( D ) Fluorescence images of cells expressing Chs5 - GFP and TGN marker Sec7 - DsRed in WT , ChAP and strains . Merged images depict the differences in Chs5 - GFP localization to the TGN , cytoplasm and distinct foci ( arrows ) in these strains . The brightness and contrast were adjusted differently in the merged images . Bar , 5 µm . ( E ) Co - localization analysis of Chs5 - GFP and TGN marker Sec7 - DsRed in WT , ChAP and strains . Mander 1 depicts the relative amount of the GFP signal co - localizing with the overall DsRed signal , and Mander 2 vice versa . Manders coefficients were obtained using the JACoP plug - in for ImageJ after background subtraction ( N = 20 ) and are given in Expanded View Table 2 .

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

(D) Fluorescence images of cells expressing Chs5-GFP and TGN marker Sec7-DsRed in WT, ChAP and strains. Merged images depict the differences in Chs5-GFP localization to the TGN, cytoplasm and distinct foci (arrows) in these strains. The brightness and contrast were adjusted differently in the merged images. Bar, 5 µm.(E) Co-localization analysis of Chs5-GFP and TGN marker Sec7-DsRed in WT, ChAP and strains. Mander 1 depicts the relative amount of the GFP signal co-localizing with the overall DsRed signal, and Mander 2 vice versa. Manders coefficients were obtained using the JACoP plug-in for ImageJ after background subtraction (N = 20) and are given in Expanded View Table 2.


( F ) Binding kinetics of Chs5 - GFP at the TGN in WT , ChAP and strains . The mean of 20 - 30 FRAP measurements from different cells is shown . Calculated parameters are shown in Table 2 . The schematic cartoons depict the predicted majority of the analyzed complexes . The violet ovals represent Chs5 dimer and circles represent ChAPs : green : Bch1 , red : Bud7 , dark blue : Bch2 , light blue : Chs6 .

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

(F) Binding kinetics of Chs5-GFP at the TGN in WT, ChAP and strains. The mean of 20-30 FRAP measurements from different cells is shown. Calculated parameters are shown in Table 2. The schematic cartoons depict the predicted majority of the analyzed complexes. The violet ovals represent Chs5 dimer and circles represent ChAPs: green:Bch1, red:Bud7, dark blue:Bch2, light blue:Chs6.


( A - B ) Overexpression of Bch2 in absence of other ChAPs results in more efficient Chs5 recruitment and stabilization at the TGN . Fluorescence images of cells expressing Chs5 - GFP ( A ) and binding kinetics of Chs5 - GFP at the TGN ( B ) in WT , BCH2 and GPD - BCH2 strains . Bar , 5 µm . The mean of 20 - 30 FRAP measurements from different cells is shown . Calculated parameters are shown in Table 2 . The schematic cartoons depict the predicted majority of the analyzed complexes ; Chs5 dimer : violet ovals , Bch2 : dark blue circles .

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

(A-B) Overexpression of Bch2 in absence of other ChAPs results in more efficient Chs5 recruitment and stabilization at the TGN. Fluorescence images of cells expressing Chs5-GFP (A) and binding kinetics of Chs5-GFP at the TGN (B) in WT, BCH2 and GPD-BCH2 strains. Bar, 5 µm. The mean of 20-30 FRAP measurements from different cells is shown. Calculated parameters are shown in Table 2. The schematic cartoons depict the predicted majority of the analyzed complexes; Chs5 dimer:violet ovals, Bch2:dark blue circles.


( C - D ) Overexpression of Chs6 in absence of other ChAPs results in more efficient Chs5 recruitment and stabilization at the TGN . ( C ) Fluorescence images of cells expressing Chs5 - GFP in WT , CHS6 and GPD - CHS6 strains . Bar , 5 µm . ( D ) Binding kinetics of Chs5 - GFP at the TGN in WT , , CHS6 and GPD - CHS6 strains . Data processing was carried out as in ( A - B ) . The schematic cartoons depict the predicted majority of the analyzed complexes ; Chs5 dimer : violet ovals , Chs6 : light blue circles .

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

(C-D) Overexpression of Chs6 in absence of other ChAPs results in more efficient Chs5 recruitment and stabilization at the TGN. (C) Fluorescence images of cells expressing Chs5-GFP in WT, CHS6 and GPD-CHS6 strains. Bar, 5 µm. (D) Binding kinetics of Chs5-GFP at the TGN in WT, , CHS6 and GPD-CHS6 strains. Data processing was carried out as in (A-B). The schematic cartoons depict the predicted majority of the analyzed complexes; Chs5 dimer: violet ovals, Chs6: light blue circles.


( E - G ) Overexpression of Chs6 rescues Chs3 export in absence of other ChAPs . ( E ) Fluorescence images of cells expressing Chs3 - GFP in WT , CHS6 and GPD - CHS6 strains . Arrows point to Chs3 - GFP signals at the bud neck . Bar , 5 µm . The inset represents a twofold magnification . ( F ) Quantification of phenotypes in ( E ) ; 100 small budded and 100 large budded cells were quantified per experiment . Average and SD of three independent biological experiments is shown .

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

(E-G) Overexpression of Chs6 rescues Chs3 export in absence of other ChAPs. (E) Fluorescence images of cells expressing Chs3-GFP in WT, CHS6 and GPD-CHS6 strains. Arrows point to Chs3-GFP signals at the bud neck. Bar, 5 µm. The inset represents a twofold magnification.(F) Quantification of phenotypes in (E); 100 small budded and 100 large budded cells were quantified per experiment. Average and SD of three independent biological experiments is shown.


( G ) Chs3 GFP GPD - CHS6 strain was sensitive to calcofluor to a similar extent as the WT strain . Plates were incubated at 30 ° C for 2 - 3 days . A representative drop test of three independent biological experiments is shown .

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

(G) Chs3 GFP GPD-CHS6 strain was sensitive to calcofluor to a similar extent as the WT strain. Plates were incubated at 30°C for 2-3 days. A representative drop test of three independent biological experiments is shown.


Fluorescence images of cells expressing Chs5 - GFP and TGN marker Sec7DsRed ( A ) and binding kinetics of Chs5 - GFP at the TGN ( B ) in WT , CHS6 and CHS6BUD7 ( A , B ) The brightness and contrast were adjusted differently in the merged images . Bar , 5 µm . The mean of 20 - 30 FRAP measurements from different cells is shown . Calculated parameters are shown in Table 2 .

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

Fluorescence images of cells expressing Chs5-GFP and TGN marker Sec7DsRed (A) and binding kinetics of Chs5-GFP at the TGN (B) in WT, CHS6 and CHS6BUD7 (A, B) The brightness and contrast were adjusted differently in the merged images. Bar, 5 µm. The mean of 20-30 FRAP measurements from different cells is shown. Calculated parameters are shown in Table 2.


Fluorescence images of cells expressing Chs5 - GFP and TGN marker Sec7DsRed ( C ) and binding kinetics of Chs5 - GFP at the TGN ( D ) in WT , or CHS6BCH2 ( C , D ) strains . The brightness and contrast were adjusted differently in the merged images . Bar , 5 µm . The mean of 20 - 30 FRAP measurements from different cells is shown . Calculated parameters are shown in Table 2 .

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

Fluorescence images of cells expressing Chs5-GFP and TGN marker Sec7DsRed (C) and binding kinetics of Chs5-GFP at the TGN (D) in WT, or CHS6BCH2 (C, D) strains. The brightness and contrast were adjusted differently in the merged images. Bar, 5 µm. The mean of 20-30 FRAP measurements from different cells is shown. Calculated parameters are shown in Table 2.


( A - D ) ChAPs are differently dependent on each other in terms of binding at the TGN . Binding kinetics of GFP - tagged Chs6 ( A ) , Bud7 ( B ) , Bch2 ( C ) and Bch1 ( D ) at the TGN in the presence ( WT ) or absence of the other ChAPs ( ) . The mean of 20 - 30 FRAP measurements from different cells is shown . Calculated parameters are shown in Table 2 .

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

(A-D) ChAPs are differently dependent on each other in terms of binding at the TGN. Binding kinetics of GFP-tagged Chs6 (A), Bud7 (B), Bch2 (C) and Bch1 (D) at the TGN in the presence (WT) or absence of the other ChAPs (). The mean of 20-30 FRAP measurements from different cells is shown. Calculated parameters are shown in Table 2.


( A - B ) Overexpression of Bch1 displaces Chs6 from the TGN and impairs plasma membrane localization of Chs6 - dependent cargo Chs3 . Fluorescence images of Chs6 - GFP ( A ) and Chs3 - GFP ( B ) in WT and GPD - BCH1 strains . Bar , 5 µm . Arrows point to Chs3 - GFP signals at the bud neck . The inset represents a twofold magnification .

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

(A-B) Overexpression of Bch1 displaces Chs6 from the TGN and impairs plasma membrane localization of Chs6-dependent cargo Chs3. Fluorescence images of Chs6-GFP (A) and Chs3-GFP (B) in WT and GPD-BCH1 strains. Bar, 5 µm. Arrows point to Chs3-GFP signals at the bud neck. The inset represents a twofold magnification.


( C ) Quantification of the phenotypes in ( B ) ; 100 small budded and 100 large budded cells were quantified per experiment . Average and SD of three independent biological experiments is shown .

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

(C) Quantification of the phenotypes in (B); 100 small budded and 100 large budded cells were quantified per experiment. Average and SD of three independent biological experiments is shown.


( D ) Chitin production is reduced upon Bch1 overexpression . Fluorescence images of chitin stained with calcofluor in Chs3 - GFP WT , chs5 and GPD - BCH1 strains .

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

(D) Chitin production is reduced upon Bch1 overexpression. Fluorescence images of chitin stained with calcofluor in Chs3-GFP WT, chs5 and GPD-BCH1 strains.


( E ) Overexpression of Bch1 does not impair plasma membrane localization of the Bch1 - dependent cargo Pin2 . Fluorescence images of Pin2 - GFP in WT and GPD - BCH1 strains .

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

(E) Overexpression of Bch1 does not impair plasma membrane localization of the Bch1-dependent cargo Pin2. Fluorescence images of Pin2-GFP in WT and GPD-BCH1 strains.


( F - I ) Overexpression of Chs6 displaces Bch1 and Chs5 from the TGN . Fluorescence images ( F - G ) and binding kinetics at the TGN ( H - I ) of cells expressing Bch1 - GFP ( F , H ) and Chs5 - GFP ( G , I ) upon overexpression of Chs6 under GPD promoter from 2μ plasmid . The insets in ( F , G ) represent two - fold magnifications . The arrows in ( F ) point to residual Bch1 staining at the TGN . The mean of 20 - 30 FRAP measurements from different cells is shown . Calculated parameters are shown in Table 2 . Size bars represent 5 µm .

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

(F-I) Overexpression of Chs6 displaces Bch1 and Chs5 from the TGN. Fluorescence images (F-G) and binding kinetics at the TGN (H-I) of cells expressing Bch1-GFP (F, H) and Chs5-GFP (G, I) upon overexpression of Chs6 under GPD promoter from 2μ plasmid. The insets in (F, G) represent two-fold magnifications. The arrows in (F) point to residual Bch1 staining at the TGN. The mean of 20-30 FRAP measurements from different cells is shown. Calculated parameters are shown in Table 2. Size bars represent 5 µm.


( A ) Overexpression of Pin2 enhances Chs5 stabilization at the TGN . Binding kinetics of Chs5 GFP in WT , bud7bch1 and bud7bch1 strain expressing Bch2 - dependent cargo Pin2 from a centromeric or a 2μ plasmid . The mean of 20 - 30 FRAP measurements from different cells is shown . Calculated parameters are shown in Table 2 .

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

(A) Overexpression of Pin2 enhances Chs5 stabilization at the TGN. Binding kinetics of Chs5 GFP in WT, bud7bch1 and bud7bch1 strain expressing Bch2-dependent cargo Pin2 from a centromeric or a 2μ plasmid. The mean of 20-30 FRAP measurements from different cells is shown. Calculated parameters are shown in Table 2.


( B ) Comparison of the expression levels of the endogenous Pin2 with the levels of Pin2 expressed from a centromeric or a 2μ plasmid . Pgk1 serves as loading control . A representative immunoblot of three independent biological experiments is shown .

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

(B) Comparison of the expression levels of the endogenous Pin2 with the levels of Pin2 expressed from a centromeric or a 2μ plasmid. Pgk1 serves as loading control. A representative immunoblot of three independent biological experiments is shown.


D , E Images of FRET efficiency in mock - treated ( - ant . A , D ) and Antimycin A - treated ( + ant . A . , E ) ubi - AT1 . 03NL wing discs at time point 2 h . A rainbow colormap is used to indicate the FRET efficiency levels . Scale bars = 50 µm . F Line graphs showing the FRET efficiency in individual mock - treated ( - ant . A , n = 26 ) and ant . A - treated ( + ant . A . , n = 19 ) ubi - AT1 . 03NL wing discs before ( 0 h ) and after 2 h of treatment . Paired t - test , ns = not significant , * * * p ≤ 0 . 001 .

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

D, E Images of FRET efficiency in mock-treated (- ant.A, D) and Antimycin A-treated (+ ant. A., E) ubi-AT1.03NL wing discs at time point 2 h. A rainbow colormap is used to indicate the FRET efficiency levels. Scale bars= 50 µm. F Line graphs showing the FRET efficiency in individual mock-treated (- ant. A, n=26) and ant. A-treated (+ ant. A., n=19) ubi-AT1.03NL wing discs before (0 h) and after 2 h of treatment. Paired t-test, ns= not significant, ***p≤0.001.


G - J Time - controlled knock - down of metabolic enzymes in the dorsal compartment of ubi - AT1 . 03NL wing discs using the apGalts driver . Images of FRET efficiency in control ( G ) , apGalts > PfkRNAi ( H ) , apGalts > GapdhRNAi ( I ) , and apGalts > Glo1RNAi ( J ) wing discs after RNAi induction for 48 h ( G ) , 120 h ( H , J ) , and 93 hours ( I ) . A rainbow colormap is used to indicate the FRET efficiency levels . Scale bars = 50 µm . K Line graphs showing the FRET efficiency in the ventral and dorsal compartment of individual control ( n = 27 ) , apGalts > PfkRNAi ( n = 7 ) , apGalts > GapdhRNAi ( n = 10 ) , and apGalts > Glo1RNAi ( n = 6 ) wing discs . Loss of Pfk , Gapdh or Glo1 in the dorsal compartment of the wing disc reduces the levels of ATP . Paired t - test , ns = not significant , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 .

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

G-J Time-controlled knock-down of metabolic enzymes in the dorsal compartment of ubi-AT1.03NL wing discs using the apGalts driver. Images of FRET efficiency in control (G), apGalts>PfkRNAi (H), apGalts>GapdhRNAi (I), and apGalts>Glo1RNAi (J) wing discs after RNAi induction for 48 h (G), 120 h (H, J), and 93 hours (I). A rainbow colormap is used to indicate the FRET efficiency levels. Scale bars = 50 µm. K Line graphs showing the FRET efficiency in the ventral and dorsal compartment of individual control (n=27), apGalts>PfkRNAi (n=7), apGalts>GapdhRNAi (n=10), and apGalts>Glo1RNAi (n=6) wing discs. Loss of Pfk, Gapdh or Glo1 in the dorsal compartment of the wing disc reduces the levels of ATP. Paired t-test, ns= not significant, **p≤0.01, ***p≤0.001.


L Time - controlled knock - down of ecdysoneless ( ecd ) in the dorsal compartment of ubi - AT1 . 03NL wing discs using apGalts > ecdRNAi . Image of FRET efficiency in apGalts > ecdRNAi wing disc 48 h after RNAi induction . A rainbow colormap is used to indicate the FRET efficiency levels . Scale bars = 50 µm . M Line graph showing the FRET in the dorsal and ventral compartment of apGalts > ecdRNAi ( n = 28 ) wing discs . Loss of Ecd in the dorsal compartment of the wing disc reduces the levels of ATP . Paired t - test , * * * p ≤ 0 . 001 .

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

L Time-controlled knock-down of ecdysoneless (ecd) in the dorsal compartment of ubi-AT1.03NL wing discs using apGalts>ecdRNAi. Image of FRET efficiency in apGalts>ecdRNAi wing disc 48 h after RNAi induction. A rainbow colormap is used to indicate the FRET efficiency levels. Scale bars = 50 µm. M Line graph showing the FRET in the dorsal and ventral compartment of apGalts>ecdRNAi (n=28) wing discs. Loss of Ecd in the dorsal compartment of the wing disc reduces the levels of ATP. Paired t-test, ***p≤0.001.


A - D Adult wing phenotype of knock - down of Gapdh ( C765 > GapdhRNAi ) , over - expression of full - length Ecd ( C765 > ecd ) , over - expression of C - terminally truncated form of Ecd ( C765 > ecdDN ) , and knock - down of ecd ( C765 > ecdRNAi ) in the whole wing disc ( see Appendix Fig S3A for the expression pattern of C765 - Gal4 ) . Images of adult wings of control ( A ) , C765 > GapdhRNAi ( A ' ) , C765 > ecd ( B ) , C765 > ecdDN ( C ) , and C765 > ecdRNAi ( D ) male flies . ( A ' ' ) Overlay of the wings shown in A and A ' . ( C ' ) Overlay of control wing and wing shown in C and C ' . Loss of Ecd throughout the wing discs produces adults with vestigal wings ( D ) . In contrast , the effects of ecdDN over - expression or Gapdh knock - down are less severe on tissue survival , giving rise to smaller wings with altered proportions . Images consist of a series of tiles that were stitched together by the microscope software . Scale bar = 500 μm . E , F Tukey box - and - whiskers plot showing quantifications of the area ( E ) and shape ( aspect ratio : major / minor axis , F ) of control , C765 > GapdhRNAi , C765 > ecd , and C765 > ecdDN wings . Lower and upper hinges correspond to the first and third quartiles , vertical lines extend to ± 1 . 5 times the interquartile range . Sample size is indicated for each perturbation . Loss of Gapdh or over - expression of EcdDN leads to smaller wings that are increased along the anterior - posterior ( A / P ) axis compared to the proximal - distal ( P / D ) axis . Statistical analysis was performed using one - way ANOVA , followed by Dunnett ' s multiple comparisons test . * * * p ≤ 0 . 001 .

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

A-D Adult wing phenotype of knock-down of Gapdh (C765>GapdhRNAi), over-expression of full-length Ecd (C765>ecd), over-expression of C-terminally truncated form of Ecd (C765>ecdDN), and knock-down of ecd (C765>ecdRNAi) in the whole wing disc (see Appendix Fig S3A for the expression pattern of C765-Gal4). Images of adult wings of control (A), C765>GapdhRNAi (A'), C765>ecd (B), C765>ecdDN (C), and C765>ecdRNAi (D) male flies. (A'') Overlay of the wings shown in A and A'. (C') Overlay of control wing and wing shown in C and C'. Loss of Ecd throughout the wing discs produces adults with vestigal wings (D). In contrast, the effects of ecdDN over-expression or Gapdh knock-down are less severe on tissue survival, giving rise to smaller wings with altered proportions. Images consist of a series of tiles that were stitched together by the microscope software. Scale bar= 500 μm. E, F Tukey box-and-whiskers plot showing quantifications of the area (E) and shape (aspect ratio: major/minor axis, F) of control, C765>GapdhRNAi, C765>ecd, and C765>ecdDN wings. Lower and upper hinges correspond to the first and third quartiles, vertical lines extend to ± 1.5 times the interquartile range. Sample size is indicated for each perturbation. Loss of Gapdh or over-expression of EcdDN leads to smaller wings that are increased along the anterior-posterior (A/P) axis compared to the proximal-distal (P/D) axis. Statistical analysis was performed using one-way ANOVA, followed by Dunnett's multiple comparisons test. ***p≤0.001.


A - B ' Time - controlled knock - down of ecd in the dorsal compartment of the wing discs ( see Fig 1C for the expression pattern of apGalts ) . IF of control ( A , A ' ) and apGalts > ecdRNAi ( B , B ' ) wing discs , stained for the Hedgehog ( Hh ) pathway components Smoothened ( Smo , A , B ) , and Ci155 ( A ' , B ' ) . Next to the images are quantifications of the respective staining in the dorsal versus ventral compartment of control ( n = 5 ) and apGalts > ecdRNAi ( n = 5 ) wing discs . Graphs show mean ( thick line ) ± SD ( thin lines ) . Dashed yellow lines indicate the position of the A / P boundary . Statistical analyses ( t - test ) revealed significant differences in Smo and Ci155 expression in the dorsal compartment between control and apGalts > ecdRNAi ( Smo : p ≤ 0 . 001 , Ci155 : p ≤ 0 . 001 ) wing discs . Thus , loss of Ecd elevates Smo levels on the plasma membrane and increases the range of Ci155 stabilization in the anterior compartment . Wing discs were analyzed 48 h after RNAi induction . Scale bars = 50 μm .

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

A-B' Time-controlled knock-down of ecd in the dorsal compartment of the wing discs (see Fig 1C for the expression pattern of apGalts). IF of control (A, A') and apGalts>ecdRNAi (B, B') wing discs, stained for the Hedgehog (Hh) pathway components Smoothened (Smo, A, B), and Ci155 (A', B'). Next to the images are quantifications of the respective staining in the dorsal versus ventral compartment of control (n=5) and apGalts>ecdRNAi (n=5) wing discs. Graphs show mean (thick line) ± SD (thin lines). Dashed yellow lines indicate the position of the A/P boundary. Statistical analyses (t-test) revealed significant differences in Smo and Ci155 expression in the dorsal compartment between control and apGalts>ecdRNAi (Smo: p≤0.001, Ci155: p≤0.001) wing discs. Thus, loss of Ecd elevates Smo levels on the plasma membrane and increases the range of Ci155 stabilization in the anterior compartment. Wing discs were analyzed 48 h after RNAi induction. Scale bars= 50 μm.


C - E ' Time - controlled knock - down of metabolic enzymes in the dorsal compartment of the wing disc . IF of apGalts > PfkRNAi , apGalts > GapdhRNAi , and apGalts > Glo1RNAi wing discs , stained for Smo ( C , D , E ) and Ci155 ( C ' , D ' , E ' ) . Next to the images are quantifications of the respective staining in the dorsal versus ventral compartment of apGalts > PfkRNAi ( n = 5 ) , apGalts > GapdhRNAi ( n = 5 ) , and apGalts > Glo1RNAi ( n = 6 ) wing discs . Graphs show mean ( thick line ) ± SD ( thin lines ) . Dashed yellow lines indicate the position of the A / P boundary . Statistical analyses ( t - test ) revealed significant differences in Smo and Ci155 expression in the dorsal compartment between control and apGalts > PfkRNAi ( Smo : p ≤ 0 . 001 , Ci155 : p ≤ 0 . 01 ) , apGalts > GapdhRNAi ( Smo , Ci155 : p ≤ 0 . 05 ) , or apGalts > Glo1RNAi ( Smo : p ≤ 0 . 001 , Ci155 : p ≤ 0 . 05 ) wing discs . Thus , loss of Pfk , Gapdh or Glo1 elevates Smo levels on the plasma membrane and increases the range of Ci155 stabilization in the anterior compartment . Wing discs were analyzed 120 h after RNAi induction . Scale bars = 50 μm .

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

C-E' Time-controlled knock-down of metabolic enzymes in the dorsal compartment of the wing disc. IF of apGalts>PfkRNAi, apGalts>GapdhRNAi, and apGalts>Glo1RNAi wing discs, stained for Smo (C, D, E) and Ci155 (C', D', E'). Next to the images are quantifications of the respective staining in the dorsal versus ventral compartment of apGalts>PfkRNAi (n=5), apGalts>GapdhRNAi (n=5), and apGalts>Glo1RNAi (n=6) wing discs. Graphs show mean (thick line) ± SD (thin lines). Dashed yellow lines indicate the position of the A/P boundary. Statistical analyses (t-test) revealed significant differences in Smo and Ci155 expression in the dorsal compartment between control and apGalts>PfkRNAi (Smo: p≤0.001, Ci155: p≤0.01), apGalts>GapdhRNAi (Smo, Ci155: p≤0.05), or apGalts>Glo1RNAi (Smo: p≤0.001, Ci155: p≤0.05) wing discs. Thus, loss of Pfk, Gapdh or Glo1 elevates Smo levels on the plasma membrane and increases the range of Ci155 stabilization in the anterior compartment. Wing discs were analyzed 120 h after RNAi induction. Scale bars= 50 μm.


A - D ' ' ' IF of NIH3T3 cells stably expressing Smo - mEos2 treated with mock ( control , A - A ' ' ' ) , the Smo agonist ( + SAG , B - B ' ' ' ) or the glycolytic inhibitors 3 - bromopyruvate ( + 3 - BP , C - C ' ' ' ) and 2 - deoxyglucose ( + 2 - DG , D - D ' ' ' ) . DAPI staining , shown in gray , was used to visualize cell nuclei . Acetylated tubulin ( AcTub ) , used as a ciliary marker , is shown in magenta , Smo - mEos2 is shown in green . Zoomed - in images of cilia marked by white arrows in A ' ' , B ' ' , C ' ' , and D ' ' are shown in A ' ' ' , B ' ' ' , C ' ' ' , and D ' ' ' . Scale bars = 5 μm , but 2 μm in A ' ' ' , B ' ' ' , C ' ' ' , and D ' ' ' . E , F Box plot showing quantification of Smo - mEos2 ciliary localization in NIH3T3 cells upon 3 - BP ( E ) and 2 - DG ( F ) treatment , respectively . Lower and upper hinges correspond to the first and third quartiles , vertical lines extend to the minimum and maximum values . Sample size is indicated for each perturbation . Treatment with glycolytic inhibitors induces Smo - mEos2 localization to the primary cilium . t - test , * * * p ≤ 0 . 001 .

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

A-D''' IF of NIH3T3 cells stably expressing Smo-mEos2 treated with mock (control, A-A'''), the Smo agonist (+ SAG, B-B''') or the glycolytic inhibitors 3-bromopyruvate (+3-BP, C-C''') and 2-deoxyglucose (+ 2-DG, D-D'''). DAPI staining, shown in gray, was used to visualize cell nuclei. Acetylated tubulin (AcTub), used as a ciliary marker, is shown in magenta, Smo-mEos2 is shown in green. Zoomed-in images of cilia marked by white arrows in A'', B'', C'', and D'' are shown in A''', B''', C''', and D'''. Scale bars= 5 μm, but 2 μm in A''', B''', C''', and D'''. E, F Box plot showing quantification of Smo-mEos2 ciliary localization in NIH3T3 cells upon 3-BP (E) and 2-DG (F) treatment, respectively. Lower and upper hinges correspond to the first and third quartiles, vertical lines extend to the minimum and maximum values. Sample size is indicated for each perturbation. Treatment with glycolytic inhibitors induces Smo-mEos2 localization to the primary cilium . t-test, ***p≤0.001.


A - B Time - controlled knock - down of ecd in the dorsal compartment of the wing discs ( see Fig 1C for the expression pattern of apGalts ) . IF of control ( A ) and apGalts > ecdRNAi ( B ) wing discs , stained for Hh . Next to the images are quantifications of the respective staining in the dorsal versus ventral compartment of control ( n = 4 ) and apGalts > ecdRNAi ( n = 5 ) wing discs . Graphs show mean ( thick line ) ± SD ( thin lines ) . Dashed yellow lines indicate the position of the A / P boundary . Statistical analysis ( t - test ) revealed no significant differences in Hh expression in the dorsal compartment between control and apGalts > ecdRNAi wing discs . Thus , loss of Ecd does not affect Hh production and spreading . Wing discs were analyzed 48 h after RNAi induction . Scale bars = 50 μm .

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

A-B Time-controlled knock-down of ecd in the dorsal compartment of the wing discs (see Fig 1C for the expression pattern of apGalts). IF of control (A) and apGalts>ecdRNAi (B) wing discs, stained for Hh. Next to the images are quantifications of the respective staining in the dorsal versus ventral compartment of control (n=4) and apGalts>ecdRNAi (n=5) wing discs. Graphs show mean (thick line) ± SD (thin lines). Dashed yellow lines indicate the position of the A/P boundary. Statistical analysis (t-test) revealed no significant differences in Hh expression in the dorsal compartment between control and apGalts>ecdRNAi wing discs. Thus, loss of Ecd does not affect Hh production and spreading. Wing discs were analyzed 48 h after RNAi induction. Scale bars= 50 μm.


C - F Time - controlled knock - down of ecd in the dorsal compartment of dispatched mutant wing discs ( dispS037707 , apGalts > ecdRNAi , see Fig 1C for the expression pattern of apGalts ) . IF of control ( C ) , apGalts > ecdRNAi ( D ) , dispS037707 ( E ) and dispS037707 , apGalts > ecdRNAi ( F ) wing discs expressing the decapentaplegic ( dpp ) reporter gene dpp - lacZ ( dppZ ) , stained for dppZ . Next to the images are quantifications of the respective stainings ( n = 4 ( C , F ) , n = 5 ( D ) , n = 3 ( E ) ) . Graphs show mean ( thick line ) ± SD ( thin lines ) . Dashed yellow lines indicate the position of the A / P boundary . Statistical analyses ( t - test ) revealed a significant difference in dppZ expression in the dorsal compartment between control and apGalts > ecdRNAi ( p ≤ 0 . 01 ) ( C vs D ) , and dispS037707 and dispS037707 , apGalts > ecdRNAi ( p ≤ 0 . 01 ) wing discs ( E vs F ) . Loss of Ecd elevates the expression of dppZ in a broader than normal stripe . However , lack of Ecd function alone is not sufficient to trigger dpp expression , which instead depends on the presence of Hh . Wing discs were analyzed 48 h after RNAi induction . Scale bars = 50 μm .

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

C-F Time-controlled knock-down of ecd in the dorsal compartment of dispatched mutant wing discs (dispS037707, apGalts>ecdRNAi, see Fig 1C for the expression pattern of apGalts). IF of control (C), apGalts>ecdRNAi (D), dispS037707 (E) and dispS037707, apGalts>ecdRNAi (F) wing discs expressing the decapentaplegic (dpp) reporter gene dpp-lacZ (dppZ), stained for dppZ. Next to the images are quantifications of the respective stainings (n=4 (C, F), n=5 (D), n=3 (E)). Graphs show mean (thick line) ± SD (thin lines). Dashed yellow lines indicate the position of the A/P boundary. Statistical analyses (t-test) revealed a significant difference in dppZ expression in the dorsal compartment between control and apGalts>ecdRNAi (p≤0.01) (C vs D), and dispS037707 and dispS037707, apGalts>ecdRNAi (p≤0.01) wing discs (E vs F). Loss of Ecd elevates the expression of dppZ in a broader than normal stripe. However, lack of Ecd function alone is not sufficient to trigger dpp expression, which instead depends on the presence of Hh. Wing discs were analyzed 48 h after RNAi induction. Scale bars= 50 μm.


G - J IF of dispS037707 ( G , I ) and dispS037707 , apGalts > ecdRNAi ( H , J ) wing discs , stained for Smo ( G , H ) and Ci155 ( I , J ) . Next to the images are quantifications of the respective stainings ( n = 2 ( G , H ) , n = 3 ( I ) , n = 4 ( J ) ) . Graphs show mean ( thick line ) ± SD ( thin lines ) . Dashed yellow lines indicate the position of the A / P boundary . Although statistical analyses ( t - test ) revealed no significant differences in Smo and Ci155 expression in the dorsal compartment between control and dispS037707 , apGalts > ecdRNAi wing discs , loss of Ecd slightly induces an increased Smo accumulation and Ci155 stabilization independently of Hh . Wing discs were analyzed 48 h after RNAi induction . Scale bars = 50 μm .

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

G-J IF of dispS037707 (G, I) and dispS037707, apGalts>ecdRNAi (H, J) wing discs, stained for Smo (G, H) and Ci155 (I, J). Next to the images are quantifications of the respective stainings (n=2 (G, H), n=3 (I), n=4 (J)). Graphs show mean (thick line) ± SD (thin lines). Dashed yellow lines indicate the position of the A/P boundary. Although statistical analyses (t-test) revealed no significant differences in Smo and Ci155 expression in the dorsal compartment between control and dispS037707, apGalts>ecdRNAi wing discs, loss of Ecd slightly induces an increased Smo accumulation and Ci155 stabilization independently of Hh. Wing discs were analyzed 48 h after RNAi induction. Scale bars= 50 μm.


A - A ' ' ' Time - controlled knock - down of ecd or Gapdh in the dorsal compartment of the wing discs ( see Fig 1C for the expression pattern of apGalts ) . The plasma membrane potential was detected using DiBAC4 ( 3 ) . The fluorescence intensity of DiBAC4 ( 3 ) increases upon plasma membrane depolarization . ( A ) The change in plasma membrane potential upon genetic perturbation was quantified as the fold change in the ratio of DiBAC4 ( 3 ) signal intensity in the dorsal to ventral compartment ( control ( n = 19 ) , apGalts > ecdRNAi ( n = 36 ) and apGalts > GapdhRNAi ( n = 18 ) wing discs ) . Error bars indicate ± SD . t - test , * p ≤ 0 . 05 , * * * p ≤ 0 . 001 . ( A ' - A ' ' ' ) Representative images of DiBAC4 ( 3 ) assay in control ( A ' ) , apGalts > ecdRNAi ( A ' ' ) , and apGalts > GapdhRNAi ( A ' ' ' ) wing discs 48 h or 120 h after RNAi induction , respectively . V / D indicates the boundary between the ventral and dorsal compartments . Next to the maximal projections are shown the sum projections of cross - sections along the A / P axis . Loss of Ecd or Gapdh alters the plasma membrane potential . Scale bars = 50 μm .

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

A-A''' Time-controlled knock-down of ecd or Gapdh in the dorsal compartment of the wing discs (see Fig 1C for the expression pattern of apGalts). The plasma membrane potential was detected using DiBAC4(3). The fluorescence intensity of DiBAC4(3) increases upon plasma membrane depolarization. (A) The change in plasma membrane potential upon genetic perturbation was quantified as the fold change in the ratio of DiBAC4(3) signal intensity in the dorsal to ventral compartment (control (n=19), apGalts>ecdRNAi (n=36) and apGalts>GapdhRNAi (n=18) wing discs). Error bars indicate ± SD. t-test, *p≤0.05, ***p≤0.001. (A'-A''') Representative images of DiBAC4(3) assay in control (A'), apGalts>ecdRNAi (A''), and apGalts>GapdhRNAi (A''') wing discs 48 h or 120 h after RNAi induction, respectively. V/D indicates the boundary between the ventral and dorsal compartments. Next to the maximal projections are shown the sum projections of cross-sections along the A/P axis. Loss of Ecd or Gapdh alters the plasma membrane potential. Scale bars= 50 μm.


A - A ' ' ' Time - controlled knock - down of ecd or Gapdh in the dorsal compartment of the wing discs ( see Fig 1C for the expression pattern of apGalts ) . The plasma membrane potential was detected using DiBAC4 ( 3 ) . The fluorescence intensity of DiBAC4 ( 3 ) increases upon plasma membrane depolarization . ( A ) The change in plasma membrane potential upon genetic perturbation was quantified as the fold change in the ratio of DiBAC4 ( 3 ) signal intensity in the dorsal to ventral compartment ( control ( n = 19 ) , apGalts > ecdRNAi ( n = 36 ) and apGalts > GapdhRNAi ( n = 18 ) wing discs ) . Error bars indicate ± SD . t - test , * p ≤ 0 . 05 , * * * p ≤ 0 . 001 . ( A ' - A ' ' ' ) Representative images of DiBAC4 ( 3 ) assay in control ( A ' ) , apGalts > ecdRNAi ( A ' ' ) , and apGalts > GapdhRNAi ( A ' ' ' ) wing

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

A-A''' Time-controlled knock-down of ecd or Gapdh in the dorsal compartment of the wing discs (see Fig 1C for the expression pattern of apGalts). The plasma membrane potential was detected using DiBAC4(3). The fluorescence intensity of DiBAC4(3) increases upon plasma membrane depolarization. (A) The change in plasma membrane potential upon genetic perturbation was quantified as the fold change in the ratio of DiBAC4(3) signal intensity in the dorsal to ventral compartment (control (n=19), apGalts>ecdRNAi (n=36) and apGalts>GapdhRNAi (n=18) wing discs). Error bars indicate ± SD. t-test, *p≤0.05, ***p≤0.001. (A'-A''') Representative images of DiBAC4(3) assay in control (A'), apGalts>ecdRNAi (A''), and apGalts>GapdhRNAi (A''') wing discs


D - E ' ' Dissipation of the plasma membrane potential upon treatment of wing discs with gramicidin A ( gA ) . ( D ) Quantification of DiBAC4 ( 3 ) signal intensity in mock - treated ( - gA , n = 7 ) and gA - treated ( + gA , n = 5 ) wing discs . ( D ' , D ' ' ) Representative images of DiBAC4 ( 3 ) assay in mock - ( D ' ) and gA - treated ( D ' ' ) wing discs . Next to the maximal projections are shown sum projections of cross - sections along the A / P axis . ( E ) Quantification of PAC - NAE signal intensity in mock - ( n = 7 ) and gA - treated ( n = 9 ) wing discs , shown as fold change from control ( mock - treated ) . ( E ' - E ' ' ) Representative images of PAC - NAE uptake in mock - ( E ' ) and gA - treated ( E ' ' ) wing discs . GA treatment strongly reduces the uptake of PAC - NAE . Error bars indicate ± SD . t - test , * p ≤ 0 . 05 , * * * p ≤ 0 . 001 . Scale bars = 50 μm .

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D-E'' Dissipation of the plasma membrane potential upon treatment of wing discs with gramicidin A (gA). (D) Quantification of DiBAC4(3) signal intensity in mock-treated (- gA, n=7) and gA-treated (+ gA, n=5) wing discs. (D', D'') Representative images of DiBAC4(3) assay in mock- (D') and gA-treated (D'') wing discs. Next to the maximal projections are shown sum projections of cross-sections along the A/P axis. (E) Quantification of PAC-NAE signal intensity in mock- (n=7) and gA-treated (n=9) wing discs, shown as fold change from control (mock-treated). (E'-E'') Representative images of PAC-NAE uptake in mock- (E') and gA-treated (E'') wing discs. GA treatment strongly reduces the uptake of PAC-NAE. Error bars indicate ± SD. t-test, *p≤0.05, ***p≤0.001. Scale bars= 50 μm.


( a ) WT and Pgam5 KO MEFs evaluated by TEM . The black arrows indicate mitochondria ; ( b ) TEM analysis after 12 h CCCP treatment ; black arrowheads indicate DMS . Inset shows a DMS with an encapsulated mitochondrion . The black arrows indicate mitochondria . Scale bar = 0 . 5 micron ;

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(a) WT and Pgam5 KO MEFs evaluated by TEM. The black arrows indicate mitochondria; (b) TEM analysis after 12 h CCCP treatment; black arrowheads indicate DMS. Inset shows a DMS with an encapsulated mitochondrion. The black arrows indicate mitochondria. Scale bar=0.5 micron;


( c ) WT and KO MEFs were treated with CCCP for 0 , 3 or 6 h and cytosolic fractions were immunoblotted with anti - LC - 3 , anti - p62 or anti - actin as indicated .

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(c) WT and KO MEFs were treated with CCCP for 0, 3 or 6 h and cytosolic fractions were immunoblotted with anti-LC-3, anti-p62 or anti-actin as indicated.


( d ) Similar to c . WT and KO MEFs were treated with CCCP for 6 and 12 h and immunoblotted for anti - COXIV , anti - Tomm20 or anti - actin as indicated . For c and d , the numbers below each lane indicate the fractional band density compared with the 0 h time point , which has been set at 1 . 0 ;

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(d) Similar to c. WT and KO MEFs were treated with CCCP for 6 and 12 h and immunoblotted for anti-COXIV, anti-Tomm20 or anti-actin as indicated. For c and d, the numbers below each lane indicate the fractional band density compared with the 0 h time point, which has been set at 1.0;


( e ) Confocal photomicrographs of mt - Keima - transduced WT and KO MEFs treated with CCCP or DMSO vehicle for 12 h ( six individual cells from each group were scanned and representative cells are shown ) ; Intracellular mt - Keima excited at 450 nm was shown in green colour , while red colour indicated the excitation by 550 nm in the same cell . Scale bar = 20 μm .

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(e) Confocal photomicrographs of mt-Keima-transduced WT and KO MEFs treated with CCCP or DMSO vehicle for 12 h (six individual cells from each group were scanned and representative cells are shown); Intracellular mt-Keima excited at 450 nm was shown in green colour, while red colour indicated the excitation by 550 nm in the same cell. Scale bar=20 μm.


( f ) Mitochondrial extracts of WT and Pgam5 KO MEFs treated with DMSO ( − ) or CCCP ( + ) for 3 h and analysed by immunoblot as above . ( Note that mouse PINK1 antibody can only detect the full - length PINK1 ) . Arrow indicates the 63 kDa form of PINK1 .

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(f) Mitochondrial extracts of WT and Pgam5 KO MEFs treated with DMSO (−) or CCCP (+) for 3 h and analysed by immunoblot as above. (Note that mouse PINK1 antibody can only detect the full-length PINK1). Arrow indicates the 63 kDa form of PINK1.


( g ) Hela cells transduced with either nonspecific ( NS ) or PGAM5 shRNA viruses were treated with CCCP for 3 h , and mitochondrial fractions were immunoblotted for the proteins indicated . Full - length ( * 63 kDa ) and cleaved ( # 54 kDa ) PINK1 are indicated . For f and g , the numbers below indicate the full - length PINK1 band density compared with the first lane that has been set at 1 .

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(g) Hela cells transduced with either nonspecific (NS) or PGAM5 shRNA viruses were treated with CCCP for 3 h, and mitochondrial fractions were immunoblotted for the proteins indicated. Full-length (*63 kDa) and cleaved (#54 kDa) PINK1 are indicated. For f and g, the numbers below indicate the full-length PINK1 band density compared with the first lane that has been set at 1.0


( h ) Confocal micrographs of Hela cells as in g were transfected with PARKIN - YFP ( green ) plasmid before CCCP treatment , then ( i ) treated with CCCP for 12 h . Mitochondria were stained with anti - Tomm20 ( red ) 4 ' , 6 - diamidino - 2 - phenylindole stained nucleus was shown in blue . Co - localization was indicated by white colour . Scale bar = 10 μm . ( j ) Quantification of the co - localization percentage of Tomm20 ( red ) with parkin ( green ) per cell by Imaris software ( n = 7 for NS and PGAM5 shRNA group separately . Results are presented as mean and s . d . values and were analysed by the student t - test ) . All the blots and images are representative of three independent experiments . DMSO , dimethylsulphoxide .

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(h) Confocal micrographs of Hela cells as in g were transfected with PARKIN-YFP (green) plasmid before CCCP treatment, then (i) treated with CCCP for 12 h. Mitochondria were stained with anti-Tomm20 (red) 4',6-diamidino-2-phenylindole stained nucleus was shown in blue. Co-localization was indicated by white colour. Scale bar=10 μm. (j) Quantification of the co-localization percentage of Tomm20 (red) with parkin (green) per cell by Imaris software (n=7 for NS and PGAM5 shRNA group separately. Results are presented as mean and s.d. values and were analysed by the student t-test). All the blots and images are representative of three independent experiments. DMSO, dimethylsulphoxide.


( a ) Mitochondria from MEFs from Parl WT ( + / + ) and KO ( − / − ) mice were treated with the indicated doses of proteinase K ( Prot . K ) for 30 min on ice and then immunoblotted for the indicated proteins . Full - length and cleaved PGAM5 bands are indicated with arrows .

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(a) Mitochondria from MEFs from Parl WT (+/+) and KO (−/−) mice were treated with the indicated doses of proteinase K (Prot.K) for 30 min on ice and then immunoblotted for the indicated proteins. Full-length and cleaved PGAM5 bands are indicated with arrows.


( b ) Pgam5 WT and KO MEFs were transduced with either nonspecific ( NS ) or Parl shRNA and either treated ( + ) or not ( − ) with CCCP for 3 h . Mitochondria were then purified and analysed by immunoblot for the indicated proteins .

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(b) Pgam5 WT and KO MEFs were transduced with either nonspecific (NS) or Parl shRNA and either treated (+) or not (−) with CCCP for 3 h. Mitochondria were then purified and analysed by immunoblot for the indicated proteins.


( c ) WT PINK1 as well as kinase dead ( KD ) or Parkinson ' s disease - associated PINK1 mutants ( C92F , A168P , H271Q , G309D , L347P , G386S , R464H ) were co - transfected into HEK293T cells with the same amount PGAM5 ( lane 1 - 9 ) or GFP ( lane 10 - 18 ) expression plasmids and total cell lysates were immunoblotting for PINK1 , PGAM5 and actin . The ratio of WT full - length ( 63 kDa ) versus cleaved ( 54 kDa ) PINK1 ( * / # ) is shown .

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(c) WT PINK1 as well as kinase dead (KD) or Parkinson's disease-associated PINK1 mutants (C92F, A168P, H271Q, G309D, L347P, G386S, R464H) were co-transfected into HEK293T cells with the same amount PGAM5 (lane 1-9) or GFP (lane 10-18) expression plasmids and total cell lysates were immunoblotting for PINK1, PGAM5 and actin. The ratio of WT full-length (63 kDa) versus cleaved (54 kDa) PINK1 (*/#) is shown.


( d ) Anti - myc immunoprecipitates ( IP ) of lysates from 293T cells transfected with combinations of myc - tagged PGAM5 and PINK1 - V5 with or without CCCP treatment and immunoblotting with anti - PINK1 or anti - myc .

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(d) Anti-myc immunoprecipitates (IP) of lysates from 293T cells transfected with combinations of myc-tagged PGAM5 and PINK1-V5 with or without CCCP treatment and immunoblotting with anti-PINK1 or anti-myc.


( e ) Four constructs expressing either : ( 1 ) no PGAM5 ( empty vector ) or ( 2 ) amino acids 1 - 98 , ( 3 ) 1 - 110 and ( 4 ) 98 - 289 truncated versions of PGAM5 ( left panel ) . Immunoblot of HEK293T cells expressing PINK1 - GFP and myc - tagged PGAM5 truncation mutants as indicated with anti - PINK1 or anti - myc for the input proteins as well as the anti - GFP IP ( right panel ) .

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(e) Four constructs expressing either: (1) no PGAM5 (empty vector) or (2) amino acids 1-98, (3) 1-110 and (4) 98-289 truncated versions of PGAM5 (left panel). Immunoblot of HEK293T cells expressing PINK1-GFP and myc-tagged PGAM5 truncation mutants as indicated with anti-PINK1 or anti-myc for the input proteins as well as the anti-GFP IP (right panel).


( f ) Co - transfection of GFP , WT PGAM5 , 2RA mutant ( R98A and R104A ) or the 2HA mutant ( H99A and H105A ) with a PINK1 expression plasmid into HEK293T cells and the input cell lysates as well as the anti - myc IP were immunoblotted with antibodies against PINK1 , PGAM5 or actin .

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(f) Co-transfection of GFP, WT PGAM5, 2RA mutant (R98A and R104A) or the 2HA mutant (H99A and H105A) with a PINK1 expression plasmid into HEK293T cells and the input cell lysates as well as the anti-myc IP were immunoblotted with antibodies against PINK1, PGAM5 or actin.


WT PINK1 was expressed in HEK293T cells with or without PGAM5 or a GFP control plasmid after ( g ) DMSO or ( h ) CCCP treatment . Cells were permeabilized with digitonin or Triton X - 100 separately , followed by treatment with the indicated amounts of trypsin on ice for 30 min . Cell lysates were immunoblotted for PINK1 , PGAM5 , Tomm20 or HSP60 as indicated . Full - length ( * 63 kDa ) and cleaved ( # 54 kDa ) PINK1 are shown .

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WT PINK1 was expressed in HEK293T cells with or without PGAM5 or a GFP control plasmid after (g) DMSO or (h) CCCP treatment. Cells were permeabilized with digitonin or Triton X-100 separately, followed by treatment with the indicated amounts of trypsin on ice for 30 min. Cell lysates were immunoblotted for PINK1, PGAM5, Tomm20 or HSP60 as indicated. Full-length (*63 kDa) and cleaved (#54 kDa) PINK1 are shown.


( a ) Body weight was measured in 5 - month - old and 12 - month - old female Pgam5 WT and KO mice .

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(a) Body weight was measured in 5-month-old and 12-month-old female Pgam5 WT and KO mice.


Behavioral measurements on 12 - month - old female WT and KO mice ( WT n = 10 , KO n = 8 ) were as follows : ( b ) Open - field tests including time spent mobile , time spent immobile , whole area distance and centre area distance ;

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Behavioral measurements on 12-month-old female WT and KO mice (WT n=10, KO n=8) were as follows: (b) Open-field tests including time spent mobile, time spent immobile, whole area distance and centre area distance;


Behavioral measurements on 12 - month - old female WT and KO mice ( WT n = 10 , KO n = 8 ) were as follows : ( c ) Gait length , gait frequency in steps / ss and step angle ; ( d ) rearing test , ( e ) akinesia test and ( f ) ladder climb test and ( g ) vertical pole test . T - turn indicates the time mice spent to turn their bodies 180 degrees from the head up start position before descending the pole . T - down indicates the time mice spent to descend to the bottom of pole . Graphs show the mean and s . d . values and were analysed by the student ' s t - test , * P0 . 05 , * * P0 . 01 , * * * P0 . 001 , NS , not significant .

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Behavioral measurements on 12-month-old female WT and KO mice (WT n=10, KO n=8) were as follows: (c) Gait length, gait frequency in steps/ss and step angle; (d) rearing test, (e) akinesia test and (f) ladder climb test and (g) vertical pole test. T-turn indicates the time mice spent to turn their bodies 180 degrees from the head up start position before descending the pole. T-down indicates the time mice spent to descend to the bottom of pole. Graphs show the mean and s.d. values and were analysed by the student's t-test, *P0.05, **P0.01, ***P0.001, NS, not significant.


High - performance liquid chromatography - electrochemical detection quantitation of ( a ) striatal DA and its metabolites ( b ) dihydroxyphenylacetic acid ( DOPAC ) ( c ) Homovanillic acid ( HVA ) and ( d ) 3MT levels at three different time points ( 1 , 2 and 12 - month - old ) in Pgam5 WT or KO mice .

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High-performance liquid chromatography-electrochemical detection quantitation of (a) striatal DA and its metabolites (b) dihydroxyphenylacetic acid (DOPAC) (c) Homovanillic acid (HVA) and (d) 3MT levels at three different time points (1, 2 and 12-month-old) in Pgam5 WT or KO mice.


( e ) Representative images of TH - immunohistochemistry from WT and KO mice . The VTA region is indicated . Stereological quantitation of ( f ) TH and ( g ) Nissl - positive neurons in the SNpc ( n = 4 mice per group ) . Error bars represent the mean ± s . e . m . , n = 4 mice per group . ( two - way analysis of variance , * * * P0 . 01 , * P0 . 05 , NS , not significant ) . mo , month old .

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(e) Representative images of TH-immunohistochemistry from WT and KO mice. The VTA region is indicated. Stereological quantitation of (f) TH and (g) Nissl-positive neurons in the SNpc (n=4 mice per group). Error bars represent the mean±s.e.m., n=4 mice per group. (two-way analysis of variance, ***P0.01, *P0.05, NS, not significant). mo, month old.


The 18 - month - old Pgam5 WT and KO mice received either water as control ( n = 4 ) , or 5 mg kg − 1 L - DOPA and 12 . 5 mg kg − 1 Benserazide ( n = 5 ) by intraperitoneal injection . Twenty minutes later , mice were subjected to the open - field test and the centre area time in seconds ( s ) as well as the centre area distance in centimeters ( cm ) were measured as described in the Materials and Methods . Graphs show the mean and s . d . values and were analysed by the student t - test , NS , no significance ; * P0 . 05 ; * * P0 . 01 .

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The 18-month-old Pgam5 WT and KO mice received either water as control (n=4), or 5 mg kg−1 L-DOPA and 12.5 mg kg−1 Benserazide (n=5) by intraperitoneal injection. Twenty minutes later, mice were subjected to the open-field test and the centre area time in seconds (s) as well as the centre area distance in centimeters (cm) were measured as described in the Materials and Methods. Graphs show the mean and s.d. values and were analysed by the student t-test, NS, no significance; *P0.05; **P0.01.


( a ) WT and dnm1Δ cells expressing Vph1 - RFP and Idp1 - GFP were grown in minimal synthetic dextrose medium and transferred to lactate medium at an initial density of OD600 0 . 08 . The cells were then sampled daily and analysed by fluorescence microscopy ( × 100 objective ) during stationary phase mitophagy in lactate medium . Scale bar , 5 μm .

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(a) WT and dnm1Δ cells expressing Vph1-RFP and Idp1-GFP were grown in minimal synthetic dextrose medium and transferred to lactate medium at an initial density of OD600 0.08. The cells were then sampled daily and analysed by fluorescence microscopy (× 100 objective) during stationary phase mitophagy in lactate medium. Scale bar, 5 μm.


( b ) WT , dnm1Δ and dnm1Δ mgm1Δ mutants were grown in SL medium as described in Methods , and samples ( 10 OD600 units ) were taken at each time point . Protein extracts were prepared and equal protein amounts ( 20 μg ) were subjected to SDS - polyacrylamide gel electrophoresis and immunoblotting with anti - GFP antibody .

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(b) WT, dnm1Δ and dnm1Δ mgm1Δ mutants were grown in SL medium as described in Methods, and samples (10 OD600 units) were taken at each time point. Protein extracts were prepared and equal protein amounts (20 μg) were subjected to SDS-polyacrylamide gel electrophoresis and immunoblotting with anti-GFP antibody.


( c ) Quantification of the percent free GFP at the day 4 time point in each of the three strains analysed in b , using four independent experiments for each strain . Error bars indicate s . d . analysis of variance , P0 . 001 ; N = 4 .

(c) Quantification of the percent free GFP at the day 4 time point in each of the three strains analysed in b, using four independent experiments for each strain. Error bars indicate s.d. analysis of variance, P0.001; N=4.


( c ) Density plot of the reproducibility of biological replicates .

(c) Density plot of the reproducibility of biological replicates.


( a ) Ratios from proteins with quantifications from all samples were log2 transformed and z - score normalized . Columns containing data from the different samples were clustered hierarchically and rows that contained protein entries were clustered by k - means . The proteins in each k - means cluster were tested for enrichment of GO CC ( indicated in red ) and GO BP ( indicated in blue ) terms .

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(a) Ratios from proteins with quantifications from all samples were log2 transformed and z-score normalized. Columns containing data from the different samples were clustered hierarchically and rows that contained protein entries were clustered by k-means. The proteins in each k-means cluster were tested for enrichment of GO CC (indicated in red) and GO BP (indicated in blue) terms.


( b ) Proteins associated with different cellular compartments were sequentially extracted and the average of their ratios were compared with averages of remaining proteins .

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(b) Proteins associated with different cellular compartments were sequentially extracted and the average of their ratios were compared with averages of remaining proteins.


( d ) GO BP enrichment analysis of proteins in the different clusters compared with the remaining proteins .

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(d) GO BP enrichment analysis of proteins in the different clusters compared with the remaining proteins.


A comparison of the SILAC - derived abundance as a function of time , for several proteins that either show differential behaviour between WT and the mutants ( Idh2 , Idp1 and Aco2 ) and proteins that behave similarly between all the genetic backgrounds tested ( Hsp78 ) . Shown are mean protein ratios ( two replicates for mutants , three replicates for WT ) , error bars indicating peptide - based relative s . d .

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

A comparison of the SILAC-derived abundance as a function of time, for several proteins that either show differential behaviour between WT and the mutants (Idh2, Idp1 and Aco2) and proteins that behave similarly between all the genetic backgrounds tested (Hsp78). Shown are mean protein ratios (two replicates for mutants, three replicates for WT), error bars indicating peptide-based relative s.d.


WT ( a ) , dnm1Δ ( b ) and atg1Δ cells ( c ) and atg32Δ cells ( d ) expressing chromosomally tagged GFP chimeras of Aco2 , Idh2 , Idp1 and Hsp78 were incubated in SL medium for 7 days , and 10 OD600 units were sampled at day 1 and day 7 and processed for immunoblotting with anti - GFP antibodies as described in the Methods section . Positive control used in c and d was from dnm1Δ cells expressing Idp1 - GFP from a plasmid at 4 - day incubation ( Fig . 1b , lane 4 ) . Release of free GFP in WT cells is reproducibly variable between these reporters , and varies between 0 ( Idh2 ) and 60 % ( Aco2 ) . ( e ) Densitometric quantification of the release of free GFP ( as % of total signal ) in four independent experiments , comparing the results between WT and dnm1Δ cells . Bars denote s . d . ( n = 4 ) .

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

WT (a), dnm1Δ (b) and atg1Δ cells (c) and atg32Δ cells (d) expressing chromosomally tagged GFP chimeras of Aco2, Idh2, Idp1 and Hsp78 were incubated in SL medium for 7 days, and 10 OD600 units were sampled at day 1 and day 7 and processed for immunoblotting with anti-GFP antibodies as described in the Methods section. Positive control used in c and d was from dnm1Δ cells expressing Idp1-GFP from a plasmid at 4-day incubation (Fig. 1b, lane 4). Release of free GFP in WT cells is reproducibly variable between these reporters, and varies between 0 (Idh2) and 60% (Aco2). (e) Densitometric quantification of the release of free GFP (as % of total signal) in four independent experiments, comparing the results between WT and dnm1Δ cells. Bars denote s.d. (n=4).


( a ) Cells expressing chromosomally tagged GFP chimeras of Aco2 , Idh2 , Idp1 and Hsp78 as well as a plasmid - borne ( pDJB12 ) , mitochondrially targeted RFP ( mtRFP ) were incubated in SL - leucine medium for 3 days and imaged daily . Aco2 and Idp1 show clear mitophagic targeting ( white arrows point at specific examples ) by day 3 , whereas Idh2 and Hsp78 show weak or no mitophagy . In addition , Idh2 and Hsp78 show different degrees of segregation relative to the mtRFP marker , whereas Idp1 - GFP and Aco2 - GFP show a near - complete co - localization with mtRFP . Scale bar , 5 μm . ( b ) Quantification of the frequency of mitophagy from a ; the percentage of cells showing mitophagic profiles in the red ( magenta bars ; plasmid - borne mtRFP ) or green ( turquoise bars ; integrated C - terminal GFP chimerae ) was calculated for each GFP chimera . Idp - GFP , red N = 76 , green N = 82 ; Aco2 - GFP , red N = 79 , green N = 82 ; Idh2 - GFP , red N = 72 , green N = 72 ; Hsp78 - GFP , red N = 70 , green N = 70 .

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

(a) Cells expressing chromosomally tagged GFP chimeras of Aco2, Idh2, Idp1 and Hsp78 as well as a plasmid-borne (pDJB12), mitochondrially targeted RFP (mtRFP) were incubated in SL-leucine medium for 3 days and imaged daily. Aco2 and Idp1 show clear mitophagic targeting (white arrows point at specific examples) by day 3, whereas Idh2 and Hsp78 show weak or no mitophagy. In addition, Idh2 and Hsp78 show different degrees of segregation relative to the mtRFP marker, whereas Idp1-GFP and Aco2-GFP show a near-complete co-localization with mtRFP. Scale bar, 5 μm. (b) Quantification of the frequency of mitophagy from a; the percentage of cells showing mitophagic profiles in the red (magenta bars; plasmid-borne mtRFP) or green (turquoise bars; integrated C-terminal GFP chimerae) was calculated for each GFP chimera. Idp-GFP, red N=76, green N=82; Aco2-GFP, red N=79, green N=82; Idh2-GFP, red N=72, green N=72; Hsp78-GFP, red N=70, green N=70.


WT and dnm1Δ cells expressing integrated Hsp78 - GFP and plasmid - borne mtRFP were grown on lactate medium for 3 days . Cells were imaged daily and the images were analysed using ImageJ as described in Methods . Graphs illustrate the distribution of pixels associated with specific values of RFP and GFP channel intensities , respectively . Diagonal concentrations of dots indicate spatial correlation between the signals . The coloured photographic panels at the bottom illustrate the increased intensity correlation of the two channels at the 3 - day time point in the dnm1Δ mutant , relative to the WT cells . Scale bar , 5 μm .

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

WT and dnm1Δ cells expressing integrated Hsp78-GFP and plasmid-borne mtRFP were grown on lactate medium for 3 days. Cells were imaged daily and the images were analysed using ImageJ as described in Methods. Graphs illustrate the distribution of pixels associated with specific values of RFP and GFP channel intensities, respectively. Diagonal concentrations of dots indicate spatial correlation between the signals. The coloured photographic panels at the bottom illustrate the increased intensity correlation of the two channels at the 3-day time point in the dnm1Δ mutant, relative to the WT cells. Scale bar, 5 μm.


B , C Serial transplantations of PR + / + and PR − / − mammary epithelia . ( B ) Fluorescence stereo microscopy of third ‐ generation mammary outgrowths derived from 8 ‐ week ‐ old PR + / + ; EGFP and PR − / − ; EGFP donor mice . ( C ) Table summarizing 3 independent serial transplant experiments with PR + / + ; EGFP and PR − / − ; EGFP . Each engrafted gland is represented by a micrograph ; black sectors represent area of fat pad filled by engrafted epithelium . Scale bar : 200 μm .

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

B, C Serial transplantations of PR+/+ and PR−/− mammary epithelia. (B) Fluorescence stereo microscopy of third‐generation mammary outgrowths derived from 8‐week‐old PR+/+; EGFP and PR−/−; EGFP donor mice. (C) Table summarizing 3 independent serial transplant experiments with PR+/+; EGFP and PR−/−; EGFP. Each engrafted gland is represented by a micrograph; black sectors represent area of fat pad filled by engrafted epithelium. Scale bar: 200 μm.


D , E Serial transplantation of RANKL + / + and RANKL − / − mammary epithelia . ( D ) Fluorescence stereo microscopy of third ‐ generation mammary outgrowths derived from 5 ‐ week ‐ old RANKL + / + ; EGFP and RANKL − / − ; EGFP donor mice . Insets : higher magnification showing side branches present in the WT control ( arrowheads ) absent from RANKL − / − ; EGFP epithelium . ( E ) Table summarizing three independent serial transplant experiments with RANKL + / + ; EGFP and RANKL − / − ; EGFP donor mice . Scale bar : 200 μm .

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

D, E Serial transplantation of RANKL+/+ and RANKL−/− mammary epithelia. (D) Fluorescence stereo microscopy of third‐generation mammary outgrowths derived from 5‐week‐old RANKL+/+; EGFP and RANKL−/−; EGFP donor mice. Insets: higher magnification showing side branches present in the WT control (arrowheads) absent from RANKL−/−; EGFP epithelium. (E) Table summarizing three independent serial transplant experiments with RANKL+/+; EGFP and RANKL−/−; EGFP donor mice. Scale bar: 200 μm.


F , G Serial transplantation of Wnt4 + / + and Wnt4 − / − mammary epithelia . ( F ) Fluorescence stereo microscopy of third ‐ generation mammary outgrowths derived from mammary buds of E12 . 5 and E13 . 5 Wnt4 + / + ; EGFP and Wnt4 − / − ; EGFP embryos . Scale bar : 200 μm . ( G ) Table summarizing three independent serial transplant experiments with Wnt4 + / + ; EGFP donor mice . Scale bar : 200 μm .

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

F, G Serial transplantation of Wnt4+/+ and Wnt4−/− mammary epithelia. (F) Fluorescence stereo microscopy of third‐generation mammary outgrowths derived from mammary buds of E12.5 and E13.5 Wnt4+/+; EGFP and Wnt4−/−; EGFP embryos. Scale bar: 200 μm. (G) Table summarizing three independent serial transplant experiments with Wnt4+/+; EGFP donor mice. Scale bar: 200 μm.


H Box plot showing the difference between percentage of reconstitution between WT and MT contralateral grafts in each transplant generation . P values were determined by Mann - Whitney U ‐ test .

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H Box plot showing the difference between percentage of reconstitution between WT and MT contralateral grafts in each transplant generation. P values were determined by Mann-Whitney U‐test.


A Epifluorescence stereo microscopy of inguinal mammary gland from a 5 ‐ day ‐ old Wnt4 : : Cre ; mT / mG female ( n = 7 ) . Scale bars : 0 . 5 mm and 0 . 1 mm ( inset ) .

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A Epifluorescence stereo microscopy of inguinal mammary gland from a 5‐day‐old Wnt4::Cre; mT/mG female (n = 7). Scale bars: 0.5 mm and 0.1 mm (inset).


B , C Histology sections of mammary glands from a 5 ‐ day ‐ old ( B ; n = 7 ) and a 10 ‐ day ‐ old ( C ; n = 5 ) Wnt4 : : Cre ; mT / mG female stained by double immunofluorescence microscopy for EGFP ( green ) and PR ( magenta , not detected ) , counterstained with DAPI ( blue ) . Scale bar : 50 mm .

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

B, C Histology sections of mammary glands from a 5‐day‐old (B; n = 7) and a 10‐day‐old (C; n = 5) Wnt4::Cre; mT/mG female stained by double immunofluorescence microscopy for EGFP (green) and PR (magenta, not detected), counterstained with DAPI (blue). Scale bar: 50 mm.


D - F EGFP ( green ) and PR ( magenta ) co ‐ immunofluorescence microscopy counterstained with DAPI ( blue ) on histology sections from mT / mG ; Wnt4 : : Cre mammary glands at different developmental stages . ( D ) TEB of a 4 ‐ week ‐ old female ( n = 4 ) ; scale bar : 30 μm . ( E ) Ducts of an 8 ‐ week ‐ old female ( n = 3 ) ; scale bar : 100 μm ; inset , scale bar : 20 μm . ( F ) Duct of a female at day 10 . 5 of pregnancy ( n = 3 ) ; scale bar : 150 μm ; inset , scale bar : 30 μm .

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D-F EGFP (green) and PR (magenta) co‐immunofluorescence microscopy counterstained with DAPI (blue) on histology sections from mT/mG; Wnt4::Cre mammary glands at different developmental stages. (D) TEB of a 4‐week‐old female (n = 4); scale bar: 30 μm. (E) Ducts of an 8‐week‐old female (n = 3); scale bar: 100 μm; inset, scale bar: 20 μm. (F) Duct of a female at day 10.5 of pregnancy (n = 3); scale bar: 150 μm; inset, scale bar: 30 μm.


G - I EGFP ( green ) , PR ( magenta ) , and p63 ( white ) triple co ‐ immunofluorescence microscopy counterstained with DAPI ( blue ) on histological sections from mT / mG ; Wnt4 : : Cre mammary glands at different developmental stages . ( G ) Ducts of 5 ‐ day ‐ old female ( n = 3 ) . ( H ) TEB of a 4 ‐ week ‐ old female ( n = 3 ) . ( I ) Duct of an 8 ‐ week ‐ old female ( n = 3 ) . Scale bars : 30 μm .

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G-I EGFP (green), PR (magenta), and p63 (white) triple co‐immunofluorescence microscopy counterstained with DAPI (blue) on histological sections from mT/mG; Wnt4::Cre mammary glands at different developmental stages. (G) Ducts of 5‐day‐old female (n = 3). (H) TEB of a 4‐week‐old female (n = 3). (I) Duct of an 8‐week‐old female (n = 3). Scale bars: 30 μm.


J - O Epifluorescence stereo microscopy of mammary glands harvested from 15 ‐ day ‐ old Wnt4 : : GFP females either WT ( n = 18 ) ( J , M ) , ERα − / − ( n = 4 ) ( K , N ) , or PR − / − ( n = 3 ) ( L , O ) . dTomato expression ( J - L ) ; EGFP expression ( M - O ) is not abrogated in ERα − / − nor PR − / − epithelia . Arrowheads mark the main duct originating from the nipple . Scale bar : 50 μm .

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

J-O Epifluorescence stereo microscopy of mammary glands harvested from 15‐day‐old Wnt4::GFP females either WT (n = 18) (J, M), ERα−/− (n = 4) (K, N), or PR−/− (n = 3) (L, O). dTomato expression (J-L); EGFP expression (M-O) is not abrogated in ERα−/− nor PR−/− epithelia. Arrowheads mark the main duct originating from the nipple. Scale bar: 50 μm.


Q Bar plots showing relative PR and Wnt4 mRNA expression normalized to CK18 mRNA in mammary organoids from 5 pubertal ( 6 weeks old ) and 3 adult ( 11 weeks old ) mice exposed for 6 h to vehicle ( C ) , 17β ‐ estradiol ( 20 nmol ) ( E2 ) , or R5020 ( 20 nmol ) ( P ) . Bars represent the mean ± SD of 3 independent experiments .

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

Q Bar plots showing relative PR and Wnt4 mRNA expression normalized to CK18 mRNA in mammary organoids from 5 pubertal (6 weeks old) and 3 adult (11 weeks old) mice exposed for 6 h to vehicle (C), 17β‐estradiol (20 nmol) (E2), or R5020 (20 nmol) (P). Bars represent the mean ± SD of 3 independent experiments.


R - X Epifluorescence stereo microscopy of contralateral mammary glands that were engrafted with Wnt4 : : GFP epithelium from 8 ‐ week ‐ old females , either PRWT ( R , T , V , X ) or PR − / − ( S , U , W ) . dTomato expression ( R , S ) ; EGFP expression ( T , U ) double epifluorescence ( V , W , X ) on contralateral engrafted glands 3 weeks after surgery when recipients were 6 weeks old . Representative result from three independent experiments . Arrowheads point to TEBs ( V , X ) or to origin of growth ( W ) . Scale bar ( R - W ) : 5 mm , ( X ) : 1 mm .

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

R-X Epifluorescence stereo microscopy of contralateral mammary glands that were engrafted with Wnt4::GFP epithelium from 8‐week‐old females, either PRWT (R, T, V, X) or PR−/− (S, U, W). dTomato expression (R, S); EGFP expression (T, U) double epifluorescence (V, W, X) on contralateral engrafted glands 3 weeks after surgery when recipients were 6 weeks old. Representative result from three independent experiments. Arrowheads point to TEBs (V, X) or to origin of growth (W). Scale bar (R-W): 5 mm, (X): 1 mm.


A - D Representative epifluorescence stereo microscopy of inguinal mammary glands from a 10 ‐ day ‐ old mT / mG ( A , C ) and MMTV : : Cre ; mT / mG ( B , D ) female ( n = 7 ) . Scale bar : 1 mm .

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A-D Representative epifluorescence stereo microscopy of inguinal mammary glands from a 10‐day‐old mT/mG (A, C) and MMTV::Cre; mT/mG (B, D) female (n = 7). Scale bar: 1 mm.


E Higher magnification of inguinal mammary gland from a 10 ‐ day ‐ old MMTV : : Cre ; mT / mG female . Scale bar : 0 . 2 mm . F Immunofluorescence microscopy for EGFP ( green ) on a mammary gland section from a 10 ‐ day ‐ old MMTV : : Cre ; mT / mG female counterstained with DAPI ( blue ) ( n = 7 ) . Scale bar : 30 μm .

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

E Higher magnification of inguinal mammary gland from a 10‐day‐old MMTV::Cre; mT/mG female. Scale bar: 0.2 mm.F Immunofluorescence microscopy for EGFP (green) on a mammary gland section from a 10‐day‐old MMTV::Cre; mT/mG female counterstained with DAPI (blue) (n = 7). Scale bar: 30 μm.


G Epifluorescence stereo microscopy of mammary glands from 10 ‐ day ‐ old littermates either MMTV : : Cre ; mT / mG Wnt4fl / + or MMTV : : Cre ; mT / mG ; Wnt4fl / fl . Scale bar : 1 mm . Arrowhead marks the main duct originating from the nipple . H Bar plot showing ratio of branching points in prepubertal Cre + ; Wnt4fl / fl ( n = 12 ) relative to Cre + ; Wnt4fl / + littermates ( n = 10 ) . Two ‐ tailed , paired Student ' s t ‐ test was used to calculate statistical significance .

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

G Epifluorescence stereo microscopy of mammary glands from 10‐day‐old littermates either MMTV::Cre; mT/mG Wnt4fl/+ or MMTV::Cre; mT/mG; Wnt4fl/fl. Scale bar: 1 mm. Arrowhead marks the main duct originating from the nipple.H Bar plot showing ratio of branching points in prepubertal Cre+;Wnt4fl/fl (n = 12) relative to Cre+;Wnt4fl/+ littermates (n = 10). Two‐tailed, paired Student's t‐test was used to calculate statistical significance.


I Fluorescence stereo microscopy of mammary glands from 35 ‐ day ‐ old littermates either MMTV : : Cre ; mT / m ; Wnt4fl / + or MMTV : : Cre ; mT / m ; Wnt4fl / fl . Note ductal elongation is delayed in Wnt4 ‐ deficient mammary epithelium compared to Wnt4fl / + control . LN : lymph node . Scale bar : 4 mm . J , K Bar plots showing number of terminal end buds ( TEBs ) ( J ) and area of mammary fat pad filled by the ductal system ( K ) . Ctrl ( Wnt4fl / wt or Wnt4fl / fl ; Cre − ) : n = 6 , fl / + ; Cre + : n = 4 , and fl / fl ; Cre + : n = 7 ) . Data are presented as the mean ± SD . Two ‐ tailed Student ' s t ‐ test was used to calculate statistical significance .

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

I Fluorescence stereo microscopy of mammary glands from 35‐day‐old littermates either MMTV::Cre; mT/m; Wnt4fl/+ or MMTV::Cre; mT/m;Wnt4fl/fl. Note ductal elongation is delayed in Wnt4‐deficient mammary epithelium compared to Wnt4fl/+ control. LN: lymph node. Scale bar: 4 mm.J, K Bar plots showing number of terminal end buds (TEBs) (J) and area of mammary fat pad filled by the ductal system (K). Ctrl (Wnt4fl/wt or Wnt4fl/fl; Cre−): n = 6, fl/+; Cre+: n = 4, and fl/fl; Cre+: n = 7). Data are presented as the mean ± SD. Two‐tailed Student's t‐test was used to calculate statistical significance.


L , M Bar plots showing BrdU incorporation index in inguinal mammary glands of 4 ‐ week ‐ old Wnt4fl / + ; Cre + ( n = 3 ) or Wnt4fl / fl ; Cre + ( n = 4 ) in the ducts ( L ) and in the TEBs ( M ) . Data are presented as the mean ± SD . Two ‐ tailed Student ' s t ‐ test was used to calculate statistical significance .

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

L, M Bar plots showing BrdU incorporation index in inguinal mammary glands of 4‐week‐old Wnt4fl/+; Cre+ (n = 3) or Wnt4fl/fl; Cre+ (n = 4) in the ducts (L) and in the TEBs (M). Data are presented as the mean ± SD. Two‐tailed Student's t‐test was used to calculate statistical significance.


A Whole ‐ mount microscopy of X ‐ gal ‐ stained Axin2 + / lacZ in E12 . 5 embryo showing β ‐ galactosidase expression in the mammary buds ( arrows ) ( n = 8 ) . Scale bar : 1 mm . Arrowheads mark mammary buds .

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A Whole‐mount microscopy of X‐gal‐stained Axin2+/lacZ in E12.5 embryo showing β‐galactosidase expression in the mammary buds (arrows) (n = 8). Scale bar: 1 mm. Arrowheads mark mammary buds.


B - F Whole ‐ mount microscopy of X ‐ gal ( blue ) ‐ and carmine alum ( red ) ‐ stained mammary glands harvested from Axin2 + / lacZ mice at distinct developmental stages . ( B ) At postnatal day 1 , β ‐ galactosidase activity detected in the nipple area ( n = 6 ) . Scale bar : 1 mm . ( C , D ) In 5 ‐ week ‐ old mammary glands , reporter activity was detected around the ducts ( small arrows ) and in the neck region of the terminal end buds ( TEBs ) ( large arrows ) ( C ) ( n = 8 ) . Scale bars : 400 μm ( C ) and 100 μm ( D ) . ( E ) At 8 . 5 day of pregnancy , reporter expression was detected in the ducts . Higher magnification ( inset ) suggests myoepithelial expression ( n = 10 ) . Scale bar : 1 mm . ( F ) Whole ‐ mount at day 14 . 5 of pregnancy : reporter activity is limited to ducts ( n = 5 ) . Scale bar : 200 μm .

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B-F Whole‐mount microscopy of X‐gal (blue)‐ and carmine alum (red)‐stained mammary glands harvested from Axin2+/lacZ mice at distinct developmental stages. (B) At postnatal day 1, β‐galactosidase activity detected in the nipple area (n = 6). Scale bar: 1 mm. (C, D) In 5‐week‐old mammary glands, reporter activity was detected around the ducts (small arrows) and in the neck region of the terminal end buds (TEBs) (large arrows) (C) (n = 8). Scale bars: 400 μm (C) and 100 μm (D). (E) At 8.5 day of pregnancy, reporter expression was detected in the ducts. Higher magnification (inset) suggests myoepithelial expression (n = 10). Scale bar: 1 mm. (F) Whole‐mount at day 14.5 of pregnancy: reporter activity is limited to ducts (n = 5). Scale bar: 200 μm.


G Histology section of Axin2 : : LacZ mammary gland at day 8 . 5 of pregnancy counterstained with nuclear red ; luminal epithelial cells show no detectable β ‐ galactosidase activity but myoepithelial cells do . Scale bar : 200 μm .

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G Histology section of Axin2::LacZ mammary gland at day 8.5 of pregnancy counterstained with nuclear red; luminal epithelial cells show no detectable β‐galactosidase activity but myoepithelial cells do. Scale bar: 200 μm.


H β ‐ galactosidase activity ( blue ) colocalizes with the myoepithelial marker p63 ( green ) detected by immunofluorescence microscopy . Arrows point to myoepithelial cells . L , lumen . Scale bar : 100 μm .

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H β‐galactosidase activity (blue) colocalizes with the myoepithelial marker p63 (green) detected by immunofluorescence microscopy. Arrows point to myoepithelial cells. L, lumen. Scale bar: 100 μm.


I Representative whole ‐ mount stereo microscopy of X ‐ gal ( blue ) ‐ and carmine alum ( magenta ) ‐ stained mammary gland biopsies taken from 14 ‐ week ‐ old Axin2 : : LacZ females collected at diestrus and estrus , respectively ( n = 3 ) . Scale bar : 200 μm .

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I Representative whole‐mount stereo microscopy of X‐gal (blue)‐ and carmine alum (magenta)‐stained mammary gland biopsies taken from 14‐week‐old Axin2::LacZ females collected at diestrus and estrus, respectively (n = 3). Scale bar: 200 μm.


J Relative Wnt4 and Axin2 mRNA expression in mammary glands from three mice in estrus versus diestrus assessed by semiquantitative qRT - PCR normalized to 18S rRNA . Two ‐ tailed , paired Student ' s t ‐ test was used to calculate statistical significance .

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J Relative Wnt4 and Axin2 mRNA expression in mammary glands from three mice in estrus versus diestrus assessed by semiquantitative qRT-PCR normalized to 18S rRNA. Two‐tailed, paired Student's t‐test was used to calculate statistical significance.


K Stereo microscopy of X ‐ gal ‐ and carmine alum ‐ stained mammary glands from ovariectomized Axin2 : : LacZ females treated for 72 h with vehicle ( n = 4 ) ( left ) , 17 ‐ β ‐ estradiol ( E2 ) ( n = 6 ) ( center ) , 17 ‐ β ‐ estradiol and progesterone ( E2 and P ) ( n = 8 ) ( right ) . Scale bar : 200 μm .

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K Stereo microscopy of X‐gal‐ and carmine alum‐stained mammary glands from ovariectomized Axin2::LacZ females treated for 72 h with vehicle (n = 4) (left), 17‐β‐estradiol (E2) (n = 6) (center), 17‐β‐estradiol and progesterone (E2 and P) (n = 8) (right). Scale bar: 200 μm.