( K ) Mobility of BACE1 carriers is significantly decreased in neurons overexpressing the AP2 - αΑ Mut compared to neurons expressing the AP2 - αΑ WT ( WT : 52 . 23 ± 4 . 16 % , Mut : 27 . 95 ± 3 . 31 , p = < 0 . 000 , WT = 26 and Mut = 29 neurons , N = 4 biological replicates ) . Data information : All graphs show mean ± SEM , statistical analysis was performed by unpaired two - tailed Student ' s t ‐ test n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

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(K) Mobility of BACE1 carriers is significantly decreased in neurons overexpressing the AP2-αΑ Mut compared to neurons expressing the AP2-αΑ WT (WT: 52.23±4.16%, Mut: 27.95±3.31, p=<0.000, WT=26 and Mut=29 neurons, N=4 biological replicates). Data information: All graphs show mean ± SEM, statistical analysis was performed by unpaired two-tailed Student's t‐test n.s.-non-significant. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.


( L ) Representative fluorescence images and corresponding kymographs from time - lapse videos of WT neurons transfected with either HA - BACE1 - GFP or HA - BACE1 - LL / AA - GFP . Scale bar : 5µm top panels , x = 2μm , y = 5s bottom panels .

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(L) Representative fluorescence images and corresponding kymographs from time-lapse videos of WT neurons transfected with either HA-BACE1-GFP or HA-BACE1-LL/AA-GFP. Scale bar: 5µm top panels, x=2μm, y=5s bottom panels.


( M ) Reduced mobility of BACE1 - LL / AA - positive vesicles , when compared to WT BACE1 ( WT : 38 . 97 ± 1 . 40 % , LL / AA : 19 . 89 ± 1 . 77 % , p < 0 . 000 , WT = 31 and LL / AA = 33 neurons , N = 4 biological replicates ) . Data information : All graphs show mean ± SEM , statistical analysis was performed by unpaired two - tailed Student ' s t ‐ test n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

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(M) Reduced mobility of BACE1-LL/AA-positive vesicles, when compared to WT BACE1 (WT: 38.97±1.40%, LL/AA: 19.89±1.77%, p<0.000, WT=31 and LL/AA=33 neurons, N=4 biological replicates). Data information: All graphs show mean ± SEM, statistical analysis was performed by unpaired two-tailed Student's t‐test n.s.-non-significant. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.


( N ) Reduced velocity of BACE1 - LL / AA - positive vesicles , when compared to WT BACE1 ( WTAntero : 0 . 38 ± 0 . 03 μm / s , LL / AAAntero : 0 . 15 ± 0 . 03 μm / s , pWTAntero vs pLL / AAAntero < 0 . 000 ; WTRetro : 0 . 39 ± 0 . 03 μm / s , LL / AARetro : 0 . 25 ± . 03 μm / s , pWTRetro vs pLL / AARetro = 0 . 000 , WT = 31 and LL / AA = 33 neurons , N = 4 biological replicates ) . Data information : All graphs show mean ± SEM , statistical analysis was performed by unpaired two - tailed Student ' s t ‐ test n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

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(N) Reduced velocity of BACE1-LL/AA-positive vesicles, when compared to WT BACE1 (WTAntero: 0.38±0.03 μm/s, LL/AAAntero: 0.15±0.03 μm/s, pWTAntero vs pLL/AAAntero<0.000; WTRetro: 0.39±0.03 μm/s, LL/AARetro: 0.25±.03 μm/s, pWTRetro vs pLL/AARetro =0.000, WT=31 and LL/AA=33 neurons, N=4 biological replicates). Data information: All graphs show mean ± SEM, statistical analysis was performed by unpaired two-tailed Student's t‐test n.s.-non-significant. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.


( A , B ) The eGFP / mCherry signal intensity ratio is significantly increased in mCherry - APP - P1 - eGFP - expressing KO neurons comparing to the WT ( WT : 0 . 94 ± 0 . 06 % , KO : 1 . 65 ± 0 . 18 , p = 0 . 000 , WT = 39 and KO = 41 neurons , N = 5 biological replicates ) . Scale bars : 2μm . Data information : All graphs show mean ± SEM , statistical analysis was performed by unpaired two - tailed Student ' s t ‐ test n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

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(A, B) The eGFP/mCherry signal intensity ratio is significantly increased in mCherry-APP-P1-eGFP-expressing KO neurons comparing to the WT (WT: 0.94±0.06%, KO: 1.65±0.18, p=0.000, WT=39 and KO=41 neurons, N=5 biological replicates). Scale bars: 2μm. Data information: All graphs show mean ± SEM, statistical analysis was performed by unpaired two-tailed Student's t‐test n.s.-non-significant. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.


Confocal images of WT and AP - 2μ ΚΟ entorhinal cortex immunostained with antibody recognizing total APP and APP - CTFs . Scale bars : 100 µm ( C )

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Confocal images of WT and AP-2μ ΚΟ entorhinal cortex immunostained with antibody recognizing total APP and APP-CTFs. Scale bars: 100 µm (C)


Confocal images of WT and AP - 2μ ΚΟ entorhinal cortex immunostained with antibody recognizing total APP and APP - CTFs . Scale bars : 20µm ( D ) .

Confocal images of WT and AP-2μ ΚΟ entorhinal cortex immunostained with antibody recognizing total APP and APP-CTFs. Scale bars: 20µm (D).


( E - G ) Levels of APP - CTFs are significantly increased in AP - 2µ KO cortex compared to the WT set to 100 % ( KOC99 : 156 . 65 ± 8 . 37 % , p = 0 . 004 , N = 4 biological replicates ; KOC83 : 154 . 79 ± 15 . 6 % , p = 0 . 012 ; N = 5 biological replicates ) . Data information : All graphs show mean ± SEM , statistical analysis was performed by one - sample Student ' s t ‐ test n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

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(E-G) Levels of APP-CTFs are significantly increased in AP-2µ KO cortex compared to the WT set to 100% (KOC99: 156.65±8.37%, p=0.004, N=4 biological replicates; KOC83: 154.79±15.6%, p=0.012; N=5 biological replicates). Data information: All graphs show mean ± SEM, statistical analysis was performed by one-sample Student's t‐test n.s.-non-significant. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.


( H ) Levels of full - length ( FL ) APP are unaltered in AP - 2µ KO cortex ( KOAPP FL : 106 . 64 ± 12 . 96 % , p = 0 . 315 , N = 6 biological replicates ) . Data information : All graphs show mean ± SEM , statistical analysis was performed by one - sample Student ' s t ‐ test n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

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(H) Levels of full-length (FL) APP are unaltered in AP-2µ KO cortex (KOAPP FL: 106.64±12.96%, p=0.315, N=6 biological replicates). Data information: All graphs show mean ± SEM, statistical analysis was performed by one-sample Student's t‐test n.s.-non-significant. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.


( I ) Levels of Nicastrin ( NCT ) are unaltered in the AP - 2µ KO

(I) Levels of Nicastrin (NCT) are unaltered in the AP-2µ KO cortex


( J , K ) Intracellular levels of Aβ1 - 40 are significantly increased in AP - 2µ KO axons compared to the WT ( WTAβ1 - 40 : 1 . 82 ± 0 . 12 , KOAβ1 - 40 : 2 . 45 ± 0 . 12 , p = 0 . 000 , WT = 24 and KO = 24 neurons , N = 3 biological replicates ) . Scale bars , 5µm . Data information : All graphs show mean ± SEM , statistical analysis was performed by unpaired two - tailed Student ' s t ‐ test n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

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(J, K) Intracellular levels of Aβ1-40 are significantly increased in AP-2µ KO axons compared to the WT (WTAβ1-40: 1.82±0.12, KOAβ1-40: 2.45±0.12, p=0.000, WT=24 and KO=24 neurons, N=3 biological replicates). Scale bars, 5µm. Data information: All graphs show mean ± SEM, statistical analysis was performed by unpaired two-tailed Student's t‐test n.s.-non-significant. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.


Intracellular levels of Aβ1 - 42 are significantly increased in AP - 2µ KO axons ( WTAβ1 - 42 : 0 . 65 ± 0 . 08 , KOAβ1 - 42 : 0 . 91 ± 0 . 10 , p = 0 . 046 , WT = 38 and KO = 38 neurons , N = 3 biological replicates ) . Scale bar : 5 µm .

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Intracellular levels of Aβ1-42 are significantly increased in AP-2µ KO axons (WTAβ1-42: 0.65±0.08, KOAβ1-42: 0.91±0.10, p=0.046, WT=38 and KO=38 neurons, N=3 biological replicates). Scale bar: 5 µm.


Intracellular levels of Aβ1 - 42 are significantly increased in AP - 2µ KO axons ( WTAβ1 - 42 : 0 . 65 ± 0 . 08 , KOAβ1 - 42 : 0 . 91 ± 0 . 10 , p = 0 . 046 , WT = 38 and KO = 38 neurons , N = 3 biological replicates ) . Scale bar : 5 µm . Data information : All graphs show mean ± SEM , statistical analysis was performed by unpaired two - tailed Student ' s t ‐ test n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

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Intracellular levels of Aβ1-42 are significantly increased in AP-2µ KO axons (WTAβ1-42: 0.65±0.08, KOAβ1-42: 0.91±0.10, p=0.046, WT=38 and KO=38 neurons, N=3 biological replicates). Scale bar: 5 µm. Data information: All graphs show mean ± SEM, statistical analysis was performed by unpaired two-tailed Student's t‐test n.s.-non-significant. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.


( N ) Significantly increased ratio of Αβ1 - 42 / Αβ1 - 40 measured by ELISA in the media of cultured AP - 2μ KO neurons compared to the WT set to 100 % ( KO : 124 . 57 ± 10 . 97 % , p = 0 . 037 , N = 6 biological replicates ) . Data information : All graphs show mean ± SEM , statistical analysis was performed by unpaired two - tailed Student ' s t ‐ test n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

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(N) Significantly increased ratio of Αβ1-42/ Αβ1-40 measured by ELISA in the media of cultured AP-2μ KO neurons compared to the WT set to 100% (KO:124.57±10.97%, p=0.037, N=6 biological replicates). Data information: All graphs show mean ± SEM, statistical analysis was performed by unpaired two-tailed Student's t‐test n.s.-non-significant. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.


Accumulation of intracellular Aβ1 − 42 in neurons expressing the AP2 - α − WT ( WT : 1 . 14 ± 0 . 18 , WT = 28 and Mut = 33 neurons , N = 4 biological replicates ) . Scale bars : 20µm , 2µm ( inserts ) .

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Accumulation of intracellular Aβ1−42 in neurons expressing the AP2-α−WT (WT: 1.14±0.18 , WT=28 and Mut=33 neurons, N=4 biological replicates). Scale bars: 20µm, 2µm (inserts).


Accumulation of intracellular Aβ1 − 42 in neurons overexpressing the AP2 - α - Mut comparing to neurons expressing the AP2 - α − WT ( WT : 1 . 14 ± 0 . 18 , Mut : 2 . 06 ± 0 . 29 , p = 0 . 011 , WT = 28 and Mut = 33 neurons , N = 4 biological replicates ) . Scale bars : 20µm , 2µm ( inserts ) . Data information : All graphs show mean ± SEM , statistical analysis was performed by unpaired two - tailed Student ' s t ‐ test n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

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Accumulation of intracellular Aβ1−42 in neurons overexpressing the AP2-α-Mut comparing to neurons expressing the AP2-α−WT (WT: 1.14±0.18, Mut: 2.06±0.29, p=0.011, WT=28 and Mut=33 neurons, N=4 biological replicates). Scale bars: 20µm, 2µm (inserts). Data information: All graphs show mean ± SEM, statistical analysis was performed by unpaired two-tailed Student's t‐test n.s.-non-significant. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.


Accumulation of intracellular Aβ1 - 42 is rescued upon BACE1 knock - down ( WTScr : 2 . 76 ± 0 . 20 WTshBACE1 : 2 . 52 ± 0 . 12 27 to 39 neurons per condition , N = 3 biological replicates ) . Scale bar : 10µm .

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Accumulation of intracellular Aβ1-42 is rescued upon BACE1 knock-down (WTScr: 2.76±0.20 WTshBACE1: 2.52±0.12 27 to 39 neurons per condition, N=3 biological replicates). Scale bar: 10µm.


Accumulation of intracellular Aβ1 - 42 is rescued upon BACE1 knock - down in AP - 2µ KO neurons ( WTScr : 2 . 76 ± 0 . 20 , KOScr : 3 . 94 ± 0 . 21 ; WTshBACE1 : 2 . 52 ± 0 . 12 ; KOshBACE1 : 2 . 74 ± 0 . 21 ; pWTScr vs pKOScr = 0 . 000 , pKOscr vs pKOshBACE1 = 000 ; pWTscr vs pWTshBACE1 = 0 . 826 ; pWTshBACE1 vs pKOshBACE1 = 0 . 863 ; 27 to 39 neurons per condition , N = 3 biological replicates ) . Scale bar : 10µm . Data information : All graphs show mean ± SEM , statistical analysis was performed by two - way ANOVA in ( T ) n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

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Accumulation of intracellular Aβ1-42 is rescued upon BACE1 knock-down in AP-2µ KO neurons (WTScr: 2.76±0.20, KOScr: 3.94±0.21; WTshBACE1: 2.52±0.12; KOshBACE1: 2.74±0.21; pWTScr vs pKOScr=0.000, pKOscr vs pKOshBACE1=000; pWTscr vs pWTshBACE1=0.826; pWTshBACE1 vs pKOshBACE1=0.863; 27 to 39 neurons per condition, N=3 biological replicates). Scale bar: 10µm. Data information: All graphs show mean ± SEM, statistical analysis was performed by two-way ANOVA in (T) n.s.-non-significant. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.


( U , V ) Levels of intracellular Aβ1 − 42 are significantly upregulated in neurons overexpressing the HA - BACE1 - LL / AA - GFP comparing to neurons expressing the HA - BACE1 - GFP ( BACE1 : 0 . 78 ± 0 . 90 , BACE1 - LL - AA : 1 . 99 ± 0 . 33 , p = 000 , 28 - 29 neurons per condition , N = 3 biological replicates ) . Scale bar , 50µm . Data information : All graphs show mean ± SEM , statistical analysis was performed by unpaired two - tailed Student ' s t ‐ test n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

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(U, V) Levels of intracellular Aβ1−42 are significantly upregulated in neurons overexpressing the HA-BACE1-LL/AA-GFP comparing to neurons expressing the HA-BACE1-GFP (BACE1: 0.78±0.90, BACE1-LL-AA: 1.99±0.33, p=000, 28-29 neurons per condition, N=3 biological replicates). Scale bar, 50µm. Data information: All graphs show mean ± SEM, statistical analysis was performed by unpaired two-tailed Student's t‐test n.s.-non-significant. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.


( A , B ) AP - 2α levels normalized to GAPDH are significantly decreased in lysates of iPSC - derived neurons from late - onset AD patients carrying the TREM2 p . R47H ( AD - TREM2 - 2 and AD - TREM2 - 4 ) variant compared to healthy controls ( CON8 and CON9 ) set to 100 % ( ADAP - 2α : 52 . 42 ± 8 . 04 % , p = 0 . 020 , N = 3 biological replicates ) . Data information : All graphs show mean ± SEM , statistical analysis was performed by one - sample Student ' s t ‐ test n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

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(A, B) AP-2α levels normalized to GAPDH are significantly decreased in lysates of iPSC-derived neurons from late-onset AD patients carrying the TREM2 p.R47H (AD-TREM2-2 and AD-TREM2-4) variant compared to healthy controls (CON8 and CON9) set to 100% (ADAP-2α: 52.42±8.04%, p=0.020, N=3 biological replicates). Data information: All graphs show mean ± SEM, statistical analysis was performed by one-sample Student's t‐test n.s.-non-significant.* indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.


( C ) Confocal images of either AP - 2 WT ( AAV - GFPCamkIIα - labelled ) or AP - 2 KO ( AAV - Cre - GFPCamkIIα - labelled ) granule neurons in the dentate gyrus of 13 - week - old mice . Scale bars : 20µm , 2µm ( inserts ) .

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(C) Confocal images of either AP-2 WT (AAV-GFPCamkIIα-labelled) or AP-2 KO (AAV-Cre-GFPCamkIIα-labelled) granule neurons in the dentate gyrus of 13-week-old mice. Scale bars: 20µm, 2µm (inserts).


( D ) Loss of AP - 2µ significantly decreases the number of spines on granule neurons ( WT : 12 . 63 ± 0 . 88 , KO : 8 . 07 ± 0 . 45 , p = 0 . 004 , N = 4 biological replicates ) . Data information : All graphs show mean ± SEM , statistical analysis was performed by unpaired two - tailed Student ' s t ‐ test n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

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(D) Loss of AP-2µ significantly decreases the number of spines on granule neurons (WT: 12.63±0.88, KO: 8.07±0.45, p=0.004, N=4 biological replicates). Data information: All graphs show mean ± SEM, statistical analysis was performed by unpaired two-tailed Student's t‐test n.s.-non-significant.* indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.


( E , F ) Overlap coefficient between the presynaptic marker Bassoon and the postsynaptic marker PSD95 is significantly decreased in the entorhinal cortex of AP - 2µ KO mice compared to the WT ( WT : 0 . 45 ± 0 . 02 , KO : 0 . 33 ± 0 . 06 , p = 0 . 034 , N = 3 biological replicates ) . Scale bar , 750nm . Data information : All graphs show mean ± SEM , statistical analysis was performed by unpaired two - tailed Student ' s t ‐ test n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

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(E, F) Overlap coefficient between the presynaptic marker Bassoon and the postsynaptic marker PSD95 is significantly decreased in the entorhinal cortex of AP-2µ KO mice compared to the WT (WT: 0.45±0.02, KO: 0.33±0.06, p=0.034, N=3 biological replicates). Scale bar, 750nm. Data information: All graphs show mean ± SEM, statistical analysis was performed by unpaired two-tailed Student's t‐test n.s.-non-significant.* indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.


Lost preference for the novel object ( nov ) comparing to the familiar object ( fam ) in AP - 2µ KO mice tested 24h after training in NOR Representative heatmaps are shown in ( G ) .

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Lost preference for the novel object (nov) comparing to the familiar object (fam) in AP-2µ KO mice tested 24h after training in NOR Representative heatmaps are shown in (G).


Lost preference for the novel object ( nov ) comparing to the familiar object ( fam ) in AP - 2µ KO mice tested 24h after training in NOR ( WTfam : 27 . 51 ± 6 . 77 , WTnov : 72 . 49 ± 6 . 77 , pWTfam vs WTnov = 0 . 000 ; KOfam : 43 . 9 ± 13 . 12 , KOnov : 59 . 91 ± 13 . 12 , pKOfam vs KOnov = 0 . 469 , N = 7 WT and 8 KO mice ) . Data information : All graphs show mean ± SEM , statistical analysis was performed by unpaired two - tailed Student ' s t ‐ test n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

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

Lost preference for the novel object (nov) comparing to the familiar object (fam) in AP-2µ KO mice tested 24h after training in NOR (WTfam: 27.51±6.77, WTnov: 72.49±6.77, pWTfam vs WTnov=0.000; KOfam: 43.9±13.12, KOnov: 59.91±13.12, pKOfam vs KOnov=0.469, N=7 WT and 8 KO mice). Data information: All graphs show mean ± SEM, statistical analysis was performed by unpaired two-tailed Student's t‐test n.s.-non-significant.* indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.


( I , J ) AP - 2µ KO mice are significantly more active in the open field compared to the WT ( WT : 1663 ± 139 . 08 % , KO : 3976 ± 802 . 30 % , p = 0 . 020 , N = 7 WT and 8 KO mice ) . Representative heatmaps are shown in ( I ) . Data information : All graphs show mean ± SEM , statistical analysis was performed by unpaired two - tailed Student ' s t ‐ test n . s . - non - significant . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

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

(I, J) AP-2µ KO mice are significantly more active in the open field compared to the WT (WT: 1663±139.08%, KO: 3976±802.30%, p=0.020, N=7 WT and 8 KO mice). Representative heatmaps are shown in (I). Data information: All graphs show mean ± SEM, statistical analysis was performed by unpaired two-tailed Student's t‐test n.s.-non-significant.* indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.


( K , L ) Overlap coefficient between Bassoon and PSD95 is rescued upon BACE1 KD in AP - 2µ KO neurons ( WTscr : 0 . 39 ± 0 . 02 ; KOscr : 0 . 24 ± 0 . 01 ; WTshBACE1 : 0 . 37 ± 0 . 02 ; KOshBACE1 : 0 . 33 ± 0 . 02 ; pWTscr vs pKOscr < 0 . 000 ; pKOscr vs pKOshBACE1 = 0 . 010 ; pWTscr vs pWTshBACE1 = 0 . 813 ; pWTshBACE1 vs pKOshBACE1 = 0 . 623 , 22 - 24 neurons for each condition , N = 3 ) . Scale bars : 10µm . 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 .

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

(K, L) Overlap coefficient between Bassoon and PSD95 is rescued upon BACE1 KD in AP-2µ KO neurons (WTscr: 0.39±0.02; KOscr: 0.24±0.01; WTshBACE1: 0.37±0.02; KOshBACE1: 0.33±0.02; pWTscr vs pKOscr<0.000; pKOscr vs pKOshBACE1=0.010; pWTscr vs pWTshBACE1=0.813; pWTshBACE1 vs pKOshBACE1=0.623, 22-24 neurons for each condition, N=3). Scale bars: 10µm. 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.


Neuronal loss is rescued in the entorhinal cortex of AP - 2µ mice haploinsufficient for BACE1 ( AP - 2WT / BACEWT : 2 . 25 ± 0 . 08 N = 9 sections from N = 3 for each genotype ) . Scale bar : 100 μm .

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

Neuronal loss is rescued in the entorhinal cortex of AP-2µ mice haploinsufficient for BACE1 (AP-2WT/BACEWT: 2.25±0.08 N=9 sections from N=3 for each genotype). Scale bar: 100 μm.


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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 / + ) . 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 .

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

(A-D) 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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 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

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

(J-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 ) 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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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 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 .

(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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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 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 .

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

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

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

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


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

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

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


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

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

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


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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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


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

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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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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..