Neuronal loss is rescued in entorhinal cortex of AP - 2µ KO mice haploinsufficient for BACE1 AP - 2KO / BACEWT : 1 . 51 ± 0 . 08 , AP - 2WT / BACEHET : 1 . 80 ± 0 . 07 , AP - 2KO / BACEHET : 2 . 00 ± 0 . 07 ; pAP - 2WT / BACE1WT vs pAP - 2KO / BACE1WT < 0 . 000 ; pAP - 2WT / BACE1WT vs pAP - 2WT / BACE1HET = 0 . 001 ; pAP - 2KO / BACE1WT vs pAP - 2KO / BACE1HET = 0 . 000 ; p AP - 2WT / BACE1HET vs AP - 2KO / BACE1HET = 0 . 268 , N = 9 sections from N = 3 for each genotype ) . Scale bar : 100 μm . Data information : graphs show mean ± , statistical was performed by two - way ANOVA n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

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

Neuronal loss is rescued in the entorhinal cortex of AP-2µ KO mice haploinsufficient for BACE1 AP-2KO/BACEWT: 1.51±0.08, AP-2WT/BACEHET: 1.80±0.07, AP-2KO/BACEHET: 2.00±0.07; pAP-2WT/BACE1WT vs pAP-2KO/BACE1WT<0.000; pAP-2WT/BACE1WT vs pAP-2WT/BACE1HET=0.001; pAP-2KO/BACE1WT vs pAP-2KO/BACE1HET=0.000; p AP-2WT/BACE1HET vs AP-2KO/BACE1HET=0.268, N=9 sections from N=3 for each genotype). Scale bar: 100 μm. Data information: All graphs show mean ± SEM, statistical analysis was performed by two-way ANOVA n.s.-non-significant.* indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.


( P , Q ) APP - CTF are rescued in entorhinal cortex of AP - 2µ KO mice haploinsufficient for BACE1 ( AP - 2WT / BACEWT : 427574 ± 24021 , AP - 2KO / BACEWT : 538895 ± 34662 , AP - 2WT / BACEHET : 389870 ± 19842 , AP - 2KO / BACEHET : 395389 ± 21853 ; pAP - 2WT / BACE1WT vs pAP - 2KO / BACE1WT = 0 . 017 ; pAP - 2KO / BACE1WT vs pAP - 2KO / BACE1HET = 0 . 001 ; p AP - 2WT / BACE1HET vs AP - 2KO / BACE1HET = 0 . 979 ; 13 - 15 sections , N = 3 mice for each genotype ) . Scale bars : 40µm . Data information : graphs show mean ± , statistical was performed by two - way ANOVA n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

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

(P, Q) APP-CTF levels are rescued in the entorhinal cortex of AP-2µ KO mice haploinsufficient for BACE1 (AP-2WT/BACEWT:427574±24021, AP-2KO/BACEWT: 538895±34662, AP-2WT/BACEHET: 389870±19842, AP-2KO/BACEHET: 395389±21853; pAP-2WT/BACE1WT vs pAP-2KO/BACE1WT=0.017; pAP-2KO/BACE1WT vs pAP-2KO/BACE1HET=0.001; p AP-2WT/BACE1HET vs AP-2KO/BACE1HET=0.979; 13-15 sections, N=3 mice for each genotype). Scale bars: 40µm. Data information: All graphs show mean ± SEM, statistical analysis was performed by two-way ANOVA n.s.-non-significant.* indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.


( A - D ) Immunostaining with dopamine antibody at late 3rd instar ( 96h AEL ) in control background ( domeMESO - GFP / + ) . inset shows DomeGFP + ( B ' ) corresponding dopamine ( C ' ) in them . ( E - L ) Similar expression of dopamine at early 3rd ( 72h AEL , E - H ) early 2nd instar ( 48h AEL , I - L ) . ( D , H L ) spectral mode of iDopamine representation with blue pixels indicating low red pixels indicating high . ( M ) Quantifications of dopamine in DomeGFP + progenitors ( domeMESO - Gal4 , UAS - GFP / + , n = 14 at 48h , n = 13 at 72h n = 16 at 96h AEL ) . Data information : is stained with Hoechst in blue , intracellular Dopamine ( iDopamine ) in red , domeMESO expression in green ( representative of progenitors in medullary zone , MZ ) . iDopamine channel has been converted to spectral mode in panels D , H L . image panels show a 20µm scale bar . inset represents a 2X zoom of region of interest ( ROIs ) marked in dashed square . AEL indicates After Egg Laying . For better representation , lobe of lymph has been represented outlined in dashed lines while MZ is outlined in yellow dashed for segregation between Dome + Dome - . in M shows median with whiskers indicating maximum minimum values , . box indicates lower upper quartiles . statistical applied is One - way ANOVA with Tukey ' s multiple comparisons . ' n ' represents number of lymph lobes analyzed , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . For better representation , lobe of lymph has been represented outlined in dashed lines while MZ is outlined in yellow dashed .

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

(A-D) Immunostaining with dopamine antibody at late 3rd instar (96h AEL) in control background (domeMESO-GFP/+). The inset shows DomeGFP+ cells (B') and the corresponding dopamine levels (C') in them. (E-L) Similar expression analysis of dopamine at early 3rd (72h AEL, E-H) and early 2nd instar (48h AEL, I-L). (D, H and L) The spectral mode of iDopamine representation with blue pixels indicating low and red pixels indicating high intensity. (M) Quantifications of dopamine levels in DomeGFP+ progenitors (domeMESO-Gal4, UAS-GFP/+, n= 14 at 48h, n=13 at 72h and n=16 at 96h AEL). Data information: DNA is stained with Hoechst in blue, intracellular Dopamine (iDopamine) in red, domeMESO expression in green (representative of progenitors in the medullary zone, MZ). The iDopamine channel has been converted to spectral mode in panels D, H and L. All image panels show a 20µm scale bar. The inset represents a 2X zoom of region of interest (ROIs) marked in white dashed square. AEL indicates After Egg Laying. For better representation, the primary lobe of the lymph gland has been represented and outlined in white dashed lines while the MZ is outlined in yellow dashed line for segregation between Dome+ and Dome- cells. The quantification in M shows median with whiskers indicating the maximum and minimum values,. box indicates the lower and upper quartiles. The statistical analysis applied 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. For better representation, the primary lobe of the lymph gland has been represented and outlined in white dashed lines while the MZ is outlined in yellow dashed line.


( B - D ' ) A temporal expression profile of TH at 48 , 72 96h AEL lymph in control background ( domeMESO - Gal4 , UAS - GFP / + ) ( E - G ' ) A temporal expression profile of Ddc at 48 , 72 96h AEL lymph in control background ( domeMESO - Gal4 , UAS - GFP / + ) . ( H ) of TH ( domeMESO - Gal4 , UAS - GFP / + , n = 18 at 48h , n = 15 , at 72h n = 23 at 96h AEL ) . ( I ) of Ddc ( domeMESO - Gal4 , UAS - GFP / + , n = 17 at 48h , n = 33 at 72h n = 38 at 96h AEL ) . Data information : is stained with Hoechst in blue , iDopamine in red , Tyrosine hydroxylase ( TH ) Dopa decarboxylase ( Ddc ) in red , domeMESO ( representative of progenitors ) expression in green , AEL indicates After Egg Laying . quantifications in H , I represents median with whiskers indicating maximum minimum values , box indicates lower upper quartiles . statistical applied in H , I , is One - way ANOVA with Tukey ' s multiple comparisons ' n ' represents number of lymph lobes analyzed , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . For better representation , lobe of lymph has been represented outlined in dashed lines while MZ is outlined in yellow dashed .

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

(B-D') A temporal expression profile of TH at 48, 72 and 96h AEL lymph glands in control background (domeMESO-Gal4, UAS-GFP/+) (E-G') A temporal expression profile of Ddc at 48, 72 and 96h AEL lymph glands in control background (domeMESO-Gal4, UAS-GFP/+). (H) Quantification of TH levels (domeMESO-Gal4, UAS-GFP/+, n=18 at 48h, n= 15, at 72h and n= 23 at 96h AEL). (I) Quantification of Ddc levels (domeMESO-Gal4, UAS-GFP/+, n= 17 at 48h, n= 33 at 72h and n= 38 at 96h AEL). Data information: DNA is stained with Hoechst in blue, iDopamine in red, Tyrosine hydroxylase (TH) and Dopa decarboxylase (Ddc) in red, domeMESO (representative of progenitors) expression in green, AEL indicates After Egg Laying. The quantifications in H, I represents the median with whiskers indicating maximum and minimum values, box indicates the lower and upper quartiles. The statistical analysis applied in H, I, 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. For better representation, the primary lobe of the lymph gland has been represented and outlined in white dashed lines while the MZ is outlined in yellow dashed line.


( J - L ) Assessment of dopamine upon knockdown of TH Ddc in progenitors using domeMESO - Gal4 , UAS - GFP . ( M ) mean of dopamine upon TH Ddc loss of function in progenitors ( domeMESO - Gal4 , UAS - GFP / + , n = 40 , domeMESO - Gal4 , UAS - GFP ; THRNAi , n = 17 , domeMESO - Gal4 , UAS - GFP ; DdcRNAi , n = 34 ) . Data information : is stained with Hoechst in blue , iDopamine in red image panels show a 20µm scale bar . quantifications in M represents median with whiskers indicating maximum minimum values , box indicates lower upper quartiles . statistical applied in M , is One - way ANOVA with Tukey ' s multiple comparisons ' n ' represents number of lymph lobes analyzed , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . For better representation , lobe of lymph has been represented outlined in dashed

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

(J-L) Assessment of dopamine levels upon the knockdown of TH and Ddc in the progenitors using domeMESO-Gal4, UAS-GFP. (M) The mean intensity quantification of dopamine levels upon TH and Ddc loss of function in the progenitors (domeMESO-Gal4, UAS-GFP/+, n=40, domeMESO-Gal4, UAS-GFP; THRNAi, n= 17, domeMESO-Gal4, UAS-GFP; DdcRNAi, n= 34). Data information: DNA is stained with Hoechst in blue, iDopamine in red All image panels show a 20µm scale bar. The quantifications in M represents the median with whiskers indicating maximum and minimum values, box indicates the lower and upper quartiles. The statistical analysis applied in M, 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. For better representation, the primary lobe of the lymph gland has been represented and outlined in white dashed lines


( N - P ) loss of progenitor TH Ddc function using domeMESO - Gal4 , UAS - GFP analysed at wandering 3rd instar , 120h AEL . Data information : is stained with Hoechst in blue , domeMESO ( representative of progenitors ) expression in green , P1 ( representative of mature plasmatocytes ) in red . image panels show a 20µm scale bar . AEL indicates After Egg Laying . For better representation , lobe of lymph has been represented outlined in dashed

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

(N-P) The loss of progenitor TH and Ddc 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. AEL indicates After Egg Laying. For better representation, the primary lobe of the lymph gland has been represented and outlined in white dashed lines


( Q ) Quantifications of lymph upon loss of TH ( domeMESO - Gal4 , UAS - GFP ; THRNAi , n = 34 ) Ddc ( domeMESO - Gal4 , UAS - GFP ; DdcRNAi , n = 56 ) when compared to stage matched control ( domeMESO - Gal4 , UAS - GFP / + , n = 90 ) . Data information : quantifications in Q represent mean with standard deviation with whiskers indicating maximum minimum values , box indicates lower upper quartiles . statistical applied in is One - way ANOVA with Tukey ' s multiple comparisons ' n ' represents number of lymph lobes analyzed , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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

(Q) Quantifications of lymph gland size upon loss of TH (domeMESO-Gal4, UAS-GFP; THRNAi, n= 34) and Ddc (domeMESO-Gal4, UAS-GFP; DdcRNAi, n= 56) when compared to stage matched control (domeMESO-Gal4, UAS-GFP/+, n=90). Data information: The quantifications in Q represent mean with standard deviation with whiskers indicating maximum and minimum values, box indicates the lower and upper quartiles. The statistical analysis applied in 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.


( R ) Quantifications of proportions of % Dome ( green bars ) , % negative ( blue bar ) , % overlap ( Dome P1 , yellow bars ) % P1 ( red bars ) . For Dome positive ( domeMESO - Gal4 , UAS - GFP / + , n = 27 , domeMESO - Gal4 , UAS - GFP ; THRNAi , n = 15 domeMESO - Gal4 , UAS - GFP ; DdcRNAi , n = 18 ) , negative overlap areas ( domeMESO - Gal4 , UAS - GFP ; THRNAi , n = 15 , domeMESO - Gal4 , UAS - GFP ; DdcRNAi , n = 18 ) P1 positive ( domeMESO - Gal4 , UAS - GFP / + , n = 27 , domeMESO - Gal4 , UAS - GFP ; THRNAi , n = 15 domeMESO - Gal4 , UAS - GFP ; DdcRNAi , n = 18 ) . Data information : quantifications in R represents mean with whiskers indicating maximum minimum values , box indicates lower upper quartiles . statistical applied in R is Two - way Anova with Tukey ' s multiple comparisons . ' n ' represents number of lymph 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 O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O 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 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 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 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-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

(R) Quantifications of the proportions of %Dome (green bars), %negative (blue bar), %overlap (Dome and P1, yellow bars) and %P1 (red bars). For Dome positive area (domeMESO-Gal4, UAS-GFP/+, n=27, domeMESO-Gal4, UAS-GFP; THRNAi, n= 15 and domeMESO-Gal4, UAS-GFP; DdcRNAi, n= 18), negative and overlap areas (domeMESO-Gal4, UAS-GFP; THRNAi, n= 15, domeMESO-Gal4, UAS-GFP; DdcRNAi, n= 18) and P1 positive area (domeMESO-Gal4, UAS-GFP/+, n=27, domeMESO-Gal4, UAS-GFP; THRNAi, n= 15 and domeMESO-Gal4, UAS-GFP; DdcRNAi, n= 18). Data information: The quantifications in R represents mean with whiskers indicating maximum and minimum values, box indicates the lower and upper quartiles. The statistical analysis applied in R 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.


( S ) of number of PPO positive crystal upon dopamine synthesis perturbation ( domeMESO - Gal4 , UAS - GFP / + , n = 29 , domeMESO - Gal4 , UAS - GFP ; THRNAi , n = 21 , domeMESO - Gal4 , UAS - GFP ; DdcRNAi , n = 23 ) . Data information : quantifications in S represent mean with standard deviation with whiskers indicating maximum minimum values , box indicates lower upper quartiles . statistical applied in S is One - way ANOVA with Tukey ' s multiple comparisons ' n ' represents number of lymph lobes analyzed , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

O O O O O O B-GENEPROD O B-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 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

(S) Quantification of the number of PPO positive crystal cells upon dopamine synthesis perturbation (domeMESO-Gal4, UAS-GFP/+, n=29, domeMESO-Gal4, UAS-GFP; THRNAi, n= 21, domeMESO-Gal4, UAS-GFP; DdcRNAi, n= 23). Data information: The quantifications in S represent mean with standard deviation with whiskers indicating maximum and minimum values, box indicates the lower and upper quartiles. The statistical analysis applied in S 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 - C ' ) Expression profile of dopamine activation receptor sensor ( DA1m ) in progenitors using domeMESO - Gal4 driver in a temporal manner - 48h , ( A - A ' ) , 72h , ( B - B ' ) 96h AEL , ( C - C ' ) . ( D ) of number DA1m + , % DA1m positive ( domeMESO - Gal4 ; DA1m , n = 10 at 96h , n = 16 at 72h n = 10 at 48h AEL ) . Data information : is stained with Hoechst in blue , lifeAct - RFP to mark progenitors in red , Dopamine receptor - reporter ( DA1m ) in green , image panels show a 20µm scale bar . AEL indicates After Egg Laying . in D represents median with whiskers indicating maximum minimum values , box indicates lower upper quartiles . statistical applied in is Two - way Anova with Tukey ' s multiple comparisons . ' n ' represents number of lymph lobes analyzed , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . For better representation , lobe of lymph has been represented outlined in dashed lines while MZ is outlined in yellow dashed .

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

(A-C') Expression profile of dopamine activation based receptor sensor (DA1m) in the progenitors using domeMESO-Gal4 driver in a temporal manner- 48h, (A-A'), 72h, (B-B') and 96h AEL, (C-C'). (D) Quantification of the number DA1m+ cells, %DA1m positive cells (domeMESO-Gal4; DA1m, n=10 at 96h, n=16 at 72h and n=10 at 48h AEL). Data information: DNA is stained with Hoechst in blue, lifeAct-RFP to mark progenitors in red, Dopamine receptor-based reporter (DA1m) in green, All image panels show a 20µm scale bar. AEL indicates After Egg Laying. The quantification in D represents the median with whiskers indicating maximum and minimum values, box indicates the lower and upper quartiles. The statistical analysis applied in 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. For better representation, the primary lobe of the lymph gland has been represented and outlined in white dashed lines while the MZ is outlined in yellow dashed line.


( E - G ' ) Temporal expression of dopamine transporter , DAT . ( H ) A plot representing trend of DAT expression at 96h AEL . ( x ) axis indicates 0µm as a beginning from region to periphery . Data information : is stained with Hoechst in blue , Dopamine transporter ( DAT ) in red , domeMESO ( representative of progenitors ) expression in green , image panels show a 20µm scale bar . AEL indicates After Egg Laying . For better representation , lobe of lymph has been represented outlined in dashed lines while MZ is outlined in yellow dashed .

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

(E-G') Temporal expression analysis of dopamine transporter, DAT. (H) A line plot representing the trend of DAT expression at 96h AEL. The distance (x) axis indicates 0µm as a line beginning from the core region to the periphery. Data information: DNA is stained with Hoechst in blue, Dopamine transporter (DAT) in red, domeMESO (representative of progenitors) expression in green, All image panels show a 20µm scale bar. AEL indicates After Egg Laying. For better representation, the primary lobe of the lymph gland has been represented and outlined in white dashed lines while the MZ is outlined in yellow dashed line.


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

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

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

(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 proportions of % Dome ( green bar ) , % negative ( blue bar ) , % overlap ( Dome P1 , yellow bar ) % P1 ( red bars ) upon loss of Dop2R DAT in progenitors . For Dome positive ( domeMESO - Gal4 , UAS - GFP / + , n = 28 , domeMESO - Gal4 , UAS - GFP ; Dop2RRNAi , n = 16 , domeMESO - Gal4 , UAS - GFP ; DATRNAi , n = 23 ) , negative ( domeMESO - Gal4 , UAS - GFP / + , n = 28 , domeMESO - Gal4 , UAS - GFP ; Dop2RRNAi , n = 16 , domeMESO - Gal4 , UAS - GFP ; DATRNAi , n = 23 ) , overlap ( domeMESO - Gal4 , UAS - GFP / + , n = 26 , domeMESO - Gal4 , UAS - GFP ; Dop2RRNAi , n = 16 , domeMESO - Gal4 , UAS - GFP ; DATRNAi , n = 25 ) P1 ( domeMESO - Gal4 , UAS - GFP / + , n = 27 , domeMESO - Gal4 , UAS - GFP ; Dop2RRNAi , n = 16 , domeMESO - Gal4 , UAS - GFP ; DATRNAi , n = 23 ) . Data information : in M represents mean , with whiskers indicating maximum minimum values , box indicates lower upper quartiles . statistical applied in M is Two - way Anova with Tukey ' s multiple comparisons . ' n ' represents number of lymph 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 O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O B-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD 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 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 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 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 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

(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 ) of number of PPO positive crystal 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 : in N represents mean with standard deviation with whiskers indicating maximum minimum values , box indicates lower upper quartiles . statistical applied in N is One - way ANOVA with Tukey ' s multiple comparisons ' n ' represents number of lymph lobes analyzed , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

O O O O O O B-GENEPROD O B-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 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

(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 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 n = 23 at 120h AEL ) . 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 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 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 domeMESO - Gal4 , UAS - GFP ; DATRNAi , n = 22 ) at 120h AEL ( domeMESO - Gal4 , UAS - GFP ; THRNAi , n = 25 , domeMESO - Gal4 , UAS - GFP ; DdcRNAi , n = 30 , domeMESO - Gal4 , UAS - GFP ; Dop2RRNAi , n = 25 domeMESO - Gal4 , UAS - GFP ; DATRNAi , n = 31 ) . Data information : represents median with whiskers representing maximum minimum values , box indicates lower upper quartiles . statistical is Two - way Anova with Tukey ' s multiple comparisons while control comparison , domeMESO - Gal4 , UAS - GFP / + , for different time points was done by One - way ANOVA with Tukey ' s multiple comparisons . ' n ' represents number of lymph 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 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 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 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 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 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 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 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 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 O O O O O O O O O O O O O O O O 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 ) of total Dome + nuclei of control ( domeMESO - Gal4 , UAS - GFP / + , n = 37 at 48h , n = 28 at 72h , n = 19 at 96h n = 23 at 120h AEL ) . 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 ) 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 of Dome - nuclei in control ( domeMESO - Gal4 , UAS - GFP / + , n = 37 at 48h , n = 28 at 72h , n = 18 at 96h , n = 23 , at 120h AEL ) . perturbation of dopamine modules in 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 ) 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 : represents median with whiskers representing maximum minimum values , box indicates lower upper quartiles . statistical applied is Two - way Anova with Tukey ' s multiple comparisons while control comparison , domeMESO - Gal4 , UAS - GFP / + , for different time points was done by One - way ANOVA with Tukey ' s multiple comparisons . ' n ' represents number of lymph lobes analyzed , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . AEL indicates After Egg

O O O O O B-GENEPROD O B-SUBCELLULAR 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 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 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-SUBCELLULAR 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 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 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 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 O O O O O O O O O O O O O O O O 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 ) of Dome + BrdU index normalised to Dome + nuclei in a time course manner with dopamine modules perturbed in 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 ) 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 ) of Dome + mitotic index normalised to Dome + nuclei in a time course manner with loss of dopamine modules in 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 ) 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 . quantifications in A B represent median with whiskers indicating maximum minimum value , box indicates lower upper quartiles statistical applied is Two - way Anova with Tukey ' s multiple comparisons . ' n ' represents number of lymph lobes analyzed , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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 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 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-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 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 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 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

(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 % G2 / M at different time points , in control dopamine perturbed backgrounds , color coding scheme to represent cycle phase as shown through FUCCI reporter is below 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 ) 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 ( ubiquitination - cycle indicator ) reporter shows G1 phase in green , S phase in red G2 / M phase in yellow . AEL indicates After Egg Laying . quantifications in in C represents mean . statistical applied is Two - way Anova with Tukey ' s multiple comparisons . domeMESO - Gal4 ; UAS - FUCCI control comparison temporally in C is analysed using One - way ANOVA with Tukey ' s multiple comparisons . ' n ' represents number of lymph 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 B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD 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 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 O O O O O O O O O O O O O O O O O O O O O O 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 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

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

O O O O O O O O B-TISSUE O O O O O O O B-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 B-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 B-SMALL_MOLECULE 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 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 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 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 B-SMALL_MOLECULE O B-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 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-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 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 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 B-TISSUE O B-TISSUE 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 ) of total upon loss of TH Ddc in 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 ) 96h AEL ( Antp - Gal4 ; UAS - GFP / + , n = 15 , Antp - Gal4 ; UAS - GFP ; THRNAi , n = 14 , Antp - Gal4 ; UAS - GFP ; DdcRNAi , n = 15 ) Data information : in L represents median with whiskers indicating maximum minimum values , box indicates lower upper quartiles . statistical applied in L is Two - way Anova with Tukey ' s multiple comparisons . ' n ' represents number of lymph lobes analyzed , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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

(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 , nodule extract . Data represents mean standard error of 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 B-TISSUE I-TISSUE 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 , nodule extract . values obtained for each of 4 measurements ( points ) together with 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 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

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

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 B-GENEPROD 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 yellowish color of non - functional nodules induced by ∆ 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 B-TISSUE 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 presence of hollow nodules with aberrant in ∆ 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 B-TISSUE I-TISSUE O B-EXP_ASSAY 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 mean of at least 30 measured after 8 post inoculation ; error bars indicate standard error of 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 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 arginine transamination network with relative enzyme activities highlighted as a color map Rows are annotated on left with abbreviated names of substrates on right with 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 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

(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 pyruvate as substrates . Substrate ( S ) consumption into products ( P ) succinate alanine , including their intermediates ( I1 , I2 ) , was determined along 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 I-SMALL_MOLECULE 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 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 ) relative metabolite abundance along reaction time - course is highlighted as a color

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY 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 with deafness - infertility syndrome lacking functional CatSper channels ( CATSPER2 - / - ) ( red ) a healthy donor ( black ) , evoked by progesterone , PGE1 , NH4Cl , or ionomycin . NH4Cl increases 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 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 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 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 ) in sperm from a healthy donor ( black ) corresponding current - voltage relationship ( right ) . voltage was stepped from - 100 mV to + 100 mV in increments of 10 mV from a holding potential of - 80 mV . Outward inward current amplitudes ( mean ± SD ) at + 100 mV - 100 mV , respectively , in CATSPER2 - / - sperm ( color code : panel B ) 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 B-CELL_TYPE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY 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 B-EXP_ASSAY I-EXP_ASSAY 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 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 of control sperm from healthy donors CATSPER2 - / - sperm form DIS using antibodies directed against CatSper 3 ( D ) or CatSper 4 ( E ) ; was labelled with DAPI ( blue ) . Scale bars represent 10 µm .

O B-EXP_ASSAY O O B-CELL_TYPE O O O B-GENEPROD O O O B-CELL_TYPE O B-DISEASE O O O O B-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD O O O O 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 anti - CatSper 3 antibody ( left ) . Axial projection of boxed region ( right ) . Scale bars represent 5 µm in xy - projections 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 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

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

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

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.


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

B-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

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


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

B-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 B-CELL_TYPE 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

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 ) capacitated sperm ( 25 mM bicarbonate ; red ; n = 232 ) determined by - 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 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 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 ) 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 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 ) , 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 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 of an optically trapped sperm , obtained at t = 0 , 55 , 155 , 185 ms . Scale bar represents 10 µm .

B-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

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 rotation frequency of trapped sperm ( each sperm is represented by a different color ) . Error bars indicate full width at half prominence of frequency peaks determined by fast Fourier . .

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

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 trapped parallel to optical axis ; were obtained at t = 0 , 95 , 165 , 205 ms . red bar indicates 360 ° rotation of tip of head . Scale bar represents 10 µm .

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

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

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 before after dragging across barrier stream . green trace indicates of fluorescein included in barrier stream . Error bars indicate full width at half prominence of frequency peaks determined by fast Fourier . Paired plot of rotation frequencies of sperm before after dragging across 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 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-CELL_TYPE 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 before after dragging from control stream containing 0 mM bicarbonate into stimulus stream containing 25 mM bicarbonate . Error bars indicate full width at half prominence of frequency peaks determined by fast Fourier . Paired plot of rotation frequencies of sperm at 0 25 mM bicarbonate .

B-EXP_ASSAY I-EXP_ASSAY O O O B-CELL_TYPE O O O O O O O O O B-SMALL_MOLECULE 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 B-CELL_TYPE 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 in presence absence of extracellular Ca2 + . Error bars indicate full width at half prominence of frequency peaks determined by fast Fourier . Paired plot of rotation frequencies in presence absence of extracellular Ca2 + .

B-EXP_ASSAY I-EXP_ASSAY O O O B-CELL_TYPE 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 B-EXP_ASSAY I-EXP_ASSAY 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 presence ( black ) 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 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 presence ( black , n = 335 ) 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 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 at t = 0 , 151 , 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 at t = 0 , 151 , 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 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 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 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 ) , efa612 / GX6w - transheterozygous mutant neurons ( G ) showed consistent dendrite pruning defects at 16 h APF , as compared to wild - type neurons ( B ) . dendrite pruning defects were fully rescued when overexpressing full - Efa6 in efa612 mutant clones ( H ) . Red arrowheads point to ddaC somas .

O O O O B-EXP_ASSAY 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 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 B-CELL_TYPE O O O O B-SUBCELLULAR O O O O O O O O O B-GENEPROD O B-GENEPROD 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 of efa612 / GX6w - mutant ddaC neurons at WP stage 16 h APF . Expression of Efa6FL ( C ) rescued pruning defects in efa612 / GX6w - mutant ddaC neurons ( B ) . Red arrowheads point to ddaC somas .

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

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

O O O O B-EXP_ASSAY O O B-CELL_TYPE 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 B-GENEPROD O O B-CELL_TYPE O O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR 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 B-SUBCELLULAR 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 of da sensory neurons expressing Efa6 - Ki - GFP ( F ) , or UAS - Efa6 - GFP driven by ppk - Gal4 ( H ) were co - stained with GFP ( Green ) Nrg ( Magenta ) at wL3 stage . ( G , I ) Confocal of epidermal expressing Efa6 - Ki - GFP ( G ) or UAS - Efa6 - GFP driven by A58 - Gal4 ( I ) were co - stained with GFP ( Green ) Dlg ( Magenta ) at wL3 stage . ddaC soma is marked by dashed lines .

O O O B-EXP_ASSAY 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 B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY O B-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 B-GENEPROD 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 proximal dendrites of ddaC neurons at 96 h AEL . horizontal arrow indicates direction toward soma , vertical arrow indicates time . In contrast to wild type ( A ) , number of EB1 - GFP comets was significantly increased in efa612 / GX6w - mutant neurons ( B ) . comet speed were significantly decreased in Efa6FL - 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 B-SUBCELLULAR O O B-CELL_TYPE 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 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 B-GENEPROD 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 of ddaC dendrites expressing tdEOS : : α - tubulin driven by ppk - Gal4 . microtubule turnover rate was significantly reduced in efa6 RNAi neurons at wL3 ( A , right panels ) WP stages ( B , right panels ) , increased in Efa6 - overexpressing neurons at wL3 stage ( C , right panels ) , compared to control neurons ( A - C , left panels ) .

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

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

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

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

O O O O B-EXP_ASSAY O O B-CELL_TYPE 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 B-CELL_TYPE O O O O O O O B-GENEPROD I-GENEPROD O O B-SUBCELLULAR 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 B-SUBCELLULAR 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 proximal dendrites of ddaC neurons at 96 h AEL .

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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 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.