) Orthogonal views of the lines shown in white in ( C - E ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-E) Single confocal planes of terminal cells expressing PH::mCherry and stained for βPS-Integrin. The outline of the cells was traced using the PH::mCherry signal and is shown as a blue dashed line. (C) Control. Blue arrowheads: βPS-Integrin signal in filopodia. (D) 1 hour at 34ºC, (E) 2 hours at 34ºC. (C''-E'') Orthogonal views of the lines shown in white in (C-E).


( F - G ) Quantification of fluorescence intensity from stained embryos of FGFR : : GFP ( F ) and of βPS - Integrin ( G ) . Data are plotted as mean + / - SD ; significance was assessed using one - way ANOVA with Dunnett ' s correction for multiple comparisons . * * p = 0 . 0022 , * * * p = 0 . 0003 , * * * * p < 0 . 0001 . Number of cells analyzed , for ( F ) : Control n = 8 , 1hr at 34ºC n = 6 , Recovery n = 8 . ( G ) . For ( G ) : Control = 7 , 1hr at 34ºC = 8 , 2hrs at 34ºC = 8 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-G) Quantification of fluorescence intensity from stained embryos of FGFR::GFP (F) and of βPS-Integrin (G). Data are plotted as mean +/- SD; significance was assessed using one-way ANOVA with Dunnett's correction for multiple comparisons. **p=0.0022, ***p=0.0003, ****p<0.0001. Number of cells analyzed, for (F): Control n=8, 1hr at 34ºC n=6, Recovery n=8. (G). For (G): Control= 7, 1hr at 34ºC= 8, 2hrs at 34ºC= 8.


High time resolution imaging of the membrane marker CD4 : : mIFP expressed under btl - gal4 . ( A ) Example of a large CD4 vesicle ( arrowhead ) rapidly moving towards the tip of the terminal cell . ( B - D ) Analysis of 28 CD4 vesicles in five terminal 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

High time resolution imaging of the membrane marker CD4::mIFP expressed under btl-gal4. (A) Example of a large CD4 vesicle (arrowhead) rapidly moving towards the tip of the terminal cell. (B-D) Analysis of 28 CD4 vesicles in five terminal cells.


( B ) Trajectories of CD4 vesicles with their original position mapped to the origin of the plot . ( C ) Rose diagram of the trajectories shown in ( B ) . ( D ) Speed of CD4 vesicles compared to speed of tube growth measured over 25 min . Data are plotted as 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

(B) Trajectories of CD4 vesicles with their original position mapped to the origin of the plot. (C) Rose diagram of the trajectories shown in (B). (D) Speed of CD4 vesicles compared to speed of tube growth measured over 25 min. Data are plotted as mean +/-SD.


( E - H ) Terminal cells expressing the membrane reporter CD4 : : mIFP together with markers for vesicles of the endocytic pathway . ( E ) Rab5 : : YFP , red arrowheads point to a Rab5 - positive vesicle ; the white arrowhead shows the same vesicle once it lost the Rab5 signal . ( F ) FYVE : : GFP , a PI3P reporter and Spin : : mRFP , a lysosomal marker . Black arrowheads point to small FYVE : : GFP vesicles and the squared area shows large ones at the tip . ( G ) Par6 : : mCherry and endogenously labelled Rab7 : : YFP . ( H ) Percentage of CD4 : : mIFP vesicles that carry the indicated markers . Data are plotted as mean + / - SD . For each marker we analyzed at least 20 minutes of cell growth with 10 - 20 timepoints . Number of cells analyzed : Par3 , n = 2 ; Rab5 , n = 3 ; FYVE : : GFP , n = 4 ; FYVE : : GFP - Spin : : RFP ( which includes single - and double - positive vesicles ) , n = 3 ; dLamp1 , n = 2 ; Rab7 , n = 5 .

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

(E-H) Terminal cells expressing the membrane reporter CD4::mIFP together with markers for vesicles of the endocytic pathway. (E) Rab5::YFP, red arrowheads point to a Rab5-positive vesicle; the white arrowhead shows the same vesicle once it lost the Rab5 signal. (F) FYVE::GFP, a PI3P reporter and Spin::mRFP , a lysosomal marker. Black arrowheads point to small FYVE::GFP vesicles and the squared area shows large ones at the tip. (G) Par6::mCherry and endogenously labelled Rab7::YFP. (H) Percentage of CD4::mIFP vesicles that carry the indicated markers. Data are plotted as mean +/-SD. For each marker we analyzed at least 20 minutes of cell growth with 10-20 timepoints. Number of cells analyzed: Par3, n=2; Rab5, n=3; FYVE::GFP, n=4; FYVE::GFP-Spin::RFP (which includes single- and double-positive vesicles), n=3; dLamp1, n=2; Rab7, n=5.


( A - F ) Terminal cells expressing CD4 : : mIFP and FYVE : : GFP under btl - gal4 . The outline of the cells was traced using the CD4 : : mIFP signal and is shown as a blue dashed line . ( A - C ) Control cell . ( D - F ) shibirets cell before 34ºC ( D ) , after 30 minutes at 34ºC ( E ) and after 30 minutes of recovery ( F ) . Red arrowheads : FYVE : : GFP puncta .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F) Terminal cells expressing CD4::mIFP and FYVE::GFP under btl-gal4. The outline of the cells was traced using the CD4::mIFP signal and is shown as a blue dashed line. (A-C) Control cell. (D-F) shibirets cell before 34ºC (D), after 30 minutes at 34ºC (E) and after 30 minutes of recovery (F). Red arrowheads: FYVE::GFP puncta.


( G - H ) Number of FYVE : : GFP vesicles in 4 control cells ( G ) and in 6 shibirets cells ( H ) . Box plots represent median , interquartile range ( IQR ) , and IQR * 1 . 5 below and above the IQR . Significance in ( H ) was assessed using one - way ANOVA with Geisser - Greenhouse correction for paired data and Dunnett ' s multiple comparisons test . * p = 0 . 0288 , * * * p = 0 . 0016 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-H) Number of FYVE::GFP vesicles in 4 control cells (G) and in 6 shibirets cells (H). Box plots represent median, interquartile range (IQR), and IQR*1.5 below and above the IQR. Significance in (H) was assessed using one-way ANOVA with Geisser-Greenhouse correction for paired data and Dunnett's multiple comparisons test. *p=0.0288, ***p=0.0016.


( I ) shrbO3 / G5 mutant cell expressing CD4 : : mIFP under btl - gal4 . Arrowhead : CD4 : : mIFP aggregation .

O O 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) shrbO3/G5 mutant cell expressing CD4::mIFP under btl-gal4. Arrowhead: CD4::mIFP aggregation.


( J - K ) Terminal cells over - expressing Shrb : : GFP under btl - gal4 together with PH : : mCherry ( J ) , or together with Spin : : RFP and CD4 : : mIFP ( K ) . Arrowheads : Shrb : : GFP accumulations .

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

(J-K) Terminal cells over-expressing Shrb::GFP under btl-gal4 together with PH::mCherry (J), or together with Spin::RFP and CD4::mIFP (K). Arrowheads: Shrb::GFP accumulations.


( L - M ) Dorsal trunk cells stained for Serp . In control embryos ( L ) , Serp is seen exclusively in the tracheal lumen . The cells themselves are not visible in these images . In embryos expressing Shrb : : GFP under btl - gal4 ( M ) , Serp is also seen inside the cells , usually in association with or surrounded by Shrb : : GFP . Boxed regions are magnified in ( L ' - M ' ) and shown as single confocal planes .

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

(L-M) Dorsal trunk cells stained for Serp. In control embryos (L), Serp is seen exclusively in the tracheal lumen. The cells themselves are not visible in these images. In embryos expressing Shrb::GFP under btl-gal4 (M), Serp is also seen inside the cells, usually in association with or surrounded by Shrb::GFP. Boxed regions are magnified in (L'-M') and shown as single confocal planes.


A , Representative images of DIV 3 + 2 cortical neurons transfected with FlucSM - EGFP ( green ) and subjected to the indicated treatments : 5 µM MG - 132 for 4 hours ; 10 nM Bafilomycin A1 ( BafA1 ) for 24 hours ; 0 . 5 µM 17 - AAG for 4 hours ; heat shock at 43 ° C for 30 min . Nuclei were labeled with DAPI ( blue ) . Insets show higher magnification of the areas outlined by the boxes .

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

A, Representative images of DIV 3+2 cortical neurons transfected with FlucSM-EGFP (green) and subjected to the indicated treatments: 5 µM MG-132 for 4 hours; 10 nM Bafilomycin A1 (BafA1) for 24 hours; 0.5 µM 17-AAG for 4 hours; heat shock at 43°C for 30 min. Nuclei were labeled with DAPI (blue). Insets show higher magnification of the areas outlined by the boxes.


D , DIV 3 + 2 cortical neurons co - transfected with FlucWT - EGFP ( green ) and HTT - Q97 - mCherry ( magenta ) and treated with 1 mM 4 - PBA ( lower row ) or vehicle control ( upper row ) from DIV 3 . Cells were stained for MAP2 ( grey ) as a neuronal marker , nuclei were labeled with DAPI ( blue ) . Arrowhead points to Fluc - EGFP foci . Corresponding cultures transfected with control HTT are shown in Fig . EV1D . E , Quantification of the fraction of double - transfected cells showing Fluc - EGFP foci . N = 4 biological replicates . Two - way ANOVA with Tukey ' s multiple comparisons test . ANOVA : HTT , * * p = 0 . 0046 ; 4 - PBA , n . s . ; HTT x 4 - PBA , n . s . Significant pairwise comparisons are indicated on the graph .

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

D, DIV 3+2 cortical neurons co-transfected with FlucWT-EGFP (green) and HTT-Q97-mCherry (magenta) and treated with 1 mM 4-PBA (lower row) or vehicle control (upper row) from DIV 3. Cells were stained for MAP2 (grey) as a neuronal marker, nuclei were labeled with DAPI (blue). Arrowhead points to Fluc-EGFP foci. Corresponding cultures transfected with control HTT are shown in Fig. EV1D. E, Quantification of the fraction of double-transfected cells showing Fluc-EGFP foci. N=4 biological replicates. Two-way ANOVA with Tukey's multiple comparisons test. ANOVA: HTT, **p=0.0046; 4-PBA, n.s.; HTT x 4-PBA, n.s. Significant pairwise comparisons are indicated on the graph. D


C , Left , Western blot of acute brain slice lysates from Fluc - EGFP mice and non - transgenic littermates . The slices were treated with 1 mM 4 - PBA or vehicle control , and subjected to heat shock at 43 ° C for 15 min , as indicated above the blot . Middle , quantification of specific luciferase activity of Fluc - EGFP upon indicated treatments , normalized to vehicle - treated slices kept at 37 ° C . Right , quantification of Fluc - EGFP protein levels in the indicated conditions . N = 3 mice . Colored asterisks indicate comparisons to the corresponding vehicle - treated 37 ° C control group ( one - sample t - test ) , black asterisk indicates comparison between 4 - PBA and vehicle - treated heat shock groups ( two - tailed t - test ) .

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

C, Left, Western blot of acute brain slice lysates from Fluc-EGFP mice and non-transgenic littermates. The slices were treated with 1 mM 4-PBA or vehicle control, and subjected to heat shock at 43°C for 15 min, as indicated above the blot. Middle, quantification of specific luciferase activity of Fluc-EGFP upon indicated treatments, normalized to vehicle-treated slices kept at 37°C. Right, quantification of Fluc-EGFP protein levels in the indicated conditions. N=3 mice. Colored asterisks indicate comparisons to the corresponding vehicle-treated 37°C control group (one-sample t-test), black asterisk indicates comparison between 4-PBA and vehicle-treated heat shock groups (two-tailed t-test).


D , Representative Western blot of hippocampal lysates from Fluc - EGFP mice at the indicated ages . Short bracket with an arrow indicates the high molecular weight species observed in older mice . Long bracket indicates the part of the lane that was used for Fluc protein quantification ( see Materials and Methods ) . Several lanes on the blot between 6 - month - old and 12 - month - old samples were digitally removed .

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

D, Representative Western blot of hippocampal lysates from Fluc-EGFP mice at the indicated ages. Short bracket with an arrow indicates the high molecular weight species observed in older mice. Long bracket indicates the part of the lane that was used for Fluc protein quantification (see Materials and Methods). Several lanes on the blot between 6-month-old and 12-month-old samples were digitally removed.


A , Cortical sections from rTg4510 : Fluc - EGFP mice ( right ) and control littermates ( left ) stained for total human tau ( HT7 , magenta ) and p - tau ( AT8 , yellow ) . Fluc - EGFP was detected by EGFP fluorescence ( green ) , nuclei were labeled with DAPI ( blue ) . Images on the right show higher magnification of the areas indicated by the boxes , with neuronal cell bodies outlined . Note the presence of EGFP foci ( arrowheads ) in the majority of neurons in rTg4510 : Fluc - EGFP mice . Occasional foci of lower relative fluorescence intensity can be observed in control mice ( arrow ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Cortical sections from rTg4510:Fluc-EGFP mice (right) and control littermates (left) stained for total human tau (HT7, magenta) and p-tau (AT8, yellow). Fluc-EGFP was detected by EGFP fluorescence (green), nuclei were labeled with DAPI (blue). Images on the right show higher magnification of the areas indicated by the boxes, with neuronal cell bodies outlined. Note the presence of EGFP foci (arrowheads) in the majority of neurons in rTg4510:Fluc-EGFP mice. Occasional foci of lower relative fluorescence intensity can be observed in control mice (arrow).