Fig. 4.

GABA catabolism via PDK activity regulates the TCA cycle in blood progenitor cells and coordinates overall lymph gland growth. (A-F) Representative lymph gland images showing PDH and PDK levels. (A,D) Controls (domeMeso-Gal4,UAS-GFP/+) showing (A) PDH and (D) PDK levels. (B,E) GatRNAi (domeMeso-Gal4,UAS-GFP;UAS-GatRNAi) and (C,F) SsadhRNAi (domeMeso-Gal4,UAS-GFP;UAS-SsadhRNAi) in progenitor cells does not reduce (B,C) PDH and (E,F) PDK levels when compared with A and D, respectively. For quantification, refer to Fig. S5A,B. (G-I) Representative lymph gland images showing pPDH on RF. (G) Control (domeMeso-Gal4,UAS-GFP/+). (H) GatRNAi (domeMeso-Gal4,UAS-GFP;UAS-GatRNAi) and (I) SsadhRNAi (domeMeso-Gal4,UAS-GFP;UAS-SsadhRNAi) lead to a reduction in pPDH levels when compared with G (control). (J-L) Representative lymph gland images showing pPDK on RF. (J) Control (domeMeso-Gal4,UAS-GFP/+). (K) GatRNAi (domeMeso-Gal4,UAS-GFP;UAS-GatRNAi) and (L) SsadhRNAi (domeMeso-Gal4,UAS-GFP;UAS-SsadhRNAi) lead to a reduction in pPDK levels when compared with J (control). (M-P) Representative lymph gland images showing ROS levels. (M) Control (domeMeso-Gal4,UAS-GFP/+). (N) GatRNAi (domeMeso-Gal4,UAS-GFP;UAS-GatRNAi) leads to an elevation in ROS levels. (O) PdhaRNAi;GatRNAi (domeMeso-Gal4,UAS-GFP;UAS-PdhaRNAi;UAS-GatRNAi) and (P) SdhARNAi;GatRNAi (domeMeso-Gal4,UAS-GFP;UAS-SdhARNAi;UAS-GatRNAi) rescue the increased ROS phenotype of N (GatRNAi). (Q-T) Succinate supplementation (SF) in (Q,R) GatRNAi (domeMeso-Gal4,UAS-GFP;UAS-GatRNAi) and (S,T) SsadhRNAi (domeMeso-Gal4,UAS-GFP;UAS-SsadhRNAi) rescues (Q,S) pPDH and (R,T) pPDK levels of (H,K) GatRNAi and (I,L) SsadhRNAi on RF. (U) Quantification of G-I,Q,S. Relative fold change in lymph gland MZ pPDH levels in domeMeso>GFP/+ (RF, control, N=5, n=51), domeMeso>GFP/GatRNAi (RF, N=5, n=32, P<0.0001), domeMeso>GFP/GatRNAi (SF, N=3, n=24, P<0.0001), domeMeso>GFP/SsadhRNAi (RF, N=5, n=47, P<0.0001), domeMeso>GFP/SsadhRNAi (SF, N=3, n=23, P=0.0007) and w1118 (SF, N=3, n=38, P<0.0001, pPDH compared with w1118, N=3, RF, n=47). (V) Quantification of J-L,R,T. Relative fold change in lymph gland MZ pPDK levels in domeMeso>GFP/+ (RF, control, N=5, n=75), domeMeso>GFP/GatRNAi (RF, N=5, n=38, P=0.0004), domeMeso>GFP/GatRNAi (SF, N=3, n=20, P=0.0006), domeMeso>GFP/SsadhRNAi (RF, N=4, n=35, P=0.0058), domeMeso>GFP/SsadhRNAi (SF, N=3, n=20, P=0.0021) and w1118 (SF, n=30, P=0.0071, pPDK compared with w1118, RF, N=3, n=30). (W) Quantification of M-P. Relative fold change in lymph gland ROS (DHE) levels in domeMeso>GFP/+(control, N=4, n=38), domeMeso>GFP/GatRNAi (N=3, n=22, P<0.0001), domeMeso>GFP/PdhaRNAi;GatRNAi (N=3, n=19, P<0.0001) and domeMeso>GFP/SdhARNAi;GatRNAi (N=4, n=26, P<0.0001). (X) Quantifications of lymph gland size in domeMeso>GFP/+ (control, N=5, n=44), domeMeso>GFP/GatRNAi (N=4, n=36, P<0.0001), domeMeso>GFP/PdhaRNAi;GatRNAi (N=5, n=50, P<0.0001), and domeMeso>GFP/SdhARNAi;GatRNAi (N=5, n=41, P<0.0001). RF, regular food; SF is succinate-supplemented food. The horizontal line indicates the median; whiskers indicate the minimum and maximum values; box indicates the lower and upper quartiles (**P<0.01, ***P<0.001, ****P<0.0001; n.s., not significant; two-way ANOVA, Tukey's multiple comparisons test). f.c., fold change. Scale bars: 20 µm. n, lymph gland lobes; N, number of experimental repeats (green dot). MZ, medullary zone. DAPI marks DNA. Comparisons for significance are with control values unless marked by horizontal lines for other respective comparisons; red bars represent rescue combinations. Lymph gland lobes are outlined with a white border and, for clarity, the background containing other tissues, such as ring gland, brain, dorsal vessel, etc., has been removed.