( I - K ) The loss of progenitor Dop2R and DAT function using domeMESO - Gal4 , UAS - GFP analysed at wandering 3rd instar , 120h AEL . Data information : DNA is stained with Hoechst in blue domeMESO ( representative of progenitors ) expression in green , P1 ( representative of mature plasmatocytes ) in red . All image panels show a 20µm scale bar . For better representation , the primary lobe of the lymph gland has been represented and outlined in white dashed

O O O O O O O O B-CELL_TYPE B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY O B-SMALL_MOLECULE O O B-GENEPROD O O O B-CELL_TYPE O B-EXP_ASSAY O O O B-GENEPROD O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O B-TISSUE O O B-TISSUE I-TISSUE O O O O O O O O

(I-K) The loss of progenitor Dop2R and DAT function using domeMESO-Gal4, UAS-GFP analysed at wandering 3rd instar, 120h AEL. Data information: DNA is stained with Hoechst in blue domeMESO (representative of progenitors) expression in green, P1 (representative of mature plasmatocytes) in red. All image panels show a 20µm scale bar. For better representation, the primary lobe of the lymph gland has been represented and outlined in white dashed lines


( L ) Quantifications of lymph gland size upon loss of Dop2R and DAT in the progenitors ( domeMESO - Gal4 , UAS - GFP / + , n = 46 , domeMESO - Gal4 , UAS - GFP ; Dop2RRNAi , n = 44 , domeMESO - Gal4 , UAS - GFP ; DATRNAi , n = 37 ) . Data information : The quantification in L represents mean with standard deviation with whiskers indicating maximum and minimum values , box indicates the lower and upper quartiles . The statistical analysis applied in L is One - way ANOVA with Tukey ' s multiple comparisons test ' n ' represents the number of lymph gland lobes analyzed , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

O O O O O B-TISSUE I-TISSUE O O O O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-TISSUE I-TISSUE I-TISSUE O O O O O O O O O O O O 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) Quantifications of lymph gland size upon loss of Dop2R and DAT in the progenitors (domeMESO-Gal4, UAS-GFP/+, n= 46, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n= 44, domeMESO-Gal4, UAS-GFP; DATRNAi, n= 37). Data information: The quantification in L represents mean with standard deviation with whiskers indicating maximum and minimum values, box indicates the lower and upper quartiles. The statistical analysis applied in L is One-way ANOVA with Tukey's multiple comparisons test 'n' represents the number of lymph gland lobes analyzed, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.


( M ) Quantifications of the proportions of % Dome ( green bar ) , % negative ( blue bar ) , % overlap ( Dome and P1 , yellow bar ) and % P1 ( red bars ) upon loss of Dop2R and DAT in the progenitors . For Dome positive area ( domeMESO - Gal4 , UAS - GFP / + , n = 28 , domeMESO - Gal4 , UAS - GFP ; Dop2RRNAi , n = 16 , domeMESO - Gal4 , UAS - GFP ; DATRNAi , n = 23 ) , negative area ( domeMESO - Gal4 , UAS - GFP / + , n = 28 , domeMESO - Gal4 , UAS - GFP ; Dop2RRNAi , n = 16 , domeMESO - Gal4 , UAS - GFP ; DATRNAi , n = 23 ) , overlap area ( domeMESO - Gal4 , UAS - GFP / + , n = 26 , domeMESO - Gal4 , UAS - GFP ; Dop2RRNAi , n = 16 , domeMESO - Gal4 , UAS - GFP ; DATRNAi , n = 25 ) and P1 area ( domeMESO - Gal4 , UAS - GFP / + , n = 27 , domeMESO - Gal4 , UAS - GFP ; Dop2RRNAi , n = 16 , domeMESO - Gal4 , UAS - GFP ; DATRNAi , n = 23 ) . Data information : The quantification in M represents mean , with whiskers indicating maximum and minimum values , box indicates the lower and upper quartiles . The statistical analysis applied in M is Two - way Anova with Tukey ' s multiple comparisons test . ' n ' represents the number of lymph gland lobes analyzed , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-TISSUE I-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(M) Quantifications of the proportions of %Dome (green bar), %negative (blue bar), %overlap (Dome and P1, yellow bar) and %P1 (red bars) upon loss of Dop2R and DAT in the progenitors. For Dome positive area (domeMESO-Gal4, UAS-GFP/+, n= 28, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n= 16, domeMESO-Gal4, UAS-GFP; DATRNAi, n= 23), negative area (domeMESO-Gal4, UAS-GFP/+, n= 28, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n= 16, domeMESO-Gal4, UAS-GFP; DATRNAi, n= 23), overlap area (domeMESO-Gal4, UAS-GFP/+, n= 26, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n= 16, domeMESO-Gal4, UAS-GFP; DATRNAi, n= 25) and P1 area (domeMESO-Gal4, UAS-GFP/+, n= 27, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n= 16, domeMESO-Gal4, UAS-GFP; DATRNAi, n= 23). Data information: The quantification in M represents mean, with whiskers indicating maximum and minimum values, box indicates the lower and upper quartiles. The statistical analysis applied in M is Two-way Anova with Tukey's multiple comparisons test. 'n' represents the number of lymph gland lobes analyzed, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.


( N ) Quantification of the number of PPO positive crystal cells upon dopamine sensing perturbation ( domeMESO - Gal4 , UAS - GFP / + , n = 29 , domeMESO - Gal4 , UAS - GFP ; Dop2RRNAi , n = 26 , domeMESO - Gal4 , UAS - GFP ; DATRNAi , n = 25 ) . Data information : The quantification in N represents mean with standard deviation with whiskers indicating maximum and minimum values , box indicates the lower and upper quartiles . The statistical analysis applied in N is One - way ANOVA with Tukey ' s multiple comparisons test ' n ' represents the number of lymph gland lobes analyzed , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

O O O O O O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O B-SMALL_MOLECULE O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-TISSUE I-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(N) Quantification of the number of PPO positive crystal cells upon dopamine sensing perturbation (domeMESO-Gal4, UAS-GFP/+, n= 29, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n= 26, domeMESO-Gal4, UAS-GFP; DATRNAi, n= 25). Data information: The quantification in N represents mean with standard deviation with whiskers indicating maximum and minimum values, box indicates the lower and upper quartiles. The statistical analysis applied in N is One-way ANOVA with Tukey's multiple comparisons test 'n' represents the number of lymph gland lobes analyzed, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.


( A ) A temporal analysis of total number of nuclei across development in control background ( domeMESO - Gal4 , UAS - GFP / + , n = 37 at 48h , n = 31 at 72h , n = 19 at 96h and n = 23 at 120h AEL ) . The loss of dopamine modules at 48h AEL ( domeMESO - Gal4 , UAS - GFP ; THRNAi , n = 19 , domeMESO - Gal4 , UAS - GFP ; DdcRNAi , n = 16 , domeMESO - Gal4 , UAS - GFP ; Dop2RRNAi , n = 30 and domeMESO - Gal4 , UAS - GFP ; DATRNAi , n = 21 ) , at 72h AEL ( domeMESO - Gal4 , UAS - GFP ; THRNAi , n = 22 , domeMESO - Gal4 , UAS - GFP ; DdcRNAi , n = 24 , domeMESO - Gal4 , UAS - GFP ; Dop2RRNAi , n = 33 and domeMESO - Gal4 , UAS - GFP ; DATRNAi , n = 27 ) , at 96h AEL ( domeMESO - Gal4 , UAS - GFP ; THRNAi , n = 21 , domeMESO - Gal4 , UAS - GFP ; DdcRNAi , n = 18 , domeMESO - Gal4 , UAS - GFP ; Dop2RRNAi , n = 20 and domeMESO - Gal4 , UAS - GFP ; DATRNAi , n = 22 ) and at 120h AEL ( domeMESO - Gal4 , UAS - GFP ; THRNAi , n = 25 , domeMESO - Gal4 , UAS - GFP ; DdcRNAi , n = 30 , domeMESO - Gal4 , UAS - GFP ; Dop2RRNAi , n = 25 and domeMESO - Gal4 , UAS - GFP ; DATRNAi , n = 31 ) . Data information : The quantification represents median with whiskers representing maximum and minimum values , box indicates the lower and upper quartiles . The statistical analysis is Two - way Anova with Tukey ' s multiple comparisons test while the control comparison , domeMESO - Gal4 , UAS - GFP / + , for different time points was done by One - way ANOVA with Tukey ' s multiple comparisons test . ' n ' represents the number of lymph gland lobes analyzed , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . AEL indicates After Egg

O O O O O O O O O O B-SUBCELLULAR O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O O 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-SMALL_MOLECULE O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O 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-TISSUE I-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) A temporal analysis of total number of nuclei across development in control background (domeMESO-Gal4, UAS-GFP/+, n= 37 at 48h, n= 31 at 72h, n= 19 at 96h and n= 23 at 120h AEL). The loss of dopamine modules at 48h AEL (domeMESO-Gal4, UAS-GFP; THRNAi, n=19, domeMESO-Gal4, UAS-GFP; DdcRNAi, n=16, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n=30 and domeMESO-Gal4, UAS-GFP; DATRNAi, n=21), at 72h AEL (domeMESO-Gal4, UAS-GFP; THRNAi, n=22, domeMESO-Gal4, UAS-GFP; DdcRNAi, n=24, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n=33 and domeMESO-Gal4, UAS-GFP; DATRNAi, n=27), at 96h AEL (domeMESO-Gal4, UAS-GFP; THRNAi, n=21, domeMESO-Gal4, UAS-GFP; DdcRNAi, n=18, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n=20 and domeMESO-Gal4, UAS-GFP; DATRNAi, n=22) and at 120h AEL (domeMESO-Gal4, UAS-GFP; THRNAi, n=25, domeMESO-Gal4, UAS-GFP; DdcRNAi, n=30, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n=25 and domeMESO-Gal4, UAS-GFP; DATRNAi, n=31). Data information: The quantification represents median with whiskers representing maximum and minimum values, box indicates the lower and upper quartiles. The statistical analysis is Two-way Anova with Tukey's multiple comparisons test while the control comparison, domeMESO-Gal4, UAS-GFP/+, for different time points was done by One-way ANOVA with Tukey's multiple comparisons test. 'n' represents the number of lymph gland lobes analyzed, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. AEL indicates After Egg Laying


( B ) Quantification of total Dome + nuclei counts of control animals ( domeMESO - Gal4 , UAS - GFP / + , n = 37 at 48h , n = 28 at 72h , n = 19 at 96h and n = 23 at 120h AEL ) . The loss of dopamine modules at 48h AEL ( domeMESO - Gal4 , UAS - GFP ; THRNAi , n = 22 , domeMESO - Gal4 , UAS - GFP ; DdcRNAi , n = 16 , domeMESO - Gal4 , UAS - GFP ; Dop2RRNAi , n = 30 , domeMESO - Gal4 , UAS - GFP ; DATRNAi , n = 21 ) , at 72h AEL ( domeMESO - Gal4 , UAS - GFP ; THRNAi , n = 22 , domeMESO - Gal4 , UAS - GFP ; DdcRNAi , n = 24 , domeMESO - Gal4 , UAS - GFP ; Dop2RRNAi , n = 32 , domeMESO - Gal4 , UAS - GFP ; DATRNAi , n = 27 ) , at 96h AEL ( domeMESO - Gal4 , UAS - GFP ; THRNAi , n = 21 , domeMESO - Gal4 , UAS - GFP ; DdcRNAi , n = 17 , domeMESO - Gal4 , UAS - GFP ; Dop2RRNAi , n = 20 , domeMESO - Gal4 , UAS - GFP ; DATRNAi , n = 22 ) and at 120h AEL ( domeMESO - Gal4 , UAS - GFP ; THRNAi , n = 24 , domeMESO - Gal4 , UAS - GFP ; DdcRNAi , n = 30 , domeMESO - Gal4 , UAS - GFP ; Dop2RRNAi , n = 25 , domeMESO - Gal4 , UAS - GFP ; DATRNAi , n = 30 ) . ( C ) A temporal analysis of Dome - nuclei counts in the control animals ( domeMESO - Gal4 , UAS - GFP / + , n = 37 at 48h , n = 28 at 72h , n = 18 at 96h , n = 23 , at 120h AEL ) . The perturbation of the dopamine modules in the progenitors at 48h AEL ( domeMESO - Gal4 , UAS - GFP ; THRNAi , n = 22 , domeMESO - Gal4 , UAS - GFP ; DdcRNAi , n = 16 , domeMESO - Gal4 , UAS - GFP ; Dop2RRNAi , n = 30 , domeMESO - Gal4 , UAS - GFP ; DATRNAi , n = 21 ) , at 72h AEL ( domeMESO - Gal4 , UAS - GFP ; THRNAi , n = 22 , domeMESO - Gal4 , UAS - GFP ; DdcRNAi , n = 24 , domeMESO - Gal4 , UAS - GFP ; Dop2RRNAi , n = 32 , domeMESO - Gal4 , UAS - GFP ; DATRNAi , n = 27 ) , at 96h AEL ( domeMESO - Gal4 , UAS - GFP ; THRNAi , n = 21 , domeMESO - Gal4 , UAS - GFP ; DdcRNAi , n = 17 , domeMESO - Gal4 , UAS - GFP ; Dop2RRNAi , n = 20 , domeMESO - Gal4 , UAS - GFP ; DATRNAi , n = 22 ) and at 120h AEL ( domeMESO - Gal4 , UAS - GFP ; THRNAi , n = 24 , domeMESO - Gal4 , UAS - GFP ; DdcRNAi , n = 30 , domeMESO - Gal4 , UAS - GFP ; Dop2RRNAi , n = 25 , domeMESO - Gal4 , UAS - GFP ; DATRNAi , n = 30 ) . Data information : The quantification represents median with whiskers representing maximum and minimum values , box indicates the lower and upper quartiles . The statistical analysis applied is Two - way Anova with Tukey ' s multiple comparisons test while the control comparison , domeMESO - Gal4 , UAS - GFP / + , for different time points was done by One - way ANOVA with Tukey ' s multiple comparisons test . ' n ' represents the number of lymph gland lobes analyzed , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . AEL indicates After Egg

O O O O O O B-GENEPROD O B-SUBCELLULAR O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O O 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-SMALL_MOLECULE O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O B-SUBCELLULAR O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O 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-SMALL_MOLECULE O O O B-CELL_TYPE O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O 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-TISSUE I-TISSUE I-TISSUE O O O O O O O O O O O O O O O O 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) Quantification of total Dome+ nuclei counts of control animals (domeMESO-Gal4, UAS-GFP/+, n= 37 at 48h, n= 28 at 72h, n= 19 at 96h and n=23 at 120h AEL). The loss of dopamine modules at 48h AEL (domeMESO-Gal4, UAS-GFP; THRNAi, n=22, domeMESO-Gal4, UAS-GFP; DdcRNAi, n=16, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n=30, domeMESO-Gal4, UAS-GFP; DATRNAi, n=21), at 72h AEL (domeMESO-Gal4, UAS-GFP; THRNAi, n=22, domeMESO-Gal4, UAS-GFP; DdcRNAi, n=24, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n=32, domeMESO-Gal4, UAS-GFP; DATRNAi, n=27), at 96h AEL (domeMESO-Gal4, UAS-GFP; THRNAi, n=21, domeMESO-Gal4, UAS-GFP; DdcRNAi, n=17, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n=20, domeMESO-Gal4, UAS-GFP; DATRNAi, n=22) and at 120h AEL (domeMESO-Gal4, UAS-GFP; THRNAi, n=24, domeMESO-Gal4, UAS-GFP; DdcRNAi, n=30, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n=25, domeMESO-Gal4, UAS-GFP; DATRNAi, n=30). (C) A temporal analysis of Dome- nuclei counts in the control animals (domeMESO-Gal4, UAS-GFP/+, n= 37 at 48h, n= 28 at 72h, n= 18 at 96h, n= 23, at 120h AEL). The perturbation of the dopamine modules in the progenitors at 48h AEL (domeMESO-Gal4, UAS-GFP; THRNAi, n=22, domeMESO-Gal4, UAS-GFP; DdcRNAi, n=16, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n=30, domeMESO-Gal4, UAS-GFP; DATRNAi, n=21), at 72h AEL (domeMESO-Gal4, UAS-GFP; THRNAi, n=22, domeMESO-Gal4, UAS-GFP; DdcRNAi, n=24, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n=32, domeMESO-Gal4, UAS-GFP; DATRNAi, n=27), at 96h AEL (domeMESO-Gal4, UAS-GFP; THRNAi, n=21, domeMESO-Gal4, UAS-GFP; DdcRNAi, n=17, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n=20, domeMESO-Gal4, UAS-GFP; DATRNAi, n=22) and at 120h AEL (domeMESO-Gal4, UAS-GFP; THRNAi, n=24, domeMESO-Gal4, UAS-GFP; DdcRNAi, n=30, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n=25, domeMESO-Gal4, UAS-GFP; DATRNAi, n=30). Data information: The quantification represents median with whiskers representing maximum and minimum values, box indicates the lower and upper quartiles. The statistical analysis applied is Two-way Anova with Tukey's multiple comparisons test while the control comparison, domeMESO-Gal4, UAS-GFP/+, for different time points was done by One-way ANOVA with Tukey's multiple comparisons test. 'n' represents the number of lymph gland lobes analyzed, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. AEL indicates After Egg Laying


( A ) Quantification of the Dome + BrdU index normalised to Dome + nuclei in a time course manner with dopamine modules perturbed in the progenitors using domeMESO - Gal4 , UAS - GFP driver . At 48h AEL ( domeMESO - Gal4 , UAS - GFP / + , n = 11 , domeMESO - Gal4 , UAS - GFP ; THRNAi , n = 13 , domeMESO - Gal4 , UAS - GFP ; DdcRNAi , n = 11 , domeMESO - Gal4 , UAS - GFP ; Dop2RRNAi , n = 15 , domeMESO - Gal4 , UAS - GFP ; DATRNAi , n = 5 ) , at 72h AEL ( domeMESO - Gal4 , UAS - GFP / + , n = 34 , domeMESO - Gal4 , UAS - GFP ; THRNAi , n = 31 , domeMESO - Gal4 , UAS - GFP ; DdcRNAi , n = 16 , domeMESO - Gal4 , UAS - GFP ; Dop2RRNAi , n = 15 , domeMESO - Gal4 , UAS - GFP ; DATRNAi , n = 27 ) and at 96h AEL ( domeMESO - Gal4 , UAS - GFP / + , n = 17 , domeMESO - Gal4 , UAS - GFP ; THRNAi , n = 28 , domeMESO - Gal4 , UAS - GFP ; DdcRNAi , n = 20 , domeMESO - Gal4 , UAS - GFP ; Dop2RRNAi , n = 17 , domeMESO - Gal4 , UAS - GFP ; DATRNAi , n = 22 ) . ( B ) Quantification of the Dome + mitotic index normalised to Dome + nuclei in a time course manner with loss of dopamine modules in the progenitors using domeMESO - Gal4 , UAS - GFP driver . At 48h AEL ( domeMESO - Gal4 , UAS - GFP / + , n = 35 , domeMESO - Gal4 , UAS - GFP ; THRNAi , n = 21 , domeMESO - Gal4 , UAS - GFP ; DdcRNAi , n = 16 , domeMESO - Gal4 , UAS - GFP ; Dop2RRNAi , n = 30 , domeMESO - Gal4 , UAS - GFP ; DATRNAi , n = 20 ) , at 72h AEL ( domeMESO - Gal4 , UAS - GFP / + , n = 27 , domeMESO - Gal4 , UAS - GFP ; THRNAi , n = 21 , domeMESO - Gal4 , UAS - GFP ; DdcRNAi , n = 23 , domeMESO - Gal4 , UAS - GFP ; Dop2RRNAi , n = 31 , domeMESO - Gal4 , UAS - GFP ; DATRNAi , n = 27 ) and at 96h AEL ( domeMESO - Gal4 , UAS - GFP / + , n = 19 , domeMESO - Gal4 , UAS - GFP ; THRNAi , n = 21 , domeMESO - Gal4 , UAS - GFP ; DdcRNAi , n = 17 , domeMESO - Gal4 , UAS - GFP ; Dop2RRNAi , n = 20 , domeMESO - Gal4 , UAS - GFP ; DATRNAi , n = 22 ) . Data information : AEL indicates After Egg Laying . The quantifications in A and B represent median with whiskers indicating maximum and minimum value , box indicates the lower and upper quartiles The statistical analysis applied is Two - way Anova with Tukey ' s multiple comparisons test . ' n ' represents the number of lymph gland lobes analyzed , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O B-GENEPROD O B-SUBCELLULAR O O O O O O B-SMALL_MOLECULE O O O O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-SUBCELLULAR O O O O O O O O B-SMALL_MOLECULE O O O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-TISSUE I-TISSUE I-TISSUE O O O O O O O O O O O O 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) Quantification of the Dome+ BrdU index normalised to Dome+ nuclei in a time course manner with dopamine modules perturbed in the progenitors using domeMESO-Gal4, UAS-GFP driver. At 48h AEL (domeMESO-Gal4, UAS-GFP/+, n=11, domeMESO-Gal4, UAS-GFP; THRNAi, n=13, domeMESO-Gal4, UAS-GFP; DdcRNAi, n=11, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n=15, domeMESO-Gal4, UAS-GFP; DATRNAi, n=5), at 72h AEL (domeMESO-Gal4, UAS-GFP/+, n=34, domeMESO-Gal4, UAS-GFP; THRNAi, n=31, domeMESO-Gal4, UAS-GFP; DdcRNAi, n=16, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n=15, domeMESO-Gal4, UAS-GFP; DATRNAi, n=27) and at 96h AEL (domeMESO-Gal4, UAS-GFP/+, n=17, domeMESO-Gal4, UAS-GFP; THRNAi, n=28, domeMESO-Gal4, UAS-GFP; DdcRNAi, n=20, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n=17, domeMESO-Gal4, UAS-GFP; DATRNAi, n=22). (B) Quantification of the Dome+ mitotic index normalised to Dome+ nuclei in a time course manner with loss of dopamine modules in the progenitors using domeMESO-Gal4, UAS-GFP driver. At 48h AEL (domeMESO-Gal4, UAS-GFP/+, n=35, domeMESO-Gal4, UAS-GFP; THRNAi, n=21, domeMESO-Gal4, UAS-GFP; DdcRNAi, n=16, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n=30, domeMESO-Gal4, UAS-GFP; DATRNAi, n=20), at 72h AEL (domeMESO-Gal4, UAS-GFP/+, n=27, domeMESO-Gal4, UAS-GFP; THRNAi, n=21, domeMESO-Gal4, UAS-GFP; DdcRNAi, n=23, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n=31, domeMESO-Gal4, UAS-GFP; DATRNAi, n=27) and at 96h AEL (domeMESO-Gal4, UAS-GFP/+, n=19, domeMESO-Gal4, UAS-GFP; THRNAi, n=21, domeMESO-Gal4, UAS-GFP; DdcRNAi, n=17, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n=20, domeMESO-Gal4, UAS-GFP; DATRNAi, n=22). Data information: AEL indicates After Egg Laying. The quantifications in A and B represent median with whiskers indicating maximum and minimum value, box indicates the lower and upper quartiles The statistical analysis applied is Two-way Anova with Tukey's multiple comparisons test. 'n' represents the number of lymph gland lobes analyzed, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.


( C ) Quantifications of % G1 , % S and % G2 / M at different time points , in control and dopamine perturbed backgrounds , color coding scheme to represent the cell cycle phase as shown through FUCCI reporter is below the graph . Across development , at 48h AEL ( domeMESO - Gal4 ; UAS - FUCCI / + , n = 34 , domeMESO - Gal4 ; UAS - FUCCI ; THRNAi , n = 13 , domeMESO - Gal4 ; UAS - FUCCI ; DdcRNAi , n = 15 , domeMESO - Gal4 ; UAS - FUCCI ; Dop2RRNAi , n = 19 , domeMESO - Gal4 ; UAS - FUCCI ; DATRNAi , n = 17 ) , at 72h AEL ( domeMESO - Gal4 ; UAS - FUCCI / + , n = 38 , domeMESO - Gal4 ; UAS - FUCCI ; THRNAi , n = 22 , domeMESO - Gal4 ; UAS - FUCCI ; DdcRNAi , n = 29 , domeMESO - Gal4 ; UAS - FUCCI ; Dop2RRNAi , n = 47 , domeMESO - Gal4 ; UAS - FUCCI ; DATRNAi , n = 29 ) and at 96h AEL ( domeMESO - Gal4 ; UAS - FUCCI / + , n = 55 , domeMESO - Gal4 ; UAS - FUCCI ; THRNAi , n = 14 , domeMESO - Gal4 ; UAS - FUCCI ; DdcRNAi , n = 19 , domeMESO - Gal4 ; UAS - FUCCI ; Dop2RRNAi , domeMESO - Gal4 ; UAS - FUCCI ; DATRNAi , n = 19 ) Data information : FUCCI ( fluorescent ubiquitination - based cell cycle indicator ) reporter shows G1 phase cells in green , S phase cells in red and G2 / M phase cells in yellow . AEL indicates After Egg Laying . The quantifications in in C represents mean . The statistical analysis applied is Two - way Anova with Tukey ' s multiple comparisons test . The domeMESO - Gal4 ; UAS - FUCCI control comparison temporally in C is analysed using One - way ANOVA with Tukey ' s multiple comparisons test . ' n ' represents the number of lymph gland lobes analyzed , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O 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-TISSUE I-TISSUE I-TISSUE O O O O O O O O O O O O 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) Quantifications of %G1, %S and %G2/M at different time points, in control and dopamine perturbed backgrounds, color coding scheme to represent the cell cycle phase as shown through FUCCI reporter is below the graph. Across development, at 48h AEL (domeMESO-Gal4; UAS-FUCCI/+, n=34, domeMESO-Gal4; UAS-FUCCI; THRNAi, n= 13, domeMESO-Gal4; UAS-FUCCI; DdcRNAi, n= 15, domeMESO-Gal4; UAS-FUCCI; Dop2RRNAi, n= 19, domeMESO-Gal4; UAS-FUCCI; DATRNAi, n= 17), at 72h AEL (domeMESO-Gal4; UAS-FUCCI/+, n=38, domeMESO-Gal4; UAS-FUCCI; THRNAi, n= 22, domeMESO-Gal4; UAS-FUCCI; DdcRNAi, n= 29, domeMESO-Gal4; UAS-FUCCI; Dop2RRNAi, n= 47, domeMESO-Gal4; UAS-FUCCI; DATRNAi, n= 29) and at 96h AEL (domeMESO-Gal4; UAS-FUCCI/+, n=55, domeMESO-Gal4; UAS-FUCCI; THRNAi, n= 14, domeMESO-Gal4; UAS-FUCCI; DdcRNAi, n= 19, domeMESO-Gal4; UAS-FUCCI; Dop2RRNAi, domeMESO-Gal4; UAS-FUCCI; DATRNAi, n= 19) Data information: FUCCI (fluorescent ubiquitination-based cell cycle indicator) reporter shows G1 phase cells in green, S phase cells in red and G2/M phase cells in yellow. AEL indicates After Egg Laying. The quantifications in in C represents mean. The statistical analysis applied is Two-way Anova with Tukey's multiple comparisons test. The domeMESO-Gal4; UAS-FUCCI control comparison temporally in C is analysed using One-way ANOVA with Tukey's multiple comparisons test. 'n' represents the number of lymph gland lobes analyzed, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.


( A - I ' ' ) Representative lymph gland images at 48 , 72 and 96h AEL depicting the lymph gland growth trajectory along with dopamine level status in the PSC and remaining lymph gland . ( A - C ' ' ) The control profile of lymph gland and PSC iDopamine levels at 48h ( A - A ' ' ) , 72h ( B - B ' ' ) and 96h ( C - C ' ' ) . ( D - I ' ' ) Downregulation of the dopamine synthesising enzymes TH and Ddc in the PSC at 48h ( D - D ' ' and G - G ' ' ) , 72h AEL ( E - E ' ' and H - H ' ' ) and at 96h AEL ( F - F ' ' and I - I ' ' ) . ( J ) Quantification of mean dopamine intensity in the PSC at upon PSC perturbation of dopamine synthesis at 48h AEL ( Antp - Gal4 ; UAS - GFP / + , n = 33 , Antp - Gal4 ; UAS - GFP ; THRNAi , n = 8 , Antp - Gal4 ; UAS - GFP ; DdcRNAi , n = 14 ) , at 72h AEL ( Antp - Gal4 ; UAS - GFP / + , n = 26 , Antp - Gal4 ; UAS - GFP ; THRNAi , n = 25 , Antp - Gal4 ; UAS - GFP ; DdcRNAi , n = 20 ) and 96h AEL ( Antp - Gal4 ; UAS - GFP / + , n = 15 , Antp - Gal4 ; UAS - GFP ; THRNAi , n = 13 , Antp - Gal4 ; UAS - GFP ; DdcRNAi , n = 17 ) . ( K ) Quantification of mean dopamine levels in the lymph gland upon PSC perturbation of dopamine synthesis at 48h AEL ( Antp - Gal4 ; UAS - GFP / + , n = 28 , Antp - Gal4 ; UAS - GFP ; THRNAi , n = 13 , Antp - Gal4 ; UAS - GFP ; DdcRNAi , n = 18 ) , at 72h AEL ( Antp - Gal4 ; UAS - GFP / + , n = 25 , Antp - Gal4 ; UAS - GFP ; THRNAi , n = 24 , Antp - Gal4 ; UAS - GFP ; DdcRNAi , n = 22 ) and 96h AEL ( Antp - Gal4 ; UAS - GFP / + , n = 15 , Antp - Gal4 ; UAS - GFP ; THRNAi , n = 16 , Antp - Gal4 ; UAS - GFP ; DdcRNAi , n = 18 ) . Data information : DNA is stained with Hoechst in blue , Antp - Gal4 ; UAS - GFP ( representative of Posterior Signaling Center , PSC ) in green , iDopamine in red . All image panels show a 20µm scale bar . AEL indicates After Egg Laying . The quantification in J , K represents the median with whiskers indicating maximum and minimum values , box indicates the lower and upper quartiles . The statistical analysis applied in J , K is Two - way Anova with Tukey ' s multiple comparisons test . The control animals Antp - Gal4 ; UAS - GFP / + were analysed by One - way Anova with Tukey ' s multiple comparisons test across time points . ' n ' represents the number of lymph gland lobes analyzed , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . For better representation , the primary lobe of the lymph gland has been represented and outlined in white dashed lines and the PSC in yellow dashed line .

O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O B-TISSUE I-TISSUE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-TISSUE I-TISSUE O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY O B-SMALL_MOLECULE O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O 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-TISSUE I-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O 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-TISSUE O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O

(A-I'') Representative lymph gland images at 48, 72 and 96h AEL depicting the lymph gland growth trajectory along with dopamine level status in the PSC and remaining lymph gland. (A-C'') The control profile of lymph gland and PSC iDopamine levels at 48h (A-A''), 72h (B-B'') and 96h (C-C''). (D-I'') Downregulation of the dopamine synthesising enzymes TH and Ddc in the PSC at 48h (D-D'' and G-G''), 72h AEL (E-E'' and H-H'') and at 96h AEL (F-F'' and I-I''). (J) Quantification of mean dopamine intensity in the PSC at upon PSC perturbation of dopamine synthesis at 48h AEL (Antp-Gal4; UAS-GFP/+, n=33, Antp-Gal4; UAS-GFP; THRNAi, n=8, Antp-Gal4; UAS-GFP; DdcRNAi, n=14), at 72h AEL (Antp-Gal4; UAS-GFP/+, n=26, Antp-Gal4; UAS-GFP; THRNAi, n=25, Antp-Gal4; UAS-GFP; DdcRNAi, n=20) and 96h AEL (Antp-Gal4; UAS-GFP/+, n=15, Antp-Gal4; UAS-GFP; THRNAi, n=13, Antp-Gal4; UAS-GFP; DdcRNAi, n=17). (K) Quantification of mean dopamine levels in the lymph gland upon PSC perturbation of dopamine synthesis at 48h AEL (Antp-Gal4; UAS-GFP/+, n=28, Antp-Gal4; UAS-GFP; THRNAi, n=13, Antp-Gal4; UAS-GFP; DdcRNAi, n=18), at 72h AEL (Antp-Gal4; UAS-GFP/+, n=25, Antp-Gal4; UAS-GFP; THRNAi, n=24, Antp-Gal4; UAS-GFP; DdcRNAi, n=22) and 96h AEL (Antp-Gal4; UAS-GFP/+, n=15, Antp-Gal4; UAS-GFP; THRNAi, n=16, Antp-Gal4; UAS-GFP; DdcRNAi, n=18). Data information: DNA is stained with Hoechst in blue, Antp-Gal4; UAS-GFP (representative of Posterior Signaling Center, PSC) in green, iDopamine in red. All image panels show a 20µm scale bar. AEL indicates After Egg Laying. The quantification in J, K represents the median with whiskers indicating maximum and minimum values, box indicates the lower and upper quartiles. The statistical analysis applied in J, K is Two-way Anova with Tukey's multiple comparisons test. The control animals Antp-Gal4; UAS-GFP/+ were analysed by One-way Anova with Tukey's multiple comparisons test across time points. 'n' represents the number of lymph gland lobes analyzed, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. For better representation, the primary lobe of the lymph gland has been represented and outlined in white dashed lines and the PSC in yellow dashed line.


( L ) Analysis of the total cell counts upon loss of TH and Ddc in the PSC at 48h AEL ( Antp - Gal4 ; UAS - GFP / + , n = 19 , Antp - Gal4 ; UAS - GFP ; THRNAi , n = 13 , Antp - Gal4 ; UAS - GFP ; DdcRNAi , n = 17 ) , at 72h AEL ( Antp - Gal4 ; UAS - GFP / + , n = 17 , Antp - Gal4 ; UAS - GFP ; THRNAi , n = 18 , Antp - Gal4 ; UAS - GFP ; DdcRNAi , n = 22 ) and 96h AEL ( Antp - Gal4 ; UAS - GFP / + , n = 15 , Antp - Gal4 ; UAS - GFP ; THRNAi , n = 14 , Antp - Gal4 ; UAS - GFP ; DdcRNAi , n = 15 ) Data information : The quantification in L represents the median with whiskers indicating maximum and minimum values , box indicates the lower and upper quartiles . The statistical analysis applied in L is Two - way Anova with Tukey ' s multiple comparisons test . ' n ' represents the number of lymph gland lobes analyzed , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-TISSUE I-TISSUE I-TISSUE O O O O O O O O O O O O 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) Analysis of the total cell counts upon loss of TH and Ddc in the PSC at 48h AEL (Antp-Gal4; UAS-GFP/+, n=19, Antp-Gal4; UAS-GFP; THRNAi, n=13, Antp-Gal4; UAS-GFP; DdcRNAi, n=17), at 72h AEL (Antp-Gal4; UAS-GFP/+, n=17, Antp-Gal4; UAS-GFP; THRNAi, n=18, Antp-Gal4; UAS-GFP; DdcRNAi, n=22) and 96h AEL (Antp-Gal4; UAS-GFP/+, n=15, Antp-Gal4; UAS-GFP; THRNAi, n=14, Antp-Gal4; UAS-GFP; DdcRNAi, n=15) Data information: The quantification in L represents the median with whiskers indicating maximum and minimum values, box indicates the lower and upper quartiles. The statistical analysis applied in L is Two-way Anova with Tukey's multiple comparisons test. 'n' represents the number of lymph gland lobes analyzed, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.


( A ) Substrate - dependent nitrogenase activity , measured through reduction of acetylene into ethylene , in isolated B . diazoefficiens bacteroids upon supplementation of malate , succinate , arginine , and nodule extract . Data represents the mean and standard error of the mean of at least eight independent replciates .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O

(A) Substrate-dependent nitrogenase activity, measured through reduction of acetylene into ethylene, in isolated B. diazoefficiens bacteroids upon supplementation of malate, succinate, arginine, and nodule extract. Data represents the mean and standard error of the mean of at least eight independent replciates.


( B ) Fold change in intracellular ATP level in isolated B . diazoefficiens bacteroids upon supplementation of succinate , arginine , and nodule extract . The values obtained for each of the 4 individual measurements ( points ) together with the regression trajectory of each experiment is shown .

O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-TISSUE I-TISSUE O 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) Fold change in intracellular ATP level in isolated B. diazoefficiens bacteroids upon supplementation of succinate, arginine, and nodule extract. The values obtained for each of the 4 individual measurements (points) together with the regression trajectory of each experiment is shown.


( A ) The aerial part of M . truncatula upon infection with S . meliloti ∆ argI1 , ∆ argI2 , ∆ argI1 , 2 and the dicarboxylate transport mutant ∆ dctAB were smaller than those inoculated with the wild type strain , highlighting the importance of arginine catabolism for nitrogen

O O O O B-TISSUE I-TISSUE O B-ORGANISM I-ORGANISM I-ORGANISM O B-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O

(A) The aerial part of M. truncatula upon infection with S. meliloti ∆argI1, ∆argI2, ∆argI1,2 and the dicarboxylate transport mutant ∆dctAB were smaller than those inoculated with the wild type strain, highlighting the importance of arginine catabolism for nitrogen fixation


( B ) Cross sections of nodules bearing S . meliloti WT or arginine catabolism mutant ∆ argI1 , ∆ argI2 reveals the yellowish color of non - functional nodules induced by the ∆ argI1 ∆ argI2 double deletion mutant .

O O O O O O B-TISSUE O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O B-TISSUE O O O O B-GENEPROD O B-GENEPROD O O O O

(B) Cross sections of nodules bearing S. meliloti WT or arginine catabolism mutant ∆argI1, ∆argI2 reveals the yellowish color of non-functional nodules induced by the ∆argI1 ∆argI2 double deletion mutant.


( C ) Bacteroid ultrastructure across nodule sections determined by scanning electron microscopy indicates the presence of hollow nodules with aberrant cell morphology in the ∆ argI1 ∆ argI2 double deletion mutant defective in arginine catabolism .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-TISSUE I-TISSUE O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O O O O O O O O

(C) Bacteroid ultrastructure across nodule sections determined by scanning electron microscopy indicates the presence of hollow nodules with aberrant cell morphology in the ∆argI1 ∆argI2 double deletion mutant defective in arginine catabolism.


( D ) Nitrogenase activity in M . truncatula nodules inoculated with S . meliloti strains defective in arginine catabolism . Data points are the mean of at least 30 plants measured after 8 weeks post inoculation ; error bars indicate standard error of the mean .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM B-TISSUE O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O

(D) Nitrogenase activity in M. truncatula nodules inoculated with S. meliloti strains defective in arginine catabolism. Data points are the mean of at least 30 plants measured after 8 weeks post inoculation; error bars indicate standard error of the mean.


( B ) Heat map representing enzymatic activities participating in the arginine transamination network with relative enzyme activities highlighted as a color map Rows are annotated on the left with the abbreviated names of the substrates and on the right with the corresponding EC numbers . Columns are annotated by corresponding enzyme names .

O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O 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) Heat map representing enzymatic activities participating in the arginine transamination network with relative enzyme activities highlighted as a color map Rows are annotated on the left with the abbreviated names of the substrates and on the right with the corresponding EC numbers. Columns are annotated by corresponding enzyme names.


( C ) Recombinant enzymes involved in arginine catabolism were combined into a single reaction mixture using arginine and pyruvate as substrates . Substrate ( S ) consumption into the products ( P ) succinate and alanine , including their intermediates ( I1 , I2 ) , was determined along the time series using LC - MS / MS .

O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O

(C) Recombinant enzymes involved in arginine catabolism were combined into a single reaction mixture using arginine and pyruvate as substrates. Substrate (S) consumption into the products (P) succinate and alanine, including their intermediates (I1,I2), was determined along the time series using LC-MS/MS.


( D ) The relative metabolite abundance along the reaction time - course is highlighted as a color

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O

(D) The relative metabolite abundance along the reaction time-course is highlighted as a color map


Representative Ca2 + signals in sperm from a patient with deafness - infertility syndrome lacking functional CatSper channels ( CATSPER2 - / - ) ( red ) and a healthy donor ( black ) , evoked by progesterone , PGE1 , NH4Cl , or ionomycin . NH4Cl increases the intracellular pH . Bar graph : Amplitudes ( n = 4 ; mean ± SD ) of Ca2 + signals in CATSPER2 - / -

O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-CELL_TYPE O O B-ORGANISM O B-DISEASE I-DISEASE I-DISEASE I-DISEASE O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-GENEPROD O O O

Representative Ca2+ signals in sperm from a patient with deafness-infertility syndrome lacking functional CatSper channels (CATSPER2-/-) (red) and a healthy donor (black), evoked by progesterone, PGE1, NH4Cl, or ionomycin. NH4Cl increases the intracellular pH. Bar graph: Amplitudes (n = 4; mean ± SD) of Ca2+ signals in CATSPER2-/- sperm


Representative monovalent CatSper currents in CATSPER2 - / - sperm ( blue , green , orange , purple , brown ) and in sperm from a healthy donor ( black ) and corresponding current - voltage relationship ( right ) . The membrane voltage was stepped from - 100 mV to + 100 mV in increments of 10 mV from a holding potential of - 80 mV . Outward and inward current amplitudes ( mean ± SD ) at + 100 mV and - 100 mV , respectively , in CATSPER2 - / - sperm ( color code : panel B ) and sperm from healthy donors ( black ) .

O O B-GENEPROD O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O B-CELL_TYPE O O O O O O O

Representative monovalent CatSper currents in CATSPER2-/- sperm (blue, green, orange, purple, brown) and in sperm from a healthy donor (black) and corresponding current-voltage relationship (right). The membrane voltage was stepped from -100 mV to +100 mV in increments of 10 mV from a holding potential of -80 mV. Outward and inward current amplitudes (mean ± SD) at +100 mV and -100 mV, respectively, in CATSPER2-/- sperm (color code: panel B) and sperm from healthy donors (black).


Representative immunocytochemical staining of control sperm from healthy donors and CATSPER2 - / - sperm form DIS patients using antibodies directed against CatSper 3 ( D ) or CatSper 4 ( E ) ; DNA was labelled with DAPI ( blue ) . Scale bars represent 10 µm .

O B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_TYPE O O O O B-GENEPROD O O O B-CELL_TYPE O B-DISEASE B-ORGANISM O O O O B-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O

Representative immunocytochemical staining of control sperm from healthy donors and CATSPER2-/- sperm form DIS patients using antibodies directed against CatSper 3 (D) or CatSper 4 (E); DNA was labelled with DAPI (blue). Scale bars represent 10 µm.


3D - STORM image in xy - projection of sperm from a healthy donor labelled with the anti - CatSper 3 antibody ( left ) . Axial projection of the boxed region ( right ) . Scale bars represent 5 µm in xy - projections and 200 nm in axial projections .

O O O B-EXP_ASSAY O O O O O B-CELL_TYPE O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

3D-STORM image in xy-projection of sperm from a healthy donor labelled with the anti-CatSper 3 antibody (left). Axial projection of the boxed region (right). Scale bars represent 5 µm in xy-projections and 200 nm in axial projections.


3D - STORM images in xy - projection of CATSPER2 - / - sperm ( left ) labelled with the anti - CatSper 3 ( upper panel ) and anti - CatSper 4 ( lower panel ) antibody . Axial projection of the boxed regions ( right ) . Scale bars represent 5 µm in xy - projections and 200 nm in axial projections .

O O O B-EXP_ASSAY O O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

3D-STORM images in xy-projection of CATSPER2-/- sperm (left) labelled with the anti-CatSper 3 (upper panel) and anti-CatSper 4 (lower panel) antibody. Axial projection of the boxed regions (right). Scale bars represent 5 µm in xy-projections and 200 nm in axial projections.


Dark - field microscopy of a single sperm ; shown are single frames obtained at t = 0 , 48 , 96 , and 136 ms . Scale bar = 25

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O

Dark-field microscopy of a single sperm; shown are single frames obtained at t = 0, 48, 96, and 136 ms. Scale bar = 25 µm


Dark - field imaging of a sperm population ; left : single frames at t = 185 , 363 , 540 , and 718 ms . Sperm selected for analysis are highlighted ( 1 - 4 ) . Right : Temporal change in the brightness ( blinking ) of sperm heads . The blue lines correspond to the time - points of the single frames . Scale bar = 25 µm .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O

Dark-field imaging of a sperm population; left: single frames at t = 185, 363, 540, and 718 ms. Sperm selected for analysis are highlighted (1-4). Right: Temporal change in the brightness (blinking) of sperm heads. The blue lines correspond to the time-points of the single frames. Scale bar = 25 µm.


Representative distribution of rotation frequencies of freely swimming non - capacitated ( 0 mM bicarbonate ; black ; n = 218 ) and capacitated sperm ( 25 mM bicarbonate ; red ; n = 232 ) determined by dark - field imaging .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-CELL_TYPE O O O B-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O

Representative distribution of rotation frequencies of freely swimming non-capacitated (0 mM bicarbonate; black; n = 218) and capacitated sperm (25 mM bicarbonate; red; n = 232) determined by dark-field imaging.


Rolling frequency ( mean ± SD ) of non - capacitated ( 0 mM bicarbonate , n = 1 , 455 ) and capacitated sperm ( 25 mM bicarbonate , n = 1 , 097 ) .

B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-CELL_TYPE O O O B-SMALL_MOLECULE O O O O O O O O

Rolling frequency (mean ± SD) of non-capacitated (0 mM bicarbonate, n = 1,455) and capacitated sperm (25 mM bicarbonate, n = 1,097).


Rolling frequency ( mean ± SD ) of freely swimming sperm in 0 ( n = 1 , 175 ) , 0 . 2 ( n = 832 ) , and 1 % ( w / v ) ( n = 599 ) methyl cellulose . .

B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-CELL_TYPE O O O O O O O O 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-SMALL_MOLECULE I-SMALL_MOLECULE O O

Rolling frequency (mean ± SD) of freely swimming sperm in 0 (n = 1,175), 0.2 (n = 832), and 1 % (w/v) (n = 599) methyl cellulose. .


Bright - field images of an optically trapped sperm cell , obtained at t = 0 , 55 , 155 , and 185 ms . Scale bar represents 10 µm .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O

Bright-field images of an optically trapped sperm cell, obtained at t = 0, 55, 155, and 185 ms. Scale bar represents 10 µm.


Representative time - course of the rotation frequency of trapped sperm ( each sperm cell is represented by a different color ) . Error bars indicate the full width at half prominence of the frequency peaks determined by the fast Fourier analysis . .

O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_TYPE O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative time-course of the rotation frequency of trapped sperm (each sperm cell is represented by a different color). Error bars indicate the full width at half prominence of the frequency peaks determined by the fast Fourier analysis..


Distribution of rotation frequencies in trapped sperm ( n = 32 ; mean frequency ± SD 6 . 0 ± 2 . 1 Hz ) .

O O B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O

Distribution of rotation frequencies in trapped sperm (n = 32; mean frequency ± SD 6.0 ± 2.1 Hz).


Image series of a sperm cell trapped parallel to the optical axis ; images were obtained at t = 0 , 95 , 165 , and 205 ms . The red bar indicates the 360 ° rotation of the tip of the head . Scale bar represents 10 µm .

B-EXP_ASSAY O O O B-CELL_TYPE O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O B-SUBCELLULAR O O O O O O O

Image series of a sperm cell trapped parallel to the optical axis; images were obtained at t = 0, 95, 165, and 205 ms. The red bar indicates the 360° rotation of the tip of the head. Scale bar represents 10 µm.


Representative distribution of rotation frequencies of freely swimming CatSper - deficient sperm incubated under non - capacitating ( 0 mM bicarbonate ; black ; n = 73 ) and capacitating conditions ( 25 mM bicarbonate ; red ; n = 272 ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O B-CELL_TYPE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O

Representative distribution of rotation frequencies of freely swimming CatSper-deficient sperm incubated under non-capacitating (0 mM bicarbonate; black; n = 73) and capacitating conditions (25 mM bicarbonate; red; n = 272).


Rolling frequency ( mean ± SD ) of freely swimming CATSPER2 - / - sperm incubated under non - capacitating ( 0 mM bicarbonate , n = 1009 ) and capacitating ( 25 mM bicarbonate , n = 946 ) conditions .

B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O

Rolling frequency (mean ± SD) of freely swimming CATSPER2-/- sperm incubated under non-capacitating (0 mM bicarbonate, n = 1009) and capacitating (25 mM bicarbonate, n = 946) conditions.


Rolling frequency ( mean ± SD ) of freely swimming CATSPER2 - / - sperm in 0 ( n = 457 ) , 0 . 2 ( n = 389 ) , and 1 % ( w / v ) ( n = 187 ) methyl cellulose .

B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O 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-SMALL_MOLECULE I-SMALL_MOLECULE O

Rolling frequency (mean ± SD) of freely swimming CATSPER2-/- sperm in 0 (n = 457), 0.2 (n = 389), and 1 % (w/v) (n = 187) methyl cellulose.


Image series of a CATSPER2 - / - sperm cell optically trapped perpendicular to the optical axis ; images were obtained at t = 0 , 75 , 150 , and 225 ms . Scale bar represents 10 µm . Image series of a CATSPER2 - / - sperm cell optically trapped parallel to the optical axis ; images were obtained at t = 0 , 60 , 195 , and 320 ms . The red bar indicates the 360 ° rotation of the tip of the head . Scale bar represents 10 µm . Representative time courses of the rotation frequencies of optically trapped CATSPER2 - / - sperm ( each sperm is represented in a different color ) . Error bars indicate the full width at half prominence of the frequency peaks determined by the fast Fourier analysis .

B-EXP_ASSAY O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O B-EXP_ASSAY O 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-GENEPROD O O O B-CELL_TYPE O O O O O O O O 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-EXP_ASSAY O O O O O B-SUBCELLULAR O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O B-CELL_TYPE O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Image series of a CATSPER2-/- sperm cell optically trapped perpendicular to the optical axis; images were obtained at t = 0, 75, 150, and 225 ms. Scale bar represents 10 µm. Image series of a CATSPER2-/- sperm cell optically trapped parallel to the optical axis; images were obtained at t = 0, 60, 195, and 320 ms. The red bar indicates the 360° rotation of the tip of the head. Scale bar represents 10 µm. Representative time courses of the rotation frequencies of optically trapped CATSPER2-/- sperm (each sperm is represented in a different color). Error bars indicate the full width at half prominence of the frequency peaks determined by the fast Fourier analysis.


Rotation frequency of a trapped sperm cell before and after dragging across the barrier stream . The green trace indicates the fluorescence of fluorescein included in the barrier stream . Error bars indicate the full width at half prominence of the frequency peaks determined by the fast Fourier analysis . Paired plot of rotation frequencies of individual sperm cells before and after dragging across the barrier stream .

B-EXP_ASSAY I-EXP_ASSAY O O O B-CELL_TYPE O O O O O O O O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O 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-CELL_TYPE O O O O O O O O O O

Rotation frequency of a trapped sperm cell before and after dragging across the barrier stream. The green trace indicates the fluorescence of fluorescein included in the barrier stream. Error bars indicate the full width at half prominence of the frequency peaks determined by the fast Fourier analysis. Paired plot of rotation frequencies of individual sperm cells before and after dragging across the barrier stream.


Rotation frequency of a trapped sperm cell before and after dragging from the control stream containing 0 mM bicarbonate into the stimulus stream containing 25 mM bicarbonate . Error bars indicate the full width at half prominence of the frequency peaks determined by the fast Fourier analysis . Paired plot of rotation frequencies of individual sperm at 0 and 25 mM bicarbonate .

B-EXP_ASSAY I-EXP_ASSAY O O O B-CELL_TYPE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O 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-CELL_TYPE O O O O O B-SMALL_MOLECULE O

Rotation frequency of a trapped sperm cell before and after dragging from the control stream containing 0 mM bicarbonate into the stimulus stream containing 25 mM bicarbonate. Error bars indicate the full width at half prominence of the frequency peaks determined by the fast Fourier analysis. Paired plot of rotation frequencies of individual sperm at 0 and 25 mM bicarbonate.


Rotation frequency of a trapped sperm cell in the presence and absence of extracellular Ca2 + . Error bars indicate the full width at half prominence of the frequency peaks determined by the fast Fourier analysis . Paired plot of rotation frequencies in the presence and absence of extracellular Ca2 + .

B-EXP_ASSAY I-EXP_ASSAY O O O B-CELL_TYPE O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE 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-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O

Rotation frequency of a trapped sperm cell in the presence and absence of extracellular Ca2+. Error bars indicate the full width at half prominence of the frequency peaks determined by the fast Fourier analysis. Paired plot of rotation frequencies in the presence and absence of extracellular Ca2+.


Fraction of motile sperm ( mean ± SD ) in a sperm population incubated in the presence ( black ) and absence ( at t = 0 ) of extracellular Ca2 + ( red ) ( n ≥ 5 ) .

O O B-EXP_ASSAY B-CELL_TYPE O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O

Fraction of motile sperm (mean ± SD) in a sperm population incubated in the presence (black) and absence (at t = 0) of extracellular Ca2+ (red) (n ≥ 5).


Distribution of rotation frequencies in populations of freely swimming sperm in the presence ( black , n = 335 ) and absence ( red , n = 224 ) of extracellular Ca2 + ( red ) .

O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O

Distribution of rotation frequencies in populations of freely swimming sperm in the presence (black, n = 335) and absence (red, n = 224) of extracellular Ca2+ (red).


Left : Bright - field image series of a freely swimming wild - type sperm cell at t = 0 , 151 , and 303 ms . Scale bar represents 10 µm . Right : Rotation frequency ( mean ± SD ) of 24 freely swimming wild - type sperm . Left : Bright - field image series of a freely swimming Catsper1 - / - sperm cell at t = 0 , 151 , and 294 ms . Scale bar represents 10 µm . Right : Rotation frequency ( mean ± SD ) of 24 freely swimming Catsper1 - / - sperm .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-CELL_TYPE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-GENEPROD O O O B-CELL_TYPE O

Left: Bright-field image series of a freely swimming wild-type sperm cell at t = 0, 151, and 303 ms. Scale bar represents 10 µm. Right: Rotation frequency (mean ± SD) of 24 freely swimming wild-type sperm. Left: Bright-field image series of a freely swimming Catsper1-/- sperm cell at t = 0, 151, and 294 ms. Scale bar represents 10 µm. Right: Rotation frequency (mean ± SD) of 24 freely swimming Catsper1-/- sperm.


B - H Live confocal images of ddaC neurons expressing mCD8 - GFP by ppk - Gal4 at WP stage or 16 h APF . Neurons expressing efa6 RNAi # 1 - # 3 ( C - E ) , efa612 mutant MARCM clones ( F ) , and efa612 / GX6w - transheterozygous mutant neurons ( G ) showed consistent dendrite pruning defects at 16 h APF , as compared to the wild - type neurons ( B ) . The dendrite pruning defects were fully rescued when overexpressing full - length Efa6 in the efa612 mutant clones ( H ) . Red arrowheads point to the ddaC somas .

O O O O B-EXP_ASSAY O O O B-CELL_TYPE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O B-CELL_TYPE O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O B-CELL_TYPE O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O B-CELL_TYPE O O O O O B-SUBCELLULAR O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O B-SUBCELLULAR O

B-H Live confocal images of ddaC neurons expressing mCD8-GFP by ppk-Gal4 at WP stage or 16 h APF. Neurons expressing efa6 RNAi #1-#3 (C-E), efa612 mutant MARCM clones (F), and efa612/GX6w- transheterozygous mutant neurons (G) showed consistent dendrite pruning defects at 16 h APF, as compared to the wild-type neurons (B). The dendrite pruning defects were fully rescued when overexpressing full-length Efa6 in the efa612 mutant clones (H). Red arrowheads point to the ddaC somas.


Live confocal images of efa612 / GX6w - mutant ddaC neurons at WP stage and 16 h APF . Expression of Efa6FL ( C ) rescued the pruning defects in efa612 / GX6w - mutant ddaC neurons ( B ) . Red arrowheads point to the ddaC somas .

O B-EXP_ASSAY O O B-GENEPROD O O O O O B-CELL_TYPE O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O B-CELL_TYPE O O O O O O O O O O B-SUBCELLULAR O

Live confocal images of efa612/GX6w- mutant ddaC neurons at WP stage and 16 h APF. Expression of Efa6FL (C) rescued the pruning defects in efa612/GX6w- mutant ddaC neurons (B). Red arrowheads point to the ddaC somas.


Live confocal images of efa612 / GX6w - mutant ddaC neurons at WP stage and 16 h APF . Expression of Efa6Nterm ( D ) , Efa6ΔPH ( F ) and Efa6Nterm + CAAX ( H ) , but not Efa6Cterm ( E ) or Efa6ΔMTED ( G ) , rescued the pruning defects in efa612 / GX6w - mutant ddaC neurons Red arrowheads point to the ddaC somas . I - J Quantification of dendrite severing defects and unpruned dendrite lengths at 16 h APF .

O B-EXP_ASSAY O O B-GENEPROD O O O O O B-CELL_TYPE O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O B-CELL_TYPE O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O

Live confocal images of efa612/GX6w- mutant ddaC neurons at WP stage and 16 h APF. Expression of Efa6Nterm (D), Efa6ΔPH (F) and Efa6Nterm+CAAX (H), but not Efa6Cterm (E) or Efa6ΔMTED (G), rescued the pruning defects in efa612/GX6w- mutant ddaC neurons Red arrowheads point to the ddaC somas. I-J Quantification of dendrite severing defects and unpruned dendrite lengths at 16 h APF.


A - D Live confocal images of ddaC neurons at WP stage and 7 h APF . Efa6FL or Efa6Nterm - overexpressing neurons ( B , C ) showed precocious pruning phenotype at 7 h APF , compared to the control and Efa6ΔMTED - overexpressing neurons ( A , D ) in which most dendrites remained attached to the soma at the same time point . Red arrowheads point to the ddaC somas . Dorsal dendrites are marked by red arrows . E Quantification of precocious pruning defects as indicated by the percentage of dorsal dendrites attached to the soma at 7 h APF .

O O O O B-EXP_ASSAY O O O B-CELL_TYPE O O O O O O O O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-CELL_TYPE O O O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O

A-D Live confocal images of ddaC neurons at WP stage and 7 h APF. Efa6FL or Efa6Nterm-overexpressing neurons (B, C) showed precocious pruning phenotype at 7 h APF, compared to the control and Efa6ΔMTED-overexpressing neurons (A, D) in which most dendrites remained attached to the soma at the same time point. Red arrowheads point to the ddaC somas. Dorsal dendrites are marked by red arrows. E Quantification of precocious pruning defects as indicated by the percentage of dorsal dendrites attached to the soma at 7 h APF.


F - I Confocal images of da sensory neurons expressing Efa6 - Ki - GFP ( F ) , or UAS - Efa6 - GFP driven by ppk - Gal4 ( H ) were co - stained with GFP ( Green ) and Nrg ( Magenta ) at wL3 stage . ( G , I ) Confocal images of the epidermal cells expressing Efa6 - Ki - GFP ( G ) or UAS - Efa6 - GFP driven by A58 - Gal4 ( I ) were co - stained with GFP ( Green ) and Dlg ( Magenta ) at wL3 stage . The ddaC soma is marked by dashed lines .

O O O B-EXP_ASSAY O O O B-CELL_TYPE I-CELL_TYPE O B-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY O O O B-CELL_TYPE I-CELL_TYPE O B-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O B-SUBCELLULAR O O O O O O

F-I Confocal images of da sensory neurons expressing Efa6-Ki-GFP (F), or UAS-Efa6-GFP driven by ppk-Gal4 (H) were co-stained with GFP (Green) and Nrg (Magenta) at wL3 stage. (G, I) Confocal images of the epidermal cells expressing Efa6-Ki-GFP (G) or UAS-Efa6-GFP driven by A58-Gal4 (I) were co-stained with GFP (Green) and Dlg (Magenta) at wL3 stage. The ddaC soma is marked by dashed lines.


A - F Representative kymographs of EB1 - GFP comets in the proximal dendrites of ddaC neurons at 96 h AEL . The horizontal arrow indicates the direction toward the soma , and the vertical arrow indicates the time . In contrast to the wild type ( A ) , the number of EB1 - GFP comets was significantly increased in efa612 / GX6w - mutant neurons ( B ) . The comet length and speed were significantly decreased in Efa6FL - and Efa6Nterm - overexpressing neurons , but not in Efa6ΔMTED - overexpression neurons ( C - F ) .

O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O B-SUBCELLULAR O O B-CELL_TYPE O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O B-CELL_TYPE O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O B-CELL_TYPE O O O O B-GENEPROD O O B-CELL_TYPE O O O O O O

A-F Representative kymographs of EB1-GFP comets in the proximal dendrites of ddaC neurons at 96 h AEL. The horizontal arrow indicates the direction toward the soma, and the vertical arrow indicates the time. In contrast to the wild type (A), the number of EB1-GFP comets was significantly increased in efa612/GX6w- mutant neurons (B). The comet length and speed were significantly decreased in Efa6FL- and Efa6Nterm-overexpressing neurons, but not in Efa6ΔMTED-overexpression neurons (C-F).


A - C Live confocal images of ddaC dendrites expressing tdEOS : : α - tubulin driven by ppk - Gal4 . The microtubule turnover rate was significantly reduced in efa6 RNAi neurons at wL3 ( A , right panels ) and WP stages ( B , right panels ) , and increased in Efa6 - overexpressing neurons at wL3 stage ( C , right panels ) , compared to the control neurons ( A - C , left panels ) .

O O O O B-EXP_ASSAY O O O B-SUBCELLULAR O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O

A-C Live confocal images of ddaC dendrites expressing tdEOS::α-tubulin driven by ppk-Gal4. The microtubule turnover rate was significantly reduced in efa6 RNAi neurons at wL3 (A, right panels) and WP stages (B, right panels), and increased in Efa6-overexpressing neurons at wL3 stage (C, right panels), compared to the control neurons (A-C, left panels).


A - H Live confocal images of ddaC neurons at WP stage and 16 h APF . Treatment with colchicine significantly suppressed the pruning defects of efa6 RNAi neurons ( A , B ) , while taxol treatment enhanced the pruning defects ( C , D ) . Removal of one copy of γTub23C ( F ) or expression of γTub23C RNAi ( H ) almost fully rescued the dendrite pruning defects in efa6 mutant ( E ) or RNAi neurons ( G ) , respectively . Red arrowheads point to the ddaC somas .

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A-H Live confocal images of ddaC neurons at WP stage and 16 h APF. Treatment with colchicine significantly suppressed the pruning defects of efa6 RNAi neurons (A, B), while taxol treatment enhanced the pruning defects (C, D). Removal of one copy of γTub23C (F) or expression of γTub23C RNAi (H) almost fully rescued the dendrite pruning defects in efa6 mutant (E) or RNAi neurons (G), respectively. Red arrowheads point to the ddaC somas.


A - F Live confocal images of ddaC neurons at WP stage and 16 h APF . stai RNAi neurons ( B ) , staiSK7 ( C ) , staiKO ( D ) mutant MARCM clones showed similar dendrite pruning defects , compared to the wild - type ddaC neurons ( A ) . Double RNAi of stai and efa6 significantly enhanced the dendrite pruning defects ( E ) , compared to their individual RNAi knockdown . Expression of stai RNAi in efa612 / GX6w - mutant background also significantly enhanced the dendrite pruning defects ( F ) , compared to the RNAi or mutant alone . Red arrowheads point to the ddaC somas .

O O O O B-EXP_ASSAY O O O B-CELL_TYPE O O O O O O O O B-GENEPROD O B-CELL_TYPE O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O B-SUBCELLULAR O O O O O O O O O O B-CELL_TYPE O O O O O O O B-GENEPROD O B-GENEPROD O O O B-SUBCELLULAR O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O

A-F Live confocal images of ddaC neurons at WP stage and 16 h APF. stai RNAi neurons (B), staiSK7 (C), staiKO (D) mutant MARCM clones showed similar dendrite pruning defects, compared to the wild-type ddaC neurons (A). Double RNAi of stai and efa6 significantly enhanced the dendrite pruning defects (E), compared to their individual RNAi knockdown. Expression of stai RNAi in efa612/GX6w- mutant background also significantly enhanced the dendrite pruning defects (F), compared to the RNAi or mutant alone. Red arrowheads point to the ddaC somas.


I - L Representative kymographs of EB1 - GFP comets driven by Gal44 - 77 in the proximal dendrites of ddaC neurons at 96 h AEL .

O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O B-SUBCELLULAR O O B-CELL_TYPE O O O O O

I-L Representative kymographs of EB1-GFP comets driven by Gal44-77 in the proximal dendrites of ddaC neurons at 96 h AEL.


D , Western blot showing AGO1x expression in three cell lines . For comparison , a parallel blot was probed with an antibody directed against canonical AGO1 . Tubulin served as loading control .

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D, Western blot showing AGO1x expression in three cell lines. For comparison, a parallel blot was probed with an antibody directed against canonical AGO1. Tubulin served as loading control.


E , F , Annotated MS / MS spectrum of peptides specific for the endogenous AGO1x , " QNAVTSLDR " , depicted in green ( E ) and " LSKPQELCHPNPEEAR " , depicted in blue ( F ) . The Mascot Ion Score ( text color corresponds to peptides marked in Fig 1C for reference ) as well as the annotated fragments ( blue = y - ions ; red = b - ions ) together with the corresponding amino acids are indicated .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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,F, Annotated MS/MS spectrum of peptides specific for the endogenous AGO1x, "QNAVTSLDR", depicted in green (E) and "LSKPQELCHPNPEEAR", depicted in blue (F). The Mascot Ion Score (text color corresponds to peptides marked in Fig 1C for reference) as well as the annotated fragments (blue = y-ions; red = b-ions) together with the corresponding amino acids are indicated.


A , Representative immunofluorescence images showing the subcellular distribution of AGO1x ( red ) relative to nuclear and cytoplasmic markers . DAPI was used to mark the nucleus ( blue ) . The co - stained subcellular marker is indicated in each panel in green . SC 35 , Lsm4 , α - Tubulin , ERP72 , p54 ( NRB ) , and Nucleolin serve as markers for nuclear speckles , cytosol and nucleus , cytosol , endoplasmic reticulum , paraspeckle and nucleolus , respectively .

O O O B-EXP_ASSAY O O O O O O B-GENEPROD O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O B-SMALL_MOLECULE O O O O O B-SUBCELLULAR O O O O O O O B-EXP_ASSAY O O O O O O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR O O O

A, Representative immunofluorescence images showing the subcellular distribution of AGO1x (red) relative to nuclear and cytoplasmic markers. DAPI was used to mark the nucleus (blue). The co-stained subcellular marker is indicated in each panel in green. SC 35, Lsm4, α-Tubulin, ERP72, p54 (NRB), and Nucleolin serve as markers for nuclear speckles, cytosol and nucleus, cytosol, endoplasmic reticulum, paraspeckle and nucleolus, respectively.


B , Mean ( + / - s . d . ) pixel intensities of AGO1x staining in nucleolus and nucleoplasm , computed from z - stack images of MDA - MB - 231 cells ( n = 20 ) . The P - value was determined using a paired two - tailed t - test .

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B, Mean (+/- s.d.) pixel intensities of AGO1x staining in nucleolus and nucleoplasm, computed from z-stack images of MDA-MB-231 cells (n=20). The P-value was determined using a paired two-tailed t-test.


C , Representative AGO1x and AGO1 blot from MDA - MB - 231 cell fractions . GAPDH and hnRNP C1 / C2 served as markers of purity of the individual fractions .

O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O O O O O O

C, Representative AGO1x and AGO1 blot from MDA-MB-231 cell fractions. GAPDH and hnRNP C1/C2 served as markers of purity of the individual fractions.


A , Top : Scheme of the sites targeted by sgRNA in the generation of AGO1x mutant cell lines . Bottom : Western analysis of lysates from mutant MDA - MB - 231 cells showing the depletion of AGO1x and the comparable total AGO1 levels across the cell lines . Three independently collected sets of cells were lysed and analysed in parallel . After calculating the levels of AGO1x and AGO1 relative to GAPDH , values corresponding to individual lanes were normalized to the average over the replicates of the control line . These numbers are indicated below the blots . The P - values of the t - tests comparing the expression AGO1x in mutant ( W1A and W6A ) and control ( CTRL ) lines were W1A - CTRL : 0 . 0008 , W6A - CTRL : 0 . 0002 , and for AGO1 W1A - CTRL : 0 . 1227 , W6A - CTRL : 0 . 1173 .

O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O B-GENEPROD O O O O B-GENEPROD 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-GENEPROD O B-GENEPROD O O B-GENEPROD O 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-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD O O O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O

A, Top: Scheme of the sites targeted by sgRNA in the generation of AGO1x mutant cell lines. Bottom: Western analysis of lysates from mutant MDA-MB-231 cells showing the depletion of AGO1x and the comparable total AGO1 levels across the cell lines. Three independently collected sets of cells were lysed and analysed in parallel. After calculating the levels of AGO1x and AGO1 relative to GAPDH, values corresponding to individual lanes were normalized to the average over the replicates of the control line. These numbers are indicated below the blots. The P-values of the t-tests comparing the expression AGO1x in mutant (W1A and W6A) and control (CTRL) lines were W1A-CTRL: 0.0008, W6A-CTRL: 0.0002, and for AGO1 W1A-CTRL: 0.1227, W6A-CTRL: 0.1173.


B , Phase contrast images of control and mutant cell lines at 24 and 48 hr after plating equal numbers ( 10 ' 000 ) of cells in individual wells of a six well plate .

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B, Phase contrast images of control and mutant cell lines at 24 and 48 hr after plating equal numbers (10'000) of cells in individual wells of a six well plate.


C , Impedance - based mean ( + / - s . d . ) cell indices at the indicated time points after seeding equal numbers of CTRL ( n = 6 ) , W1A ( n = 5 ) and W6A ( n = 5 ) cells . From 6 hours on , there is a statistically significant difference between control and the two mutant cell lines ( P < 0 . 001 , two tailed t - test ) . D , Impedance - based mean ( + / - s . d . ) cell indices as a function of time for control ( n = 3 ) , W1A ( n = 4 ) and W6A ( n = 3 ) cells grown in electronically monitored Boyden chambers . From 12 hours on , there is a statistical significant difference between control and the two mutants ( P < 0 . 005 , two tailed t - test ) .

O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Impedance-based mean (+/- s.d.) cell indices at the indicated time points after seeding equal numbers of CTRL (n=6), W1A (n=5) and W6A (n=5) cells. From 6 hours on, there is a statistically significant difference between control and the two mutant cell lines (P < 0.001, two tailed t-test). D, Impedance-based mean (+/- s.d.) cell indices as a function of time for control (n=3), W1A (n=4) and W6A (n=3) cells grown in electronically monitored Boyden chambers. From 12 hours on, there is a statistical significant difference between control and the two mutants (P < 0.005, two tailed t-test).


E , Mean ( + / - s . d . ) number of colonies obtained in a soft agar colony formation assay of control , W1A and W6A mutant cell lines ( all n = 4 , P - value determined using an unpaired two - tailed t - test ) . F , Mean ( + / - s . d . ) number of spheres obtained from a sphere formation assay of control , W1A and W6A mutant cell lines ( all n = 8 , P - values computed as in E ) .

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E, Mean (+/- s.d.) number of colonies obtained in a soft agar colony formation assay of control, W1A and W6A mutant cell lines (all n=4, P-value determined using an unpaired two-tailed t-test). F, Mean (+/- s.d.) number of spheres obtained from a sphere formation assay of control, W1A and W6A mutant cell lines (all n=8, P-values computed as in E).


G , AGO1x staining of tissue sections from breast cancers , classified as low - proliferating or high - proliferating based on Ki - 67 staining . The upper and lower panel represents two different tissue sections . H , Mean ( + / - s . d . ) percentage of AGO1x positive cells in breast tumors with low ( n = 7 ) and high ( n = 9 ) proliferation index , assessed by the expression level of Ki - 67 . P - value was determined using an unpaired two - tailed t - test .

O O B-GENEPROD B-EXP_ASSAY O O O O B-DISEASE I-DISEASE O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O 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-GENEPROD O O O B-DISEASE I-DISEASE O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O

G, AGO1x staining of tissue sections from breast cancers, classified as low-proliferating or high-proliferating based on Ki-67 staining. The upper and lower panel represents two different tissue sections. H, Mean (+/- s.d.) percentage of AGO1x positive cells in breast tumors with low (n=7) and high (n=9) proliferation index, assessed by the expression level of Ki-67. P-value was determined using an unpaired two-tailed t-test.


A , Scatter plot of mean log2 mRNA fold - changes in the mutant cell lines versus control ( n = 3 ) . Shown are also the Pearson correlation coefficient and respective P - value . The dashed line indicates equal change in the two mutant lines .

O O O O O O O O O O O O O O O O O O O O O O O O O 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, Scatter plot of mean log2 mRNA fold-changes in the mutant cell lines versus control (n=3). Shown are also the Pearson correlation coefficient and respective P-value. The dashed line indicates equal change in the two mutant lines.


C , Mean ( + / - s . d . ) activity of STAT1 / IRF1 / IRF2 / IRF3 / IRF9 transcription factor motifs estimated by ISMARA ( Balwierz et al , 2014 ) ( n = 3 ) . Shown are the P - values ( * * * P < 0 . 001 ) determined by Dunn ' s multiple comparisons test post hoc and the non - parametric Kruskal - Wallis test ( P < 0 . 0001 ) .

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C, Mean (+/- s.d.) activity of STAT1/IRF1/IRF2/IRF3/IRF9 transcription factor motifs estimated by ISMARA (Balwierz et al, 2014) (n=3). Shown are the P-values (*** P < 0.001) determined by Dunn's multiple comparisons test post hoc and the non-parametric Kruskal-Wallis test (P < 0.0001).


D , Mean ( + / - s . e . m . ) log2 mRNA expression fold - changes ( inferred from RNA - Seq ) of genes involved in the interferon alpha response and apoptosis in the two mutant cell lines relative to control ( n = 3 ) . Multiple - testing - corrected P - values for Wald tests comparing fold - changes with respect to control are depicted above each bar ( * * P < 0 . 01 , * * * P < 0 . 001 ) .

O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Mean (+/- s.e.m.) log2 mRNA expression fold-changes (inferred from RNA-Seq) of genes involved in the interferon alpha response and apoptosis in the two mutant cell lines relative to control (n=3). Multiple-testing-corrected P-values for Wald tests comparing fold-changes with respect to control are depicted above each bar (** P < 0.01, *** P < 0.001).


E , Mean ( + / - s . d . ) expression fold - changes of the corresponding proteins ( if detected in the proteomics data ) in the two mutant cell lines ( W1A , n = 2 ; W6A , n = 1 ) compared to Control ( n = 2 ) . Multiple - testing - corrected P - values for unpaired two - tailed t - tests ( proteomics ) comparing fold - changes with respect to control are depicted above each bar ( * P < 0 . 05 , * * 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 B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O O O O O O O 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, Mean (+/- s.d.) expression fold-changes of the corresponding proteins (if detected in the proteomics data) in the two mutant cell lines (W1A, n=2; W6A, n=1) compared to Control (n=2). Multiple-testing-corrected P-values for unpaired two-tailed t-tests (proteomics) comparing fold-changes with respect to control are depicted above each bar (* P < 0.05, ** P < 0.01, *** P < 0.001).


F , Representative result of the apoptosis assay using AnnexinV and propidium iodide ( PI ) staining in the control and the two mutant cell lines ( left ) . The percentage of cells in each quadrant is depicted for each cell line . Quantification of the mean ( + / - s . d . ) percentage of apoptotic cells ( Q1 + Q2 , AnnexinV + ) across the different cell lines ( n = 4 ) ( right ) . Shown is the P - value determined by unpaired two - tailed t - test .

O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O 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, Representative result of the apoptosis assay using AnnexinV and propidium iodide (PI) staining in the control and the two mutant cell lines (left). The percentage of cells in each quadrant is depicted for each cell line. Quantification of the mean (+/- s.d.) percentage of apoptotic cells (Q1+Q2, AnnexinV+) across the different cell lines (n=4) (right). Shown is the P-value determined by unpaired two-tailed t-test.


G , Impedance - based mean ( + / - s . d . ) cell index values at the indicated time points of growth after seeding equal numbers of control , W1A and W6A cells , upon treatment with DMSO or Ruxolitinib . For the DMSO treatment : Control ( n = 4 ) , W1A ( n = 3 ) and W6A ( n = 3 ) . For the Ruxolitinib treatment : Control ( n = 6 ) , W1A ( n = 3 ) and W6A ( n = 4 ) . From 6 hours after DMSO treatment , there is a statistically significant difference between control and the two mutants ( P < 0 . 05 , two tailed t - test ) , whereas no statistically significant difference is found after Ruxolitinib treatment .

O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O 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-SMALL_MOLECULE O O O 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-SMALL_MOLECULE O O O O O O O O O O O O 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-SMALL_MOLECULE O O

G, Impedance-based mean (+/- s.d.) cell index values at the indicated time points of growth after seeding equal numbers of control, W1A and W6A cells, upon treatment with DMSO or Ruxolitinib. For the DMSO treatment: Control (n=4), W1A (n=3) and W6A (n=3). For the Ruxolitinib treatment: Control (n=6), W1A (n=3) and W6A (n=4). From 6 hours after DMSO treatment, there is a statistically significant difference between control and the two mutants (P < 0.05, two tailed t-test), whereas no statistically significant difference is found after Ruxolitinib treatment.


B , Mean ( + / - s . e . m . ) log2 mRNA fold - changes of dsRNAs sensors ( from panel A ) in the two mutant cell lines compared to control ( n = 3 ) inferred from RNA - Seq . Multiple - testing - corrected P - values for Wald tests comparing fold - changes with respect to control are depicted above each bar ( * * * 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O 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, Mean (+/- s.e.m.) log2 mRNA fold-changes of dsRNAs sensors (from panel A) in the two mutant cell lines compared to control (n=3) inferred from RNA-Seq. Multiple-testing-corrected P-values for Wald tests comparing fold-changes with respect to control are depicted above each bar (*** P < 0.001).


C , Mean ( + / - s . d . ) fold - changes of corresponding proteins ( see B ) , if detected in the proteomics data from the two mutant ( W1A , n = 2 ; W6A , n = 1 ) lines relative to control ( n = 2 ) . Multiple - testing - corrected P - values for unpaired two - tailed t - tests ( proteomics ) comparing fold - changes with respect to control are depicted above each bar ( * P < 0 . 05 , * * 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 B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O O O O O O O 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, Mean (+/- s.d.) fold-changes of corresponding proteins (see B), if detected in the proteomics data from the two mutant (W1A, n=2; W6A, n=1) lines relative to control (n=2). Multiple-testing-corrected P-values for unpaired two-tailed t-tests (proteomics) comparing fold-changes with respect to control are depicted above each bar (* P < 0.05, ** P < 0.01, *** P < 0.001).


D , Western blot detection of phosphorylated eIF2α ( Ser51 ) and total eIF2α levels in cell lysates of control and mutant MDA - MB - 231 cells . GAPDH and Tubulin serve as loading control for phosphorylated eIF2α and total eIF2α blots respectively . Numbers indicated below the blots represent the band intensities of target proteins normalized to the loading controls , with the value in the control line taken as baseline of 1 .

O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY B-GENEPROD O O O O O B-GENEPROD O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O O B-EXP_ASSAY B-GENEPROD O O B-GENEPROD B-EXP_ASSAY O O O O O O B-EXP_ASSAY O O 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, Western blot detection of phosphorylated eIF2α (Ser51) and total eIF2α levels in cell lysates of control and mutant MDA-MB-231 cells. GAPDH and Tubulin serve as loading control for phosphorylated eIF2α and total eIF2α blots respectively. Numbers indicated below the blots represent the band intensities of target proteins normalized to the loading controls, with the value in the control line taken as baseline of 1.


E , Mean ( + / - s . d . ) fold - changes in phosphorylated peptide ( phosphorylation sites are indicated in brackets ) levels derived from a proteomic analysis of phosphopeptides in the two mutant lines ( W1A , n = 2 ; W6A , n = 2 ) compared to control ( n = 1 ) . Multiple - testing - corrected P - values for Wald tests ( transcriptomics ) or unpaired two - tailed t - tests ( proteomics ) comparing fold - changes with respect to control are depicted above each bar ( * * P < 0 . 01 ) .

O O O O O O O O O O O O O O O O B-EXP_ASSAY O O B-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY 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, Mean (+/- s.d.) fold-changes in phosphorylated peptide (phosphorylation sites are indicated in brackets) levels derived from a proteomic analysis of phosphopeptides in the two mutant lines (W1A, n=2; W6A, n=2) compared to control (n=1). Multiple-testing-corrected P-values for Wald tests (transcriptomics) or unpaired two-tailed t-tests (proteomics) comparing fold-changes with respect to control are depicted above each bar (** P < 0.01).


F , Quantification of the relative abundance ( mean + / - s . e . m . ) of distinct dsRNA foci in each cell type . 3D images were projected on a single plane , non - overlapping foci were counted from 100 cells of each type , and the numbers in three abundance bins ( < 1 , 1 - 5 , > 5 ) are shown .

O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O 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, Quantification of the relative abundance (mean+/- s.e.m.) of distinct dsRNA foci in each cell type . 3D images were projected on a single plane, non-overlapping foci were counted from 100 cells of each type, and the numbers in three abundance bins (<1, 1-5, >5) are shown.


G , Representative immunofluorescence image of the control and mutant MDA - MB - 231 cells stained with AGO1x ( green ) and J2 antibody ( red ) and DAPI ( blue ) to mark the nucleus . Loss of AGO1x signal in mutants also confirms the efficacy of CRISPR - induced mutations .

O O O B-EXP_ASSAY O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-SUBCELLULAR O O O B-GENEPROD O O O O O O O O O O O O O

G, Representative immunofluorescence image of the control and mutant MDA-MB-231 cells stained with AGO1x (green) and J2 antibody (red) and DAPI (blue) to mark the nucleus. Loss of AGO1x signal in mutants also confirms the efficacy of CRISPR-induced mutations.


Concentration of recovered RNA ( mean + / - s . e . m . ) measured by Nanodrop ™ ( H ) Multiple testing corrected P - values from unpaired two - tailed t - tests are depicted above each comparison ( * P < 0 . 05 , * * * 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

Concentration of recovered RNA (mean +/- s.e.m.) measured by Nanodrop™ (H) Multiple testing corrected P-values from unpaired two-tailed t-tests are depicted above each comparison (* P < 0.05, *** P < 0.001).