Figure 3A . Co - IP analysis of the interaction of Aster - C with COPA and COPG in response to starvation and AA stimulation using FLAG - DEPDC5 as a negative control in HEK293T cells transiently expressing the indicated proteins . Anti - FLAG antibody was used for immunoprecipitation . Black arrow indicates the COPG protein . B . Co - IP analysis of the interaction of COPA with mTOR in HEK293T cells transiently expressing GFP - COPA in response to starvation and AA stimulation . Anti - GFP antibody was used for immunoprecipitation . Black arrow indicates the COPG protein . C . Co - IP analysis of the interaction of COPA with mTOR in C2C12 - Vector and Aster - C KO cells in response to starvation and AA stimulation . Anti - COPA antibody was used for immunoprecipitation . D . Confocal imaging analysis depicting the co - localization of YFP - mTOR ( yellow ) with GFP - COPA ( green ) and lysosomes ( red ) in live C2C12 - Vector and Aster - C KO cells in response to nutrient starvation and re - stimulation by AA . Arrows highlight co - localization of mTOR , COPA and lysosomes . Scale bar , 20 μm . E and F . Statistical analysis of the Pearson ' s correlation coefficient of mTOR / Lysosome ( E ) and COPA / Lysosome ( F ) in Figure 3D ( n = 10 - 12 cells per group ) . Data are represented as mean ± SD . * * * p < 0 . 001 by one - way ANOVA .

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

Figure 3A. Co-IP analysis of the interaction of Aster-C with COPA and COPG in response to starvation and AA stimulation using FLAG-DEPDC5 as a negative control in HEK293T cells transiently expressing the indicated proteins. Anti-FLAG antibody was used for immunoprecipitation. Black arrow indicates the COPG protein.B. Co-IP analysis of the interaction of COPA with mTOR in HEK293T cells transiently expressing GFP-COPA in response to starvation and AA stimulation. Anti-GFP antibody was used for immunoprecipitation. Black arrow indicates the COPG protein.C. Co-IP analysis of the interaction of COPA with mTOR in C2C12-Vector and Aster-C KO cells in response to starvation and AA stimulation. Anti-COPA antibody was used for immunoprecipitation.D. Confocal imaging analysis depicting the co-localization of YFP-mTOR (yellow) with GFP-COPA (green) and lysosomes (red) in live C2C12-Vector and Aster-C KO cells in response to nutrient starvation and re-stimulation by AA. Arrows highlight co-localization of mTOR, COPA and lysosomes. Scale bar, 20 μm. E and F. Statistical analysis of the Pearson's correlation coefficient of mTOR/Lysosome (E) and COPA/Lysosome (F) in Figure 3D (n=10-12 cells per group). Data are represented as mean ± SD. ***p<0.001 by one-way ANOVA.


Figure 4Time - lapse confocal imaging analysis of the co - localization of YFP - mTOR ( red ) with GFP - COPA ( green ) and lysosomes ( blue ) in live C2C12 cells under nutrient starvation ( 0 min ) , and in response to AA re - stimulation for indicated time ( 0 . 5 min , 2 min , 6 min and 8 min ) . Arrows highlight COPA puncta that were co - localized with mTOR or COPA puncta alone prior to their association with mTOR and lysosomes . . Scale bar , 5 μm .

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

Figure 4Time-lapse confocal imaging analysis of the co-localization of YFP-mTOR (red) with GFP-COPA (green) and lysosomes (blue) in live C2C12 cells under nutrient starvation (0 min), and in response to AA re-stimulation for indicated time (0.5 min, 2 min, 6 min and 8 min). Arrows highlight COPA puncta that were co-localized with mTOR or COPA puncta alone prior to their association with mTOR and lysosomes.. Scale bar, 5 μm.


Figure 5A . Confocal imaging analysis depicting the lysosomal association of mTOR in response to AA stimulation in the presence or absence of BFA ( 10 μM ) or Exo - 2 ( 10 μM ) in live C2C12 cells transiently expressing YFP - mTOR and stained with Lysotracker Red . Scale bar , 20 μm . Western blot analysis of mTORC1 activity in C2C12 cells in CM , in response to nutrient starvation and AA stimulation in the presence or absence of BFA ( 10 μM ) ( B ) C . Western blot analysis of mTORC1 activity in C2C12 cells in CM , in response to nutrient starvation and AA stimulation in the presence or absence of Exo - 2 ( 10 μM ) ( C ) . D . Western blot analysis of mTORC1 activity in C2C12 - Vector and ARF1 KO cells in CM , in response to nutrient starvation and AA stimulation . E . Confocal imaging analysis depicting COPA puncta ( green ) formation and co - localization with lysosomes ( red ) in live C2C12 cells transiently expressing GFP - COPA and stained with Lysotracker Red in response to nutrient starvation and AA stimulation in the presence or absence of BFA ( 10 μM ) or Exo - 2 ( 10 μM ) . Arrows highlight co - localization of COPA with lysosomes . Scale bar , 5 μm . F . Statistical analysis of the Pearson ' s correlation coefficient of COPA and lysosomes in Figure 5E ( n = 10 - 15 cells per group ) . Data are represented as mean ± SD . * * * p < 0 . 001 by one - way ANOVA .

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

Figure 5A. Confocal imaging analysis depicting the lysosomal association of mTOR in response to AA stimulation in the presence or absence of BFA (10 μM) or Exo-2 (10 μM) in live C2C12 cells transiently expressing YFP-mTOR and stained with Lysotracker Red. Scale bar, 20 μm.Western blot analysis of mTORC1 activity in C2C12 cells in CM, in response to nutrient starvation and AA stimulation in the presence or absence of BFA (10 μM) (B)C. Western blot analysis of mTORC1 activity in C2C12 cells in CM, in response to nutrient starvation and AA stimulation in the presence or absence of Exo-2 (10 μM) (C).D. Western blot analysis of mTORC1 activity in C2C12-Vector and ARF1 KO cells in CM, in response to nutrient starvation and AA stimulation.E. Confocal imaging analysis depicting COPA puncta (green) formation and co-localization with lysosomes (red) in live C2C12 cells transiently expressing GFP-COPA and stained with Lysotracker Red in response to nutrient starvation and AA stimulation in the presence or absence of BFA (10 μM) or Exo-2 (10 μM). Arrows highlight co-localization of COPA with lysosomes. Scale bar, 5 μm.F. Statistical analysis of the Pearson's correlation coefficient of COPA and lysosomes in Figure 5E (n=10-15 cells per group). Data are represented as mean ± SD. ***p<0.001 by one-way ANOVA.


Figure 6A . Co - IP analysis of the interaction of Aster - C with COPA , MYH10 , WDR24 and Mios in HEK293T cells transiently expressing the indicated proteins in response to treatment with brefeldin A ( BFA , 10 μM ) and / or blebbistatin ( BBS , 10 μM ) , inhibitors of ARFs and MYH10 , respectively . Anti - FLAG antibody was used for immunoprecipitation . B . Western blot analysis of mTORC1 activity in wild type ( WT ) and MYH10 KO MEF cells cultured in CM , or in response to starvation and AA stimulation . C . Western blot analysis of mTORC1 activity in MYH10 KO MEF cells with stably re - expressed exogenous mCherry - MYH10 in response to starvation and AA stimulation . MYH10 KO MEF cells were transfected with mCherry - MYH10 and selected with G418 for 2 weeks . D . Confocal imaging analysis depicting COPA puncta ( green ) formation and co - localization with mTOR ( red ) in live C2C12 cells transiently expression GFP - COPA and YFP - mTOR in response to nutrient starvation in the presence or absence of BBS ( 10 µM ) . Arrows highlight the co - localization of mTOR with COPA . Scale bar , 20 μm . E . Statistical analysis of the Pearson ' s correlation coefficient of mTOR and COPA in Figure 6D ( n = 10 - 12 cells per group ) . Data are represented as mean ± SD . * * * p < 0 . 001 by student ' s t test .

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

Figure 6A. Co-IP analysis of the interaction of Aster-C with COPA, MYH10, WDR24 and Mios in HEK293T cells transiently expressing the indicated proteins in response to treatment with brefeldin A (BFA, 10 μM) and/or blebbistatin (BBS, 10 μM), inhibitors of ARFs and MYH10, respectively. Anti-FLAG antibody was used for immunoprecipitation.B. Western blot analysis of mTORC1 activity in wild type (WT) and MYH10 KO MEF cells cultured in CM, or in response to starvation and AA stimulation.C. Western blot analysis of mTORC1 activity in MYH10 KO MEF cells with stably re-expressed exogenous mCherry-MYH10 in response to starvation and AA stimulation. MYH10 KO MEF cells were transfected with mCherry-MYH10 and selected with G418 for 2 weeks.D. Confocal imaging analysis depicting COPA puncta (green) formation and co-localization with mTOR (red) in live C2C12 cells transiently expression GFP-COPA and YFP-mTOR in response to nutrient starvation in the presence or absence of BBS (10 µM). Arrows highlight the co-localization of mTOR with COPA. Scale bar, 20 μm.E. Statistical analysis of the Pearson's correlation coefficient of mTOR and COPA in Figure 6D (n=10-12 cells per group). Data are represented as mean ± SD. ***p<0.001 by student's t test.


Figure 7A . Confocal imaging analysis depicting mTOR puncta ( green ) formation on actin filaments ( red ) in COS - 7 cells transiently expressing YFP - mTOR in response to AA stimulation in the presence or absence of latrunculin B ( LA - B , 100 nM ) . Actin filaments were stained using BODIPY ™ 558 / 568 - Phalloidin . Arrows indicated the association of mTOR with actin filaments . Scale bar , 5 μm . B . Immunostaining of endogenous mTOR and LAMP1 in C2C12 cells under amino acids starvation ( - AA ) , or in response to AA stimulation ( + AA ) in the presence or absence of LA - B ( 100 nM ) . Scale bar , 20 μm . C . Western blot analysis of mTORC1 activity in C2C12 cells in CM , in response to nutrient starvation and AA stimulation in the presence or absence of LA - B ( 100 nM ) . D . Co - IP analysis of the interaction of Aster - C with COPA and MYH10 in the presence or absence of LA - B ( 100 nM ) or cytochalasin D ( Cyto - D , 250 nM ) in HEK293T cells transiently expressing FLAG - Aster - C . Anti - FLAG antibody was used for immunoprecipitation .

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

Figure 7A. Confocal imaging analysis depicting mTOR puncta (green) formation on actin filaments (red) in COS-7 cells transiently expressing YFP-mTOR in response to AA stimulation in the presence or absence of latrunculin B (LA-B, 100 nM). Actin filaments were stained using BODIPY™ 558/568-Phalloidin. Arrows indicated the association of mTOR with actin filaments. Scale bar, 5 μm.B. Immunostaining of endogenous mTOR and LAMP1 in C2C12 cells under amino acids starvation (-AA), or in response to AA stimulation (+AA) in the presence or absence of LA-B (100 nM). Scale bar, 20 μm.C. Western blot analysis of mTORC1 activity in C2C12 cells in CM, in response to nutrient starvation and AA stimulation in the presence or absence of LA-B (100 nM).D. Co-IP analysis of the interaction of Aster-C with COPA and MYH10 in the presence or absence of LA-B (100 nM) or cytochalasin D (Cyto-D, 250 nM) in HEK293T cells transiently expressing FLAG-Aster-C. Anti-FLAG antibody was used for immunoprecipitation.


Figure 1A - Distribution of Casp3 + cells in rostral and ventral domains of an E11 . 5 Rx - Dicer mutant embryo . NCx , neocortex ; OB , olfactory bulb ; Spt , septum . B , C - Marker analysis of Casp3 + cells in the rostral telencephalon of E12 . 5 Rx - Dicer mutant embryos . Most apoptotic cells are Pax6 + RGCs ( solid arrowheads ) and not Tbr1 + neurons ( open arrowheads ) . N = 3 replicates per marker . D , E - Distribution and abundance of apoptotic cells ( Casp3 + ) in the rostral telencephalic primordium of control and Rx - Dicer mutant embryos at the indicated ages . Dotted line indicates basal surface , dashed line apical surface . N = 3 replicates per genotype and age . F , G - Distribution and abundance of mitoses ( PH3 + ) and neurons ( Tuj1 ) in the rostral telencephalic primordium of control and Rx - Dicer mutant embryos at the indicated ages . Dotted line indicates basal surface , dashed line apical surface . N = 3 - 4 replicates per genotype and age . No significant differences were found between control and mutant embryos in apical nor basal mitoses .

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

Figure 1A - Distribution of Casp3+ cells in rostral and ventral domains of an E11.5 Rx-Dicer mutant embryo. NCx, neocortex; OB, olfactory bulb; Spt, septum.B,C - Marker analysis of Casp3+ cells in the rostral telencephalon of E12.5 Rx-Dicer mutant embryos. Most apoptotic cells are Pax6+ RGCs (solid arrowheads) and not Tbr1+ neurons (open arrowheads). N = 3 replicates per marker.D,E - Distribution and abundance of apoptotic cells (Casp3+) in the rostral telencephalic primordium of control and Rx-Dicer mutant embryos at the indicated ages. Dotted line indicates basal surface, dashed line apical surface. N = 3 replicates per genotype and age.F,G - Distribution and abundance of mitoses (PH3+) and neurons (Tuj1) in the rostral telencephalic primordium of control and Rx-Dicer mutant embryos at the indicated ages. Dotted line indicates basal surface, dashed line apical surface. N = 3-4 replicates per genotype and age. No significant differences were found between control and mutant embryos in apical nor basal mitoses.


Figure 2A - DAPI stain of sagittal sections through the rostral telencephalon of E17 . 5 control and Rx - Dicer mutant littermates showing the neocortex ( NCx ) and olfactory bulb ( OB ; dashed line ) . B - Expression pattern of Grm1 mRNA in OB ( dotted line ) of control and Rx - Dicer mutants . C - Quantification of OB perimeter ( mean ± SEM ; symbols indicate values for individual embryos ) ; t - test , * * * P < 0 . 001 . N = 14 replicates per genotype . D - Immunostains of E17 . 5 control and Rx - Dicer mutant brains showing the distribution of progenitor cells ( Ki67 , red ) and neurons ( Tuj1 , green ) . Arrowheads indicate rosettes . Sp , septum . E - Detail of a rosette from an E17 . 5 Rx - Dicer mutant embryo displaying typical features : closed apical surface ( dotted line ) with PH3 + apical mitoses ( white arrowhead ) and basal mitoses , surrounded by Tuj1 + neurons ( green ) . Dashed line indicates the basal border of the rosette . F - Rostral half of an Rx - Dicer mutant E17 . 5 brain immunostained for Pax6 , clarified and segmented to reveal rosettes ( yellow ) . C , caudal ; R , rostral . G , H - BrdU incorporation and cell cycle re - entry analysis with the progenitor cell marker Ki67 at E17 . 5 , in the rostral cortex of control embryos compared to rostral rosettes of Rx - Dicer mutants . Dotted lines indicate apical surface , dashed lines indicate basal border of VZ . Arrowheads indicate double - positive cells . Data in histograms are mean ± SEM , symbols indicate values for individual embryos ; n ≥ 3 embryos ; X2 - test , * P < 0 . 05 , * * * P < 0 . 001 . I - Apical lumen of rosettes immunostained against apical complex proteins ( Par3 , β - Catenin ) , primary cilia ( Arl13b ) , apical mitoses ( PhVim ) and neurons ( Tuj1 ) . J - Coronal section through the rostral telencephalon of an E14 . 5 Rx - Dicer mutant embryo illustrating the high abundance and location of rosettes ( arrows ) , as revealed by the distribution of mitoses ( PH3 ) and neurons ( Tuj1 ) . K - S - Analysis of the regional identity of rosettes in E14 . 5 Rx - Dicer mutants . ( K ) Schema of normal transcription factor expression patterns defining telencephalic regional identity . Expression of Ngn2 , Tbr2 and Pax6 identifies rosettes in the rostro - dorsal telencephalon as having dorsal identity ( L - N , P ) ; Gsx2 , Dlx2 and Dlx5 identify rosettes in the ventral telencephalon as having ventral identity ( O , Q ) ; Nkx2 . 1 identifies MGE rosettes as being normotopic ( R ) ; absence of Gsx2 in LGE rosette cells identifies them as ectopic ( S ) . Tuj1 labels neurons . In ( L , M ) , arrowheads indicate the border between dorsal and ventral territories , and dotted line indicates the outer border of the telencephalon . LGE , lateral ganglionic eminence ; MGE , medial ganglionic eminence ; P , pallium ; SP , subpallium .

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

Figure 2A - DAPI stain of sagittal sections through the rostral telencephalon of E17.5 control and Rx-Dicer mutant littermates showing the neocortex (NCx) and olfactory bulb (OB; dashed line).B - Expression pattern of Grm1 mRNA in OB (dotted line) of control and Rx-Dicer mutants. C - Quantification of OB perimeter (mean ± SEM; symbols indicate values for individual embryos); t-test, ***P < 0.001. N = 14 replicates per genotype. D - Immunostains of E17.5 control and Rx-Dicer mutant brains showing the distribution of progenitor cells (Ki67, red) and neurons (Tuj1, green). Arrowheads indicate rosettes. Sp, septum.E - Detail of a rosette from an E17.5 Rx-Dicer mutant embryo displaying typical features: closed apical surface (dotted line) with PH3+ apical mitoses (white arrowhead) and basal mitoses, surrounded by Tuj1+ neurons (green). Dashed line indicates the basal border of the rosette.F - Rostral half of an Rx-Dicer mutant E17.5 brain immunostained for Pax6, clarified and segmented to reveal rosettes (yellow). C, caudal; R, rostral.G,H - BrdU incorporation and cell cycle re-entry analysis with the progenitor cell marker Ki67 at E17.5, in the rostral cortex of control embryos compared to rostral rosettes of Rx-Dicer mutants. Dotted lines indicate apical surface, dashed lines indicate basal border of VZ. Arrowheads indicate double-positive cells. Data in histograms are mean ± SEM, symbols indicate values for individual embryos; n ≥ 3 embryos; X2-test, *P < 0.05, ***P < 0.001.I - Apical lumen of rosettes immunostained against apical complex proteins (Par3, β-Catenin), primary cilia (Arl13b), apical mitoses (PhVim) and neurons (Tuj1).J - Coronal section through the rostral telencephalon of an E14.5 Rx-Dicer mutant embryo illustrating the high abundance and location of rosettes (arrows), as revealed by the distribution of mitoses (PH3) and neurons (Tuj1). K-S - Analysis of the regional identity of rosettes in E14.5 Rx-Dicer mutants. (K) Schema of normal transcription factor expression patterns defining telencephalic regional identity. Expression of Ngn2, Tbr2 and Pax6 identifies rosettes in the rostro-dorsal telencephalon as having dorsal identity (L-N,P); Gsx2, Dlx2 and Dlx5 identify rosettes in the ventral telencephalon as having ventral identity (O,Q); Nkx2.1 identifies MGE rosettes as being normotopic (R); absence of Gsx2 in LGE rosette cells identifies them as ectopic (S). Tuj1 labels neurons. In (L,M), arrowheads indicate the border between dorsal and ventral territories, and dotted line indicates the outer border of the telencephalon. LGE, lateral ganglionic eminence; MGE, medial ganglionic eminence; P, pallium; SP, subpallium.


Figure 3A - Coronal section through the rostral telencephalon of an E14 . 5 Rx - Dicer mutant embryo displaying the three types of rosettes . B - High magnification of rosettes in Rx - Dicer mutants at E12 . 5 ( Type 1 ) , E14 . 5 ( Type 2 ) and E17 . 5 ( Type 3 ) , immunostained for PH3 and Tuj1 . Dashed line indicates ventricular surface ; arrowheads indicate basal mitoses ; arrows indicate a stream of apical mitoses connecting the lumen of the rosette with the lumen of the telencephalic ventricle . C , D - Abundance of rosette types at the indicated ages ( C ) , and rostro - caudal distribution of total rosette abundance per age , independent of type ( D ) . n ≥ 2 brains per age . F , G - Distribution of neurons ( Tuj1 + cells ) and apical adherens junction protein Par3 in the OB primordium of control and Rx - Dicer mutant embryos at the indicated ages . Dotted lines indicate basal border , dashed lines indicate apical border . White arrowheads indicate ectopic neurons and / or absence of Par3 . Arrow in ( G ) indicates accumulation of Par3 at the lumen of a nascent rosette .

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

Figure 3A - Coronal section through the rostral telencephalon of an E14.5 Rx-Dicer mutant embryo displaying the three types of rosettes.B - High magnification of rosettes in Rx-Dicer mutants at E12.5 (Type 1), E14.5 (Type 2) and E17.5 (Type 3), immunostained for PH3 and Tuj1. Dashed line indicates ventricular surface; arrowheads indicate basal mitoses; arrows indicate a stream of apical mitoses connecting the lumen of the rosette with the lumen of the telencephalic ventricle.C,D - Abundance of rosette types at the indicated ages (C), and rostro-caudal distribution of total rosette abundance per age, independent of type (D). n ≥ 2 brains per age.F,G - Distribution of neurons (Tuj1+ cells) and apical adherens junction protein Par3 in the OB primordium of control and Rx-Dicer mutant embryos at the indicated ages. Dotted lines indicate basal border, dashed lines indicate apical border. White arrowheads indicate ectopic neurons and/or absence of Par3. Arrow in (G) indicates accumulation of Par3 at the lumen of a nascent rosette.


Figure 4A , B - Changes in expression levels of miRNAs between control and Rx - Dicer mutants at E11 . 5 in the rostral telencephalon . Red dots indicate statistically differentially expressed miRNAs according to FDR correction of P values based on the Wald statistic ( DESeq2 analysis ) ( B ) . Dashes lines indicate stringency limits in evaluation of results . D , E - Changes in mRNA expression levels between control and Rx - Dicer mutants at E11 . 5 in the rostral telencephalon . Statistically differentially expressed mRNAs according to FDR correction of P values based on the Wald statistic ( DESeq2 analysis ; Adj . P < 0 . 1 and FC > 1 . 25 ) are highlighted in red , and the three with the highest Adj . P value are identified by name . Blue dot indicates Irs2 : FC = 1 . 748 , Adj . P = 5 . 95e - 08 . G - Heatmaps of relative expression levels of DEGs related to apoptosis ( top ) and proliferation ( bottom ) . Rows correspond to independent biological replicates . Indicated are some example genes . H - Enrichment plots from GSEA for MSigDB Hallmark Neurogenesis ( NES = 2 . 98 ; p = 0 ; Adj . P = 0 . 00045 ) , Regulation of cell proliferation ( NES = 2 . 40 ; p = 0 ; Adj . P = 0 . 0033 ) , Positive regulation of cell adhesion ( NES = 2 . 06 ; p = 0 . 0058 ; Adj . P = 0 . 013 ) , Regulation of response to stress ( NES = 2 . 50 ; p = 0 ; Adj . P = 0 . 0023 ) , Positive regulation of cell death ( NES = 1 . 84 ; p = 0 . 0179 ; Adj . P = 0 . 0326 ) , Apoptotic signaling pathway ( NES = 1 . 59 ; p = 0 . 047 ; Adj . P = 0 . 084 ) . I , J - ISH stains of Irs2 mRNA , and qPCR for Irs2 mRNA and let - 7 - 5p miRNA , in the rostral telencephalon of control and Rx - Dicer mutant embryos . Dashed line indicates basal border , dotted line indicates apical surface . Arrowheads indicate area with the greatest increase in Irs2 expression . BG , basal ganglia ; H , hippocampus ; NCx , neocortex ; OB , olfactory bulb ; Spt , septum . Histograms represent mean ± SEM ( logarithmic scale ) ; symbols in plots indicate values for individual embryos ; t - test , * P < 0 . 05 , * * P < 0 . 01 . N = 6 - 9 replicates per group . Scale bar , 100 µm .

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

Figure 4A,B - Changes in expression levels of miRNAs between control and Rx-Dicer mutants at E11.5 in the rostral telencephalon. Red dots indicate statistically differentially expressed miRNAs according to FDR correction of P values based on the Wald statistic (DESeq2 analysis) (B). Dashes lines indicate stringency limits in evaluation of results.D,E - Changes in mRNA expression levels between control and Rx-Dicer mutants at E11.5 in the rostral telencephalon. Statistically differentially expressed mRNAs according to FDR correction of P values based on the Wald statistic (DESeq2 analysis; Adj. P < 0.1 and FC > 1.25) are highlighted in red, and the three with the highest Adj. P value are identified by name. Blue dot indicates Irs2: FC=1.748, Adj. P = 5.95e-08.G - Heatmaps of relative expression levels of DEGs related to apoptosis (top) and proliferation (bottom). Rows correspond to independent biological replicates. Indicated are some example genes.H - Enrichment plots from GSEA for MSigDB Hallmark Neurogenesis (NES = 2.98; p = 0; Adj. P = 0.00045), Regulation of cell proliferation (NES = 2.40; p=0; Adj. P = 0.0033), Positive regulation of cell adhesion (NES = 2.06; p = 0.0058; Adj. P = 0.013), Regulation of response to stress (NES = 2.50; p = 0; Adj. P = 0.0023), Positive regulation of cell death (NES = 1.84; p = 0.0179; Adj. P = 0.0326), Apoptotic signaling pathway (NES = 1.59; p= 0.047; Adj. P = 0.084).I,J - ISH stains of Irs2 mRNA, and qPCR for Irs2 mRNA and let-7-5p miRNA, in the rostral telencephalon of control and Rx-Dicer mutant embryos. Dashed line indicates basal border, dotted line indicates apical surface. Arrowheads indicate area with the greatest increase in Irs2 expression. BG, basal ganglia; H, hippocampus; NCx, neocortex; OB, olfactory bulb; Spt, septum. Histograms represent mean ± SEM (logarithmic scale); symbols in plots indicate values for individual embryos; t-test, *P <0.05, **P <0.01. N = 6-9 replicates per group. Scale bar, 100 µm.


Figure 5A - C - Immunostains for activated p53 ( red ) in the rostral telencephalon of control and Rx - Dicer mutant embryos at E12 . 5 , and quantification of positive cells ( C ) . Dashed lines indicate apical side , dotted lines indicate basal side . N = 4 replicates per genotype . Scale bar , 50µm . D - L - Rostral telencephalon of E12 . 5 embryos of the indicated genotypes immunostained for the detection of apoptotic cells ( Casp3 ) , neurons ( Tuj1 ) and the apical complex protein Par3 , and quantification of cells positive for Casp3 ( H ) . N = 4 - 7 replicates per genotype . Scale bar , 50 µm . M - O - Sagittal sections through the rostral telencephalon of E17 . 5 embryos of the indicated genotypes . Note the rosettes and general disorganization in Rx - Dicer single mutants , and complete rescue of this phenotype in Rx - Dicer - p53 double mutants . NCx , neocortex ; OB , olfactory bulb . Scale bar , 1 mm .

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

Figure 5A-C - Immunostains for activated p53 (red) in the rostral telencephalon of control and Rx-Dicer mutant embryos at E12.5, and quantification of positive cells (C). Dashed lines indicate apical side, dotted lines indicate basal side. N = 4 replicates per genotype. Scale bar, 50µm.D-L - Rostral telencephalon of E12.5 embryos of the indicated genotypes immunostained for the detection of apoptotic cells (Casp3), neurons (Tuj1) and the apical complex protein Par3, and quantification of cells positive for Casp3 (H). N = 4-7 replicates per genotype. Scale bar, 50 µm.M-O - Sagittal sections through the rostral telencephalon of E17.5 embryos of the indicated genotypes. Note the rosettes and general disorganization in Rx-Dicer single mutants, and complete rescue of this phenotype in Rx-Dicer-p53 double mutants. NCx, neocortex; OB, olfactory bulb. Scale bar, 1 mm.


Figure 6A - Relative Irs2 mRNA levels in HEK cells upon transfection with Gfp - or Irs2 - encoding plasmids . Mean ± SEM ; t - test , * * * * P < 0 . 0001 . N = 6 replicates per group . B - D - Distribution of neurons ( Tuj1 , red ) , GFP ( green ) and mitoses ( PH3 , white ) in the rostral telencephalon of wild - type E14 . 5 or E17 . 5 mouse embryos , as indicated , electroporated at E12 . 5 with the indicated plasmid combinations . Dashed line indicates the apical surface . Insets show the same field of view , with location and extent of electroporations . Note the overlap of rosettes ( arrows ) with ectopic neurons at the apical surface ( arrowheads ) and increased basal mitoses , upon overexpression of Irs2 ( C ) . Asterisk indicates absence of malformation in nearby cortex . At E17 . 5 ( D ) GFP + rosettes remain in deep cortical layers ( arrowheads ) . E - H - Details of rosettes ( large arrows in E ; dashed lines in F - H ) from Irs2 + Gfp - overexpressing E14 . 5 embryos immunostained as indicated . Small arrows indicate apical mitoses , arrowheads indicate basal mitoses ( F , G ) and Par3 + apical surface at the centre of rosettes ( H ) , dotted lines indicate apical surface , dashed lines indicate basal side of rosettes . Inset in ( H ) is at the same scale . I - M - Distribution of neurons ( Tuj1 , red ) , GFP ( green ) and mitotic ( PH3 , white ) or apoptotic ( Casp3 , white ) cells in the rostral telencephalon of wild - type E13 . 5 embryos electroporated at E12 . 5 with the indicated plasmids , and quantification of Casp3 + cells ( M ) . Dashed line indicates apical surface , dotted lines indicate borders of the cortical plate ( CP ) . Nascent rosettes ( arrows ) are next to ectopic ventricular neurons ( arrowheads in K ' ) and basal mitoses , without significant apoptosis in VZ ( L ' , M ) . Mean ± SEM ; t - test ; ns , not significant , * P < 0 . 05 . N = 3 - 6 replicates per group . N - Distribution of neurons ( Tuj1 , red ) in the rostral telencephalon of wild - type E14 . 5 embryos electroporated at E12 . 5 with a combination of plasmids encoding Irs2 , let - 7a , let - 7b and let - 7c , and Gfp . Dashed line indicates apical surface . Formation of rosettes in Irs2 - expressing embryos is rescued by overexpression of let - 7 . Inset shows the same field of view , with location and extent of electroporation .

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

Figure 6A - Relative Irs2 mRNA levels in HEK cells upon transfection with Gfp- or Irs2-encoding plasmids. Mean ± SEM; t-test, ****P < 0.0001. N = 6 replicates per group.B-D - Distribution of neurons (Tuj1, red), GFP (green) and mitoses (PH3, white) in the rostral telencephalon of wild-type E14.5 or E17.5 mouse embryos, as indicated, electroporated at E12.5 with the indicated plasmid combinations. Dashed line indicates the apical surface. Insets show the same field of view, with location and extent of electroporations. Note the overlap of rosettes (arrows) with ectopic neurons at the apical surface (arrowheads) and increased basal mitoses, upon overexpression of Irs2 (C). Asterisk indicates absence of malformation in nearby cortex. At E17.5 (D) GFP+ rosettes remain in deep cortical layers (arrowheads).E-H - Details of rosettes (large arrows in E; dashed lines in F-H) from Irs2+Gfp-overexpressing E14.5 embryos immunostained as indicated. Small arrows indicate apical mitoses, arrowheads indicate basal mitoses (F,G) and Par3+ apical surface at the centre of rosettes (H), dotted lines indicate apical surface, dashed lines indicate basal side of rosettes. Inset in (H) is at the same scale.I-M - Distribution of neurons (Tuj1, red), GFP (green) and mitotic (PH3, white) or apoptotic (Casp3, white) cells in the rostral telencephalon of wild-type E13.5 embryos electroporated at E12.5 with the indicated plasmids, and quantification of Casp3+ cells (M). Dashed line indicates apical surface, dotted lines indicate borders of the cortical plate (CP). Nascent rosettes (arrows) are next to ectopic ventricular neurons (arrowheads in K') and basal mitoses, without significant apoptosis in VZ (L',M). Mean ± SEM; t-test; ns, not significant, *P < 0.05. N = 3-6 replicates per group. N - Distribution of neurons (Tuj1, red) in the rostral telencephalon of wild-type E14.5 embryos electroporated at E12.5 with a combination of plasmids encoding Irs2, let-7a, let-7b and let-7c, and Gfp. Dashed line indicates apical surface. Formation of rosettes in Irs2-expressing embryos is rescued by overexpression of let-7. Inset shows the same field of view, with location and extent of electroporation.


Figure 7A - Frequency of rosette formation in the experimental conditions indicated . n , number of embryos per condition . B - G - Distribution of neurons ( Tuj1 , red ) , GFP ( green ) and mitoses ( PH3 , white ) in the rostral telencephalon of wild - type E14 . 5 mouse embryos electroporated at E12 . 5 with the indicated plasmid combinations . Inset shows the same field of view , with location and extent of electroporations . In ( C ) and ( E ) , arrows indicate rosettes and arrowheads indicate neuronal ventricular ectopias . ( D ) and ( F ) are high magnification details of individual rosettes ( dashed lines ) , where arrowheads indicate apical and basal mitoses . Loss of endogenous let - 7 drives the formation of rosettes and ventricular neuronal ectopias ( C ) , which is rescued with the additional loss of Irs2 ( G ) .

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

Figure 7A - Frequency of rosette formation in the experimental conditions indicated. n, number of embryos per condition.B-G - Distribution of neurons (Tuj1, red), GFP (green) and mitoses (PH3, white) in the rostral telencephalon of wild-type E14.5 mouse embryos electroporated at E12.5 with the indicated plasmid combinations. Inset shows the same field of view, with location and extent of electroporations. In (C) and (E), arrows indicate rosettes and arrowheads indicate neuronal ventricular ectopias. (D) and (F) are high magnification details of individual rosettes (dashed lines), where arrowheads indicate apical and basal mitoses. Loss of endogenous let-7 drives the formation of rosettes and ventricular neuronal ectopias (C), which is rescued with the additional loss of Irs2 (G).


Figure 8B - G - Stains and quantifications of human cerebral organoids electroporated with the indicated construct combinations , to reveal the frequency of electroporated cells remaining as progenitors ( % GFP + Ki67 + / GFP + ) . Histograms represent mean ± SEM ; symbols in plots indicate values for individual organoids ; X2 - test , * P < 0 . 05 . N = 8 - 11 replicates per group . Dashed lines indicate ventricular surface , solid arrowheads indicate Ki67 + / GFP + cells , open arrowheads indicate Ki67 - / GFP + cells . Scale bars , 50 µm .

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

Figure 8B-G - Stains and quantifications of human cerebral organoids electroporated with the indicated construct combinations, to reveal the frequency of electroporated cells remaining as progenitors (%GFP+Ki67+/GFP+). Histograms represent mean ± SEM; symbols in plots indicate values for individual organoids; X2-test, *P < 0.05. N = 8-11 replicates per group. Dashed lines indicate ventricular surface, solid arrowheads indicate Ki67+/GFP+ cells, open arrowheads indicate Ki67-/GFP+ cells. Scale bars, 50 µm.


Figure 1q - RT - PCR reveals that miR - 181a / b silencing leads to upregulation of miR - 181a / b predicted targets in SH - SY5Y cells . N = 3 independent experiments . miR - 181 - mimic transfection specifically inhibits Luciferase activity of constructs containing WT 3 ' - UTR predicted target sequences . Point mutations ( mut ) in miR - 181a / b binding sites abolish Luciferase repression in all cases apart from PPARGC1A . Data are normalized to negative mimic transfection ( dashed line ) . N = 6 independent experiments . q - RT - PCR reveals upregulation of miR - 181a / b targets in the eyes of miR - 181a / b - 1 - / - vs . miR - 181a / b - 1 + / + animals . n ≥ 5 animals / genotype . miR - 181a / b - 1 - / - mice show increased mtDNA content vs . miR - 181a / b - 1 + / + mice as measured by q - PCR . N = 4 animals / genotype . WB analysis ( left panel ) reveals increased levels of mitochondrial proteins in the eyes of miR - 181a / b - 1 - / - ( - / - ) vs . miR - 181a / b - 1 + / + ( + / + ) mice ( quantified in the right panel ) . Data are normalized to either p115 or Gapdh . N ≥ 3 animals / genotype . Please note that all compared bands from + / + and - / - samples are from the same blots which were cropped and shown split for the sake of data presentation clarity . WB analysis on mitochondrial and cytosolic fractions ( left panel ) show increased levels of p62 and Parkin in mitochondrial fraction from the eye of - / - vs . + / + mice ( quantified in the right panel ) . Data are normalized to Ndufb8 or Gapdh for mitochondrial and cytosolic fractions , respectively . N = 3 animals / genotype . Cell death analysis shows that miR - 181a / b silencing protects SH - SY5Y cells from FCCP treatment . N ≥ 7 independent experiments .

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

Figure 1q-RT-PCR reveals that miR-181a/b silencing leads to upregulation of miR-181a/b predicted targets in SH-SY5Y cells. N=3 independent experiments.miR-181-mimic transfection specifically inhibits Luciferase activity of constructs containing WT 3'-UTR predicted target sequences. Point mutations (mut) in miR-181a/b binding sites abolish Luciferase repression in all cases apart from PPARGC1A. Data are normalized to negative mimic transfection (dashed line). N=6 independent experiments.q-RT-PCR reveals upregulation of miR-181a/b targets in the eyes of miR-181a/b-1-/- vs. miR-181a/b-1+/+ animals. n≥5 animals/genotype.miR-181a/b-1-/- mice show increased mtDNA content vs. miR-181a/b-1+/+ mice as measured by q-PCR. N=4 animals/genotype.WB analysis (left panel) reveals increased levels of mitochondrial proteins in the eyes of miR-181a/b-1-/- (-/-) vs. miR-181a/b-1+/+ (+/+) mice (quantified in the right panel). Data are normalized to either p115 or Gapdh. N≥3 animals/genotype. Please note that all compared bands from +/+ and -/- samples are from the same blots which were cropped and shown split for the sake of data presentation clarity.WB analysis on mitochondrial and cytosolic fractions (left panel) show increased levels of p62 and Parkin in mitochondrial fraction from the eye of -/- vs. +/+ mice (quantified in the right panel). Data are normalized to Ndufb8 or Gapdh for mitochondrial and cytosolic fractions, respectively. N=3 animals/genotype.Cell death analysis shows that miR-181a/b silencing protects SH-SY5Y cells from FCCP treatment. N≥7 independent experiments.


Figure 2TUNEL assays on eye and brain sections of stage ( st ) 30 medakafish control and MO - injected embryos reveal a decrease in the number of apoptotic cells in both hccs - MO / miR - 181a / b - MO - and cox7b - MO / miR - 181a / b - MO - injected compared to hccs - MO - and cox7b - MO - injected embryos . Scale bars are 20μm . TUNEL - positive cells / eye . n ≥ 5 eyes for each model . Caspase assays show restored levels of caspase - 3 and caspase - 9 activities in hccs - MO / miR - 181a / b - MO - and cox7b - MO / miR - 181a / b - MO - injected embryos with respect to hccs - MO - and cox7b - MO - injected embryos . n ≥ 5 embryos for each model .

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

Figure 2TUNEL assays on eye and brain sections of stage (st)30 medakafish control and MO-injected embryos reveal a decrease in the number of apoptotic cells in both hccs-MO/miR-181a/b-MO- and cox7b-MO/miR-181a/b-MO-injected compared to hccs-MO- and cox7b-MO-injected embryos. Scale bars are 20μm.TUNEL-positive cells/eye. n≥5 eyes for each model.Caspase assays show restored levels of caspase-3 and caspase-9 activities in hccs-MO/miR-181a/b-MO- and cox7b-MO/miR-181a/b-MO-injected embryos with respect to hccs-MO- and cox7b-MO-injected embryos. n≥5 embryos for each model.


Figure 3Representative images of st30 medaka embryos injected with hccs - MO ( B ) alone or co - injected with miR - 181a / b - MOs Co - injection of miR - 181a / b - MOs rescues microphthalmia and microcephaly in both hccs - MO embryos . hccs - MO / miR - 181a / b - MO - b - MO - injected embryos were treated with Baf - A1 , PD98059 or HA14 - 1 . Baf - A1 treatment counteracts the protective effect of miR - 181a / b downregulation in hccs - MO / miR - 181a / b - MO embryos PD98059 treatment does not interfere with the modulation of the MLS phenotype mediated by miR - 181a / b downregulation HA14 - 1 treatment counteracts the effect of miR - 181a / b downregulation in the hccs - MO / miR - 181a / b - MO model Scale bars are 100μm . Representative images of st30 medaka embryos injected with cox7b - MO ( H ) alone or co - injected with miR - 181a / b - MOs Co - injection of miR - 181a / b - MOs rescues microphthalmia and microcephaly in cox7b - MO embryos . cox7b - MO / miR - 181a / b - MO - injected embryos were treated with Baf - A1 , PD98059 or HA14 - 1 . Baf - A1 treatment counteracts the protective effect of miR - 181a / b downregulation in cox7b - MO / miR - 181a / b - MO embryos PD98059 treatment does not interfere with the modulation of the MLS phenotype mediated by miR - 181a / b downregulation Scale bars are 100μm . Percentage of embryos with or without MLS phenotype in the different conditions in hccs - MO embryos . N ≥ 300 embryos / conditions . Percentage of embryos with or without MLS phenotype in the different conditions in cox7b - MO ( N ) embryos . N ≥ 300 embryos / conditions .

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

Figure 3Representative images of st30 medaka embryos injected with hccs-MO (B) alone or co-injected with miR-181a/b-MOs Co-injection of miR-181a/b-MOs rescues microphthalmia and microcephaly in both hccs-MO embryos. hccs-MO/miR-181a/b-MO- b-MO-injected embryos were treated with Baf-A1, PD98059 or HA14-1. Baf-A1 treatment counteracts the protective effect of miR-181a/b downregulation in hccs-MO/miR-181a/b-MO embryos PD98059 treatment does not interfere with the modulation of the MLS phenotype mediated by miR-181a/b downregulation HA14-1 treatment counteracts the effect of miR-181a/b downregulation in the hccs-MO/miR-181a/b-MO model Scale bars are 100μm.Representative images of st30 medaka embryos injected with cox7b-MO (H) alone or co-injected with miR-181a/b-MOs Co-injection of miR-181a/b-MOs rescues microphthalmia and microcephaly in cox7b-MO embryos. cox7b-MO/miR-181a/b-MO-injected embryos were treated with Baf-A1, PD98059 or HA14-1. Baf-A1 treatment counteracts the protective effect of miR-181a/b downregulation in cox7b-MO/miR-181a/b-MO embryos PD98059 treatment does not interfere with the modulation of the MLS phenotype mediated by miR-181a/b downregulation Scale bars are 100μm.Percentage of embryos with or without MLS phenotype in the different conditions in hccs-MO embryos. N≥300 embryos/conditions.Percentage of embryos with or without MLS phenotype in the different conditions in cox7b-MO (N) embryos. N≥300 embryos/conditions.


Figure 4Immunofluorescence analysis with anti - NeuN antibody in the retina of miR - 181a / b - 1 + / + and miR - 181a / b - 1 - / - mice intravitreally injected with Rotenone or DMSO . RGCs are preserved in miR - 181a / b - 1 - / - rotenone - injected mice with respect to controls at both one and two weeks after injection . Scale bars are 50μm . Number of RGCs / mm ( indicated as % ) at one - and two - weeks post - injection . N = 10 . NADH dehydrogenase histochemical reaction on retinal sections of miR - 181a / b - 1 + / + and miR - 181a / b - 1 - / - mice intravitreally injected with Rotenone or DMSO . At one week post - injection , NADH dehydrogenase activity is lost in RGCs ( GCL , areas within dashed lines ) of miR - 181a / b - 1 + / + Rotenone - injected eyes , while it is preserved in those of miR - 181a / b - 1 - / - Rotenone - injected eyes . GCL , Ganglion Cells Layer ONL ; INL , Inner Nuclear Layer ; Outer Nuclear Layer . Scale bars are 50μm . Immunofluorescence analysis , with an anti - CoxIV antibody , in the retina of miR - 181a / b - 1 + / + and miR - 181a / b - 1 - / - mice , injected with either Rotenone or DMSO , showed preserved mitochondria in miR - 181a / b - 1 - / - rotenone - injected eyes at one - week post - injection . Dashed boxes indicate the area of magnifications shown in right panels . Scale bars are 10μm . Graphical representation of the results of the optokinetic tracking assays reported as fold change of cycles / degree . Visual acuity is preserved in miR - 181a / b - 1 - / - rotenone - injected mice with respect to controls at both one and two weeks after injection . N = 10 .

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

Figure 4Immunofluorescence analysis with anti-NeuN antibody in the retina of miR-181a/b-1+/+ and miR-181a/b-1-/- mice intravitreally injected with Rotenone or DMSO. RGCs are preserved in miR-181a/b-1-/- rotenone-injected mice with respect to controls at both one and two weeks after injection. Scale bars are 50μm.Number of RGCs/mm (indicated as %) at one- and two-weeks post-injection. N=10.NADH dehydrogenase histochemical reaction on retinal sections of miR-181a/b-1+/+ and miR-181a/b-1-/- mice intravitreally injected with Rotenone or DMSO. At one week post-injection, NADH dehydrogenase activity is lost in RGCs (GCL, areas within dashed lines) of miR-181a/b-1+/+ Rotenone-injected eyes, while it is preserved in those of miR-181a/b-1-/- Rotenone-injected eyes. GCL, Ganglion Cells Layer ONL; INL, Inner Nuclear Layer; Outer Nuclear Layer. Scale bars are 50μm.Immunofluorescence analysis, with an anti-CoxIV antibody, in the retina of miR-181a/b-1+/+ and miR-181a/b-1-/- mice, injected with either Rotenone or DMSO, showed preserved mitochondria in miR-181a/b-1-/- rotenone-injected eyes at one-week post-injection. Dashed boxes indicate the area of magnifications shown in right panels. Scale bars are 10μm.Graphical representation of the results of the optokinetic tracking assays reported as fold change of cycles/degree. Visual acuity is preserved in miR-181a/b-1-/- rotenone-injected mice with respect to controls at both one and two weeks after injection. N=10.


Figure 5Immunofluorescence analysis with anti - NeuN antibody in the retina of WT , Ndufs4 - / - , Ndufs4 - / - / miR - 181a / b - 1 - / - and miR - 181a / b - 1 - / - mice . RGCs are preserved in Ndufs4 - / - / miR - 181a / b - 1 - / - with respect to Ndufs4 - / - mice . Scale bars are 50μm . Number of RGCs / mm ( % ) . N = 5 . Graphical representation of the results of the optokinetic tracking assays reported as fold change of cycles / degree . Visual acuity is preserved in Ndufs4 - / - / miR - 181a / b - 1 - / - with respect to Ndufs4 - / - mice . N ≥ 8 . Electroretinographic analysis reveals amelioration of b - wave patterns in Ndufs4 - / - / miR - 181a / b - 1 - / - with respect to Ndufs4 - / - mice . N ≥ 5 .

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Figure 5Immunofluorescence analysis with anti-NeuN antibody in the retina of WT, Ndufs4-/-, Ndufs4-/-/miR-181a/b-1-/- and miR-181a/b-1-/- mice. RGCs are preserved in Ndufs4-/-/miR-181a/b-1-/- with respect to Ndufs4-/- mice. Scale bars are 50μm.Number of RGCs/mm (%). N=5.Graphical representation of the results of the optokinetic tracking assays reported as fold change of cycles/degree. Visual acuity is preserved in Ndufs4-/-/miR-181a/b-1-/- with respect to Ndufs4-/- mice. N≥8.Electroretinographic analysis reveals amelioration of b-wave patterns in Ndufs4-/-/miR-181a/b-1-/- with respect to Ndufs4-/-mice. N≥5.


Figure 6Electron microscopy analysis shows amelioration of mitochondria morphology and increase of mitochondrial number in Ndufs4 - / - / miR - 181a / b - 1 - / - vs . Ndufs4 - / - mice . Scale bars are 1μm . The quantitative increase of mitochondria is reported in C as number of mitochondria / RGC , at p30 and p55 . N ≥ 2 animals / genotype . Ndufs4 - / - / miR - 181a / b - 1 - / - mice show increased mtDNA content vs . Ndufs4 - / - mice as measured by q - PCR . N ≥ 4 animals / genotype . Biochemical activity of MRC complexes I , II and IV normalized by the percentage of citrate synthase ( CS ) activity in Ndufs4 - / - / miR - 181a / b - 1 - / - vs . Ndufs4 - / - mice . N ≥ 3 animals / genotype . p ‐ values were calculated by two tailed Student ' s t - test ; Error bars are SEM .

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

Figure 6Electron microscopy analysis shows amelioration of mitochondria morphology and increase of mitochondrial number in Ndufs4-/-/miR-181a/b-1-/- vs. Ndufs4-/- mice. Scale bars are 1μm. The quantitative increase of mitochondria is reported in C as number of mitochondria/RGC, at p30 and p55. N≥2 animals/genotype.Ndufs4-/-/miR-181a/b-1-/- mice show increased mtDNA content vs. Ndufs4-/- mice as measured by q-PCR. N≥4 animals/genotype.Biochemical activity of MRC complexes I, II and IV normalized by the percentage of citrate synthase (CS) activity in Ndufs4-/-/miR-181a/b-1-/- vs. Ndufs4-/- mice. N≥3 animals/genotype. p‐values were calculated by two tailed Student's t-test; Error bars are SEM.


Figure 2Tap - 1 is required for the secretion of TseL . Pellet ( P ) and supernatant ( S ) of bacterial cultures were analyzed by SDS - PAGE . The result of immunoblotting with antisera against TseL and Hcp and purified antibodies against DnaK is shown . The asterisk marks an unspecific band , detected by the antiserum against TseL , present in the pre - immunized serum . One representative experiment of three independent experiments is shown . Tap - 1 is required for TseL - mediated killing . Wild - type C6706 or C6706 lacking tsiV1 were exposed to the indicated V52 mutants in a killing assay . The y - axis indicates the number of surviving C6706 . Tap - 1 is required for TseL function . Killing assays in which V52Δtap - 1 was mixed with wild - type C6706 or mutants lacking tsiV1 , tsiV2 , or tsiV3 . The y - axis indicates surviving C6706 .

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Figure 2Tap-1 is required for the secretion of TseL. Pellet (P) and supernatant (S) of bacterial cultures were analyzed by SDS-PAGE. The result of immunoblotting with antisera against TseL and Hcp and purified antibodies against DnaK is shown. The asterisk marks an unspecific band, detected by the antiserum against TseL, present in the pre-immunized serum. One representative experiment of three independent experiments is shown.Tap-1 is required for TseL-mediated killing. Wild-type C6706 or C6706 lacking tsiV1 were exposed to the indicated V52 mutants in a killing assay. The y-axis indicates the number of surviving C6706.Tap-1 is required for TseL function. Killing assays in which V52Δtap-1 was mixed with wild-type C6706 or mutants lacking tsiV1, tsiV2, or tsiV3. The y-axis indicates surviving C6706.


Figure 3TseL secretion depends on VgrG - 1 . Western blot analysis of pellet ( P ) and supernatant ( S ) samples of the indicated strains . Samples were immunoblotted with antisera against TseL and Hcp and purified antibodies against DnaK . The unspecific band detected by rabbit serum is marked with an asterisk . One representative of three experiments is shown . VgrG - 1 is required for TseL - mediated killing . Killing assay with wild - type V52 or indicated mutants and wild - type C6706 or a mutant lacking the immunity gene tsiV1 . The y - axis indicates surviving C6706 . VasX - and VgrG - 3 - mediated killing but not TseL - mediated killing occurs in the absence of VgrG - 1 . V52ΔvgrG - 1 was mixed in a killing assay with wild - type C6706 or mutants lacking immunity genes tsiV1 , tsiV2 , or tsiV3 ( specific for TseL , VasX , and VgrG - 3 , respectively ) . The y - axis indicates surviving C6706 .

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

Figure 3TseL secretion depends on VgrG-1. Western blot analysis of pellet (P) and supernatant (S) samples of the indicated strains. Samples were immunoblotted with antisera against TseL and Hcp and purified antibodies against DnaK. The unspecific band detected by rabbit serum is marked with an asterisk. One representative of three experiments is shown.VgrG-1 is required for TseL-mediated killing. Killing assay with wild-type V52 or indicated mutants and wild-type C6706 or a mutant lacking the immunity gene tsiV1. The y-axis indicates surviving C6706.VasX- and VgrG-3-mediated killing but not TseL-mediated killing occurs in the absence of VgrG-1. V52ΔvgrG-1 was mixed in a killing assay with wild-type C6706 or mutants lacking immunity genes tsiV1, tsiV2, or tsiV3 (specific for TseL, VasX, and VgrG-3, respectively). The y-axis indicates surviving C6706.


Figure 4A , B Interactions between Tap - 1 and TseL ( A ) or VgrG - 1 ( B ) . Shown are immunoblots of lysates ( total ) and immunoprecipitates with anti - FLAG affinity beads ( IP : FLAG ) of V52Δtap - 1 ( A ) or V52 vgrG - 1 : : myc ( B ) transformed with empty vector or a plasmid encoding His - tagged or FLAG - tagged Tap - 1 . C Interaction between TseL and VgrG - 1 . Shown are immunoblots of lysates ( total ) and immunoprecipitates with an anti - FLAG affinity beads ( IP : FLAG ) of V52 or V52Δtap - 1 transformed with empty vector or a plasmid encoding HIS - tagged or FLAG - tagged VgrG - 1 .

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

Figure 4A, B Interactions between Tap-1 and TseL (A) or VgrG-1 (B). Shown are immunoblots of lysates (total) and immunoprecipitates with anti-FLAG affinity beads (IP:FLAG) of V52Δtap-1 (A) or V52 vgrG-1::myc (B) transformed with empty vector or a plasmid encoding His-tagged or FLAG-tagged Tap-1.C Interaction between TseL and VgrG-1. Shown are immunoblots of lysates (total) and immunoprecipitates with an anti-FLAG affinity beads (IP:FLAG) of V52 or V52Δtap-1 transformed with empty vector or a plasmid encoding HIS-tagged or FLAG-tagged VgrG-1.


Figure 5Specificity of the C - terminal Tap - 1 segment . Killing assay in which the lack of tap - 1 in V52 is complemented by empty vector ( control ) or one of two different alleles of tap - 1 . The ability to kill in a TseL - dependent manner is indicated by the logarithm of surviving C6706ΔtsiV1 . Specificity of N - terminal segment of Tap - 1 . Killing assay in which the lack of tap - 1 in 1587 is complemented by empty vector ( control ) or one of two different alleles of tap - 1 . The ability to facilitate killing by the effector A55 _ 1502 is indicated by the logarithm of surviving 1587ΔA55 _ 1501 - 03 .

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

Figure 5Specificity of the C-terminal Tap-1 segment. Killing assay in which the lack of tap-1 in V52 is complemented by empty vector (control) or one of two different alleles of tap-1. The ability to kill in a TseL-dependent manner is indicated by the logarithm of surviving C6706ΔtsiV1.Specificity of N-terminal segment of Tap-1. Killing assay in which the lack of tap-1 in 1587 is complemented by empty vector (control) or one of two different alleles of tap-1. The ability to facilitate killing by the effector A55_1502 is indicated by the logarithm of surviving 1587ΔA55_1501-03.


Figure 2 ( A ) The time courses of normalized FRET ratio ( Mean ± SEM ) of calcium biosensor before and after shockwave stimulation in HBSS Ca2 + medium with ( yellow line , n = 6 , N = 3 ) or without the Piezo1 expression ( black line , n = 6 , N = 3 ) . ( B ) The representative FRET / ECFP ratio images of calcium biosensor in Piezo1 - expressed HEK cells before ( top left ) and after ( top right ) LIS stimulation . Phase image with laser initiating point ( left ) and fluorescent image indicating Piezo1 expression ( right ) were also shown on the bottom panels ( Scale bar , 40 μm ) . Color scale bars in the figures are to show the FRET / ECFP ratios , with cold and hot colors representing low and high ratios , respectively . ( C ) The time course of normalized FRET ratio ( Mean ± SEM ) of calcium biosensor in the Piezo1 - expressing HEK cells before and after multiple shockwave stimulations in HBSS Ca2 + medium ( n = 6 , N = 3 ) .

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

Figure 2(A) The time courses of normalized FRET ratio (Mean ± SEM) of calcium biosensor before and after shockwave stimulation in HBSS Ca2+ medium with (yellow line, n = 6, N = 3) or without the Piezo1 expression (black line, n = 6, N = 3). (B) The representative FRET/ECFP ratio images of calcium biosensor in Piezo1-expressed HEK cells before (top left) and after (top right) LIS stimulation. Phase image with laser initiating point (left) and fluorescent image indicating Piezo1 expression (right) were also shown on the bottom panels (Scale bar, 40 μm). Color scale bars in the figures are to show the FRET/ECFP ratios, with cold and hot colors representing low and high ratios, respectively. (C) The time course of normalized FRET ratio (Mean ± SEM) of calcium biosensor in the Piezo1-expressing HEK cells before and after multiple shockwave stimulations in HBSS Ca2+ medium (n = 6, N = 3).


Figure 3 ( A ) The representative ECFP / FRET ratio images of FAK biosensor in Piezo1 overexpressed HEK cells before ( left ) and after ( right ) shockwave stimulation ( Scale bar , 40 μm ) . Color scale bars in the figures are to show the ECFP / FRET ratios , with cold and hot colors representing low and high ratios , respectively . ( B ) The time courses of normalized ECFP / FRET ratio ( Mean ± SEM ) of FAK FRET biosensor before and after shockwave stimulation in HBSS Ca2 + media with ( green line , n = 16 , N = 3 ) or without Piezo1 ( black line , n = 10 , N = 3 ) . ( C ) The percentage change of FRET ratio of the FAK FRET biosensor in HEK cells with or without Piezo1 after shockwave stimulation in HBSS Ca2 + medium . * P < 0 . 05 from student two - tailed t - test .

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Figure 3(A) The representative ECFP/FRET ratio images of FAK biosensor in Piezo1 overexpressed HEK cells before (left) and after (right) shockwave stimulation (Scale bar, 40 μm). Color scale bars in the figures are to show the ECFP/FRET ratios, with cold and hot colors representing low and high ratios, respectively. (B) The time courses of normalized ECFP/FRET ratio (Mean ± SEM) of FAK FRET biosensor before and after shockwave stimulation in HBSS Ca2+ media with (green line, n = 16, N = 3) or without Piezo1 (black line, n = 10, N = 3). (C) The percentage change of FRET ratio of the FAK FRET biosensor in HEK cells with or without Piezo1 after shockwave stimulation in HBSS Ca2+ medium. *P < 0.05 from student two-tailed t-test.


Figure 4 ( A ) The representative ECFP / FRET ratio images of FAK biosensor in Piezo1 expressing HEK cells before and after 25 μM Yoda1 stimulation ( Scale bar , 10 μm ) . Color scale bars in the figures indicate ECFP / FRET ratios , with cold and hot colors representing low and high ratios , respectively . ( B ) The time courses of normalized FRET ratio ( Mean ± SEM ) of FAK FRET biosensor before and after Yoda1 stimulation with ( pink line , n = 10 , N = 3 ) or without Piezo1 ( black line , n = 11 , N = 3 ) . The grey line represents the DMSO control group ( n = 8 , N = 3 ) . ( C ) The representative FRET / ECFP ratio images of the calcium biosensor in the Piezo1 - expressing HEK cells before and after 25 μM Yoda1 stimulation ( Scale bar , 10 μm ) . Color scale bars in the figures indicate FRET / ECFP ratios , with cold and hot colors representing low and high ratios , respectively . ( D ) The time courses of normalized FRET ratio ( Mean ± SEM ) of calcium biosensor in the Piezo1 - expressing HEK cells ( purple line , n = 9 , N = 3 ) before and after 25 μM Yoda1 stimulation in culture medium .

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

Figure 4(A) The representative ECFP/FRET ratio images of FAK biosensor in Piezo1 expressing HEK cells before and after 25 μM Yoda1 stimulation (Scale bar, 10 μm). Color scale bars in the figures indicate ECFP/FRET ratios, with cold and hot colors representing low and high ratios, respectively. (B) The time courses of normalized FRET ratio (Mean ± SEM) of FAK FRET biosensor before and after Yoda1 stimulation with (pink line, n = 10, N = 3) or without Piezo1 (black line, n = 11, N = 3). The grey line represents the DMSO control group (n = 8, N = 3).(C) The representative FRET/ECFP ratio images of the calcium biosensor in the Piezo1-expressing HEK cells before and after 25 μM Yoda1 stimulation (Scale bar, 10 μm). Color scale bars in the figures indicate FRET/ECFP ratios, with cold and hot colors representing low and high ratios, respectively. (D) The time courses of normalized FRET ratio (Mean ± SEM) of calcium biosensor in the Piezo1-expressing HEK cells (purple line, n = 9, N = 3) before and after 25 μM Yoda1 stimulation in culture medium.


Figure 7 ( A ) & ( B ) , The time courses ( Mean ± SEM ) ( A ) and representative images ( Scale bar , 10 μm ) of FRET ratio of SHP2 biosensor ( Y542F ) before and after 25 μM or 0 . 5 μM Yoda1 stimulation in cells with or without Piezo1 overexpression ( red bar , n = 5 , N = 3 ; blue bar , n = 5 , N = 3 ; purple bar , n = 11 , N = 3 ; green bar , n = 5 , N = 3 ) .

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Figure 7(A)&(B), The time courses (Mean ± SEM) (A) and representative images (Scale bar, 10 μm) of FRET ratio of SHP2 biosensor (Y542F) before and after 25 μM or 0.5 μM Yoda1 stimulation in cells with or without Piezo1 overexpression (red bar, n = 5, N=3; blue bar, n = 5, N=3; purple bar, n = 11, N=3; green bar, n = 5, N=3).


Figure 1B ) Expression of the 21U sensor ( left ) and DAPI staining ( right ) of gonad arms in the indicated genetic backgrounds . Gonads are outlined by a dashed line . The mCherry signal is represented in pseudo - colours [ LUT fire ( ImageJ ) ] to reflect differences in the intensity of the signal . Number of animals analysed and with indicated phenotype are given in the panel . Animals not showing the activation of the 21U sensor ( + ) were still silenced , and animals that did not show the silenced 21U sensor ( RNAe ) state were expressing weakly , comparable to the 21U sensor ( + ) . Scale bar : 25 μm . C ) Tc1 reversion assay in different genetic backgrounds . All the strains tested carried the unc - 22 : : Tc1 ( st136 ) allele . Tc1 excision can result in restoration of unc - 22 function , which can be scored visually . Negative control = unc - 22 : : Tc1 ( st136 ) in a wild type background ; positive control = wago - 1 / - 2 / - 3 . Two independent experiments per strain are represented . D ) Crossing scheme to address the re - initiation of silencing of the 21U sensor . A mut - 7 mutant male expressing the 21U sensor is crossed with either a wild type ( left ) , prg - 1 ( middle ) or pid - 2 ( right ) mutant hermaphrodite . Their F1 offspring was scored for expression of the 21U sensor by microscopy . Three states of expression were scored , and represented in a pie chart . The three expression states are exemplified by representative images at the bottom : OFF ( left ) , FAINT ( right ) or ON ( middle ) . DIC images are shown above the fluorescence panels . Gonads are outlined by a dashed line . Scale bar : 25 µm . E ) Crossing scheme to address if maternal 21U RNAs are sufficient to re - initiate the silencing of the 21U sensor . A pid - 1 mutant male expressing the 21U sensor is crossed with a hermaphrodite , heterozygous for the same mutation . All their F1 offspring inherit a pool of 21U RNAs from the hermaphrodite , but in 50 % of the F1 , which is pid - 1 homozygous mutant , no zygotic PID - 1 is present , hence no zygotic 21U RNAs can be made . The silencing or expression of the 21U sensor in the pid - 1 homozygous mutant F1 has been scored by microscopy , and depicted in a pie chart . At the bottom , a representative image of an animal carrying a silenced 21U sensor ( lower : mCherry signal ; upper : DAPI staining ) in pid - 1 mutant offspring . Gonads are outlined by a dashed line . Scale bar : 25 μm . F ) Crossing scheme to test the role of PID - 2 in re - initiating the silencing of the 21U sensor , mediated by maternally provided 21U RNAs only . The expression of the 21U sensor in the F1 has been scored by microscopy , and depicted in a pie chart . White arrow heads indicate the many arrayed oocytes , typical of a feminized germline . DIC and fluorescence image of a representative animal are shown at the bottom . Gonads are outlined by a dashed line . Scale bar : 25 μm .

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

Figure 1B) Expression of the 21U sensor (left) and DAPI staining (right) of gonad arms in the indicated genetic backgrounds. Gonads are outlined by a dashed line. The mCherry signal is represented in pseudo-colours [LUT fire (ImageJ)] to reflect differences in the intensity of the signal. Number of animals analysed and with indicated phenotype are given in the panel. Animals not showing the activation of the 21U sensor(+) were still silenced, and animals that did not show the silenced 21U sensor(RNAe) state were expressing weakly, comparable to the 21U sensor(+). Scale bar: 25 μm.C) Tc1 reversion assay in different genetic backgrounds. All the strains tested carried the unc-22::Tc1(st136) allele. Tc1 excision can result in restoration of unc-22 function, which can be scored visually. Negative control = unc-22::Tc1(st136) in a wild type background; positive control = wago-1/-2/-3. Two independent experiments per strain are represented.D) Crossing scheme to address the re-initiation of silencing of the 21U sensor. A mut-7 mutant male expressing the 21U sensor is crossed with either a wild type (left), prg-1 (middle) or pid-2 (right) mutant hermaphrodite. Their F1 offspring was scored for expression of the 21U sensor by microscopy. Three states of expression were scored, and represented in a pie chart. The three expression states are exemplified by representative images at the bottom: OFF (left), FAINT (right) or ON (middle). DIC images are shown above the fluorescence panels. Gonads are outlined by a dashed line. Scale bar: 25 µm.E) Crossing scheme to address if maternal 21U RNAs are sufficient to re-initiate the silencing of the 21U sensor. A pid-1 mutant male expressing the 21U sensor is crossed with a hermaphrodite, heterozygous for the same mutation. All their F1 offspring inherit a pool of 21U RNAs from the hermaphrodite, but in 50% of the F1, which is pid-1 homozygous mutant, no zygotic PID-1 is present, hence no zygotic 21U RNAs can be made. The silencing or expression of the 21U sensor in the pid-1 homozygous mutant F1 has been scored by microscopy, and depicted in a pie chart. At the bottom, a representative image of an animal carrying a silenced 21U sensor (lower: mCherry signal; upper: DAPI staining) in pid-1 mutant offspring. Gonads are outlined by a dashed line. Scale bar: 25 μm.F) Crossing scheme to test the role of PID-2 in re-initiating the silencing of the 21U sensor, mediated by maternally provided 21U RNAs only. The expression of the 21U sensor in the F1 has been scored by microscopy, and depicted in a pie chart. White arrow heads indicate the many arrayed oocytes, typical of a feminized germline. DIC and fluorescence image of a representative animal are shown at the bottom. Gonads are outlined by a dashed line. Scale bar: 25 μm.


Figure 2The 22G RNAs mapping to the 21U sensor transgene were identified from small RNA sequencing data , and read density was plotted over the transgene , which is schematically depicted at the bottom . The aggregated results of three replicates is shown for each indicated genotype in the different panels . The shading , in grey , represents the standard deviation of the read density from the three replicates . ( + ) means that the sensor was detectably expressed . ( RNAe ) means that the sensor was crossed into the respective mutant background in an RNAe state ( i . e . its silencing was PRG - 1 - independent ) , and its expression remained undetectable by microscopy .

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

Figure 2The 22G RNAs mapping to the 21U sensor transgene were identified from small RNA sequencing data, and read density was plotted over the transgene, which is schematically depicted at the bottom. The aggregated results of three replicates is shown for each indicated genotype in the different panels. The shading, in grey, represents the standard deviation of the read density from the three replicates. (+) means that the sensor was detectably expressed. (RNAe) means that the sensor was crossed into the respective mutant background in an RNAe state (i.e. its silencing was PRG-1-independent), and its expression remained undetectable by microscopy.


Figure 3A ) MA - plot of log2 fold changes ( Y - axis ) versus the mean of normalized counts of 22G RNAs ( X - axis ) for pid - 2 ( xf23 ) mutants , compared to wild type . Red dots : genes with adjusted p - value < 0 . 05 and fold - change > 2 . Blue dots : genes with adjusted p - value < 0 . 05 and fold - change < - 2 . B ) Heatmap displaying overlap significance between different gene sets and genes that either up - or down - regulated in pid - 2 ( xf23 ) mutants . Significance was tested with Fisher ' s exact test and p - values adjusted with the Benjamini - Hochberg Procedure . Colour scheme represents the odds ratio of overlaps representing the strength of association . N . S . : not significant . C - D ) Cumulative 22G coverage along the gene body of Mutator and CSR - 1 targets . Values represent 22G coverage normalized to the total coverage of each gene , for wild type ( N2 ) and pid - 2 ( xf23 ) mutants . The lines represent the average of biological replicates , whereas the shading represents the standard deviation of biological replicates . a . u . : arbitrary units .

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

Figure 3A) MA-plot of log2 fold changes (Y-axis) versus the mean of normalized counts of 22G RNAs (X-axis) for pid-2(xf23) mutants, compared to wild type. Red dots: genes with adjusted p-value < 0.05 and fold-change > 2. Blue dots: genes with adjusted p-value < 0.05 and fold-change < -2.B) Heatmap displaying overlap significance between different gene sets and genes that either up- or down-regulated in pid-2(xf23) mutants. Significance was tested with Fisher's exact test and p-values adjusted with the Benjamini-Hochberg Procedure. Colour scheme represents the odds ratio of overlaps representing the strength of association. N.S.: not significant.C-D) Cumulative 22G coverage along the gene body of Mutator and CSR-1 targets. Values represent 22G coverage normalized to the total coverage of each gene, for wild type (N2) and pid-2(xf23) mutants. The lines represent the average of biological replicates, whereas the shading represents the standard deviation of biological replicates. a.u.: arbitrary units.


Figure 4A ) Volcano plot representing the enrichment of proteins interacting with PID - 2 , isolated by immunoprecipitation of eGFP : : PID - 2 protein , followed by quantitative label - free mass spectrometry . As control , eGFP IPs were performed on protein extracts from wild - type , non - transgenic animals . For each IP - MS experiment , quadruplicates were measured and analysed . The dashed line reflects a significance threshold of p - value < 0 . 05 at two - fold enrichment . Volcano plot representing the enrichment of proteins interacting with PID - 2 , isolated by immunoprecipitation of eGFP : : PID - 2 protein , followed by quantitative label - free mass spectrometry . In this experiment , the endogenous PID - 2 protein was immunoprecipitated , and pid - 2 ( xf23 ) mutant protein extracts were used as control . Volcano plot , representing the enrichment of proteins interacting with PID - 4 , by immunoprecipitating endogenously tagged PID - 4 protein . IPs from protein extracts from wild - type , non - tagged animals were used as control . Volcano plot , representing the enrichment of proteins interacting with PID - 5 , by immunoprecipitating endogenously tagged PID - 5 protein . IPs from protein extracts from wild - type , non - tagged animals were used as control .

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

Figure 4A) Volcano plot representing the enrichment of proteins interacting with PID-2, isolated by immunoprecipitation of eGFP::PID-2 protein, followed by quantitative label-free mass spectrometry. As control, eGFP IPs were performed on protein extracts from wild-type, non-transgenic animals. For each IP-MS experiment, quadruplicates were measured and analysed. The dashed line reflects a significance threshold of p-value < 0.05 at two-fold enrichment.Volcano plot representing the enrichment of proteins interacting with PID-2, isolated by immunoprecipitation of eGFP::PID-2 protein, followed by quantitative label-free mass spectrometry. In this experiment, the endogenous PID-2 protein was immunoprecipitated, and pid-2(xf23) mutant protein extracts were used as control.Volcano plot , representing the enrichment of proteins interacting with PID-4, by immunoprecipitating endogenously tagged PID-4 protein. IPs from protein extracts from wild-type, non-tagged animals were used as control.Volcano plot, representing the enrichment of proteins interacting with PID-5, by immunoprecipitating endogenously tagged PID-5 protein. IPs from protein extracts from wild-type, non-tagged animals were used as control.


Figure 5A ) Expression of the 21U sensor in the indicated genetic backgrounds . Gonads are outlined by a dashed line . The mCherry signal is represented in pseudo - colours [ LUT fire ( ImageJ ) ] to reflect differences in the intensity of the signal . The panels on the right are DIC images of the respective animals . Scale bar : 25 μm . B ) 22G RNA coverage of the 21U sensor ( RNAe ) in the indicated genetic backgrounds . C ) MA - plot of log2 fold changes ( on the Y - axis ) versus the mean of normalized counts of 22G RNAs ( on the X - axis ) for pid - 4 mutants , compared to wild type . Red dots : genes with adjusted p - value < 0 . 05 and fold - change > 2 . Blue dots : genes with adjusted p - value < 0 . 05 and fold - change < - 2 . D ) Heatmap displaying overlap significance between different gene sets and genes that either up - or down - regulated in pid - 4 mutants . Colour scheme represents the odds ratio of overlaps representing the strength of association . MA - plot of log2 fold changes ( on the Y - axis ) versus the mean of normalized counts of 22G RNAs ( on the X - axis ) , compared to wild type . Red dots : genes with adjusted p - value < 0 . 05 and fold - change > 2 . Blue dots : genes with adjusted p - value < 0 . 05 and fold - change < - 2 . E ) for pid - 5 mutantHeatmap displaying overlap significance between different gene sets and genes that either up - or down - regulated in F ) pid - 5 mutantMA - plot of log2 fold changes ( on the Y - axis ) versus the mean of normalized counts of 22G RNAs ( on the X - axis ) compared to wild type . Red dots : genes with adjusted p - value < 0 . 05 and fold - change > 2 . Blue dots : genes with adjusted p - value < 0 . 05 and fold - change < - 2 . G ) for pid - 4 ; pid - 5 double mutantHeatmap displaying overlap significance between different gene sets and genes that either up - or down - regulated in H ) pid - 4 ; pid - 5 double mutantI , J ) Line plots representing fertility over generations for the indicated genetic backgrounds at 25oC . Six L2 - L3 larvae for each of the indicated backgrounds were hand - picked to a fresh plate every four or six days , counting as two or three generations , respectively , until no larvae were present on the plate to be picked . * : two plates of N2 were contaminated and were excluded from the assay .

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

Figure 5A) Expression of the 21U sensor in the indicated genetic backgrounds. Gonads are outlined by a dashed line. The mCherry signal is represented in pseudo-colours [LUT fire (ImageJ)] to reflect differences in the intensity of the signal. The panels on the right are DIC images of the respective animals. Scale bar: 25 μm.B) 22G RNA coverage of the 21U sensor(RNAe) in the indicated genetic backgrounds.C) MA-plot of log2 fold changes (on the Y-axis) versus the mean of normalized counts of 22G RNAs (on the X-axis) for pid-4 mutants, compared to wild type. Red dots: genes with adjusted p-value < 0.05 and fold-change > 2. Blue dots: genes with adjusted p-value < 0.05 and fold-change < -2.D) Heatmap displaying overlap significance between different gene sets and genes that either up- or down-regulated in pid-4 mutants. Colour scheme represents the odds ratio of overlaps representing the strength of association.MA-plot of log2 fold changes (on the Y-axis) versus the mean of normalized counts of 22G RNAs (on the X-axis) , compared to wild type. Red dots: genes with adjusted p-value < 0.05 and fold-change > 2. Blue dots: genes with adjusted p-value < 0.05 and fold-change < -2. E) for pid-5 mutantHeatmap displaying overlap significance between different gene sets and genes that either up- or down-regulated in F) pid-5 mutantMA-plot of log2 fold changes (on the Y-axis) versus the mean of normalized counts of 22G RNAs (on the X-axis) compared to wild type. Red dots: genes with adjusted p-value < 0.05 and fold-change > 2. Blue dots: genes with adjusted p-value < 0.05 and fold-change < -2. G) for pid-4;pid-5 double mutantHeatmap displaying overlap significance between different gene sets and genes that either up- or down-regulated in H) pid-4;pid-5 double mutantI, J) Line plots representing fertility over generations for the indicated genetic backgrounds at 25oC. Six L2-L3 larvae for each of the indicated backgrounds were hand-picked to a fresh plate every four or six days, counting as two or three generations, respectively, until no larvae were present on the plate to be picked. *: two plates of N2 were contaminated and were excluded from the assay.