Glycolytic Disruption Triggers Interorgan Signaling to Nonautonomously Restrict Drosophila Larval Growth

Abstract

Drosophila larval growth requires efficient conversion of dietary nutrients into biomass. Lactate Dehydrogenase (Ldh) and Glycerol-3-phosphate dehydrogenase (Gpdh1) support larval biosynthetic metabolism by maintaining NAD⁺/NADH redox balance and promoting glycolytic flux. Consistent with the cooperative functions of Ldh and Gpdh1, the loss of both enzymes, but neither single enzyme, induces a developmental arrest. However, Ldh and Gpdh1 exhibit complex and often mutually exclusive expression patterns, suggesting that the Gpdh1; Ldh double mutant lethal phenotype could be mediated nonautonomously. Here we find that the developmental arrest displayed by the double mutants extends beyond simple metabolic disruption and instead stems, in part, from changes in systemic growth factor signaling. Specifically, we demonstrate that this synthetic lethality is linked to the upregulation of Upd3, a cytokine involved in the Jak/Stat signaling pathway. Moreover, we demonstrate that either loss of the Upd3 or dietary administration of the steroid hormone 20-hydroxyecdysone (20E) rescue the synthetic lethal phenotype of Gpdh1; Ldh double mutants. Together, these findings demonstrate that metabolic disruptions within a single tissue can nonautonomously modulate interorgan signaling to ensure synchronous developmental growth.

Keywords: Drosophila melanogaster; ecdysone; glycolysis; interorgan communication; metabolism.

Figure 1.. Ldh and Gpdh1 expression patterns are complex and non-strictly overlapping.

Representative confocal images of second instar larval tissues expressing Ldh-GFP^Genomic and immuno-stained with aGpdh1 antibody. DAPI is shown in blue, Ldh-GFP and Gpdh1 are represented in green and magenta, respectively. The rightmost panel displays the merged images of Ldh-GFP and Gpdh1 staining. (A-A”’) Malpighian tubules, (B-B”’) gut, (C-C”’) ventral side of CNS, (D-D”’) magnification of the outlined region of interest in (C-C”’), and (E-E”’) muscles. Arrows denote non-overlapping Ldh expression. The scale bar represents 40 μM. The scale bar in (A) applies to all panels.

Figure 2.. Tissue-specific loss of Ldh and Gpdh1 induces systemic growth defects.

Growth and development of both control (Mef2R-Gal4 and UAS-Ldh-RNAi strains) and mutant strains (Gpdh1^A10/B18 mutants, Mef2R-Ldh-RNAi, and Gpdh1^A10/B18; Mef2R-Ldh-RNAi) were monitored throughout larval development. (A) Representative images of L2 larvae (60 hr AEL) from the indicated genotypes. The scale bar represents 1 mm. (B-C) Quantification of (B) larval length and (C) time to pupation from the indicated genotypes. All experiments were repeated a minimum of three times. n≥5 biological replicates for (B, C). Error bars represent standard deviation; *P<0.05, ***P<0.0001. P-values were calculated using the Mann-Whitney test. (D-G) Representative images of salivary glands dissected from the indicated genotypes. DAPI is shown in blue. The scale bar represents 40 μM. The scale bar in (D) applies to (E).

Figure 3.. An upd3 mutation suppresses the synthetic lethal phenotype of Gpdh1; Ldh double mutants.

(A-B) Venn diagrams showing the overlap between the number of genes that were either (A) downregulated or (B) upregulated in either single mutant (Gpdh1^A10/B18 and Ldh^16/17) as well as the double mutant (Gpdh1^A10/B18; Ldh^16/17) relative to the respective heterozygous controls strains. Associated tables in (A, B) list secreted factors that are only dysregulated in the Gpdh1^A10/B18; Ldh^16/17 double mutant. (C) Representative larval images of the indicated genotypes. The scale bars represent 1 mm and apply to all panels. (D) Quantification of the larval size for the indicated genotypes. (E) A significant number of male and female triple mutant pupae (upd3^Δ; Ldh^16/17; Gpdh1^A10/B18) successfully eclosed whereas all Gpdh1^A10/B18; Ldh^16/17 died during metamorphosis. The scale bar represents 1mm and applies to all panels. (F) The rate of eclosion was quantified for pupae of the indicated genotypes. Note that no eclosion was observed among vials containing Gpdh1^A10/B18; Ldh^16/17 pupae. All experiments are repeated a minimum of three times. n≥5 biological replicates. Error bars represent standard deviation; **P<0.05. P-values were calculated by using a Mann-Whitney test.

Figure 4.. Dietary supplementation with 20E suppresses the synthetic lethal phenotype of Gpdh1; Ldh double mutants.

(A-D) Representative confocal images of Stat-GFP expression in the CNS of Gpdh1^A10/B18; Ldh^16/17 double mutants as compared to the heterozygous control (Gpdh1^A10/+; Ldh^16/+) and single mutant strains (Gpdh1^A10/B18 and Ldh^16/17). DAPI is shown in blue and Stat-GFP expression in green. The scale bar in (A) represents 40 μM and applies to (B-D). (E) A graph illustrating the percent of Gpdh1^A10/B18; Ldh^16/17 double mutant that pupariate when raised on yeast-molasses agar that contains either ecdysone or the solvent (ethanol) control. All experiments are repeated a minimum of three times. n=9 biological replicates. Error bars represent standard deviation. **P<0.05. P value was calculated using a Mann-Whitney test.