Fig. 3: Global and local ICWs are regulated by gap junction differently.

A Both global and local ICWs are present in the larval fat body. Organism-level global ICWs traveling from larval head to tail are only observed in vivo. Conversely, local ICWs, initiated and propagated randomly in the fat body cells, are evident in both in vitro and in vivo experimental settings. B, C Disruption of intercellular gap junctions by Inx2 knockdown significantly scattered the Ca²⁺ activities in ex vivo cultured fat body. The dynamic Ca²⁺ activities were highlighted in the lower panel by removing the constant background signal. N = 4, 4 fat bodies (C). D, E Inx2 knockdown does not affect the magnitude or period of global Ca²⁺ waves in vivo, while in AkhR mutants both global and local Ca²⁺ waves are completely blocked. Meanwhile, overexpressing AKH in the fat body led to a random propagation of Ca²⁺ waves. Larvae were kept under starved conditions to trigger the release of endogenous AKH. E Quantification of the traveling velocity of the global and local Ca²⁺ waves. N = 6, 6, 6 fat bodies in vivo, 8 fat bodies ex vivo. F, G The proportion of cells with Ca²⁺ activities in the fat body of free-behaving WT and Inx2 RNAi larvae were quantified. N = 6, 6 independent biological replicates (G). H, I Inx2 knockdown completely scattered the Ca²⁺ waves in the dissected adult fly fat bodies. The dynamic Ca²⁺ activities were highlighted in the lower panel by removing the constant background signal. N = 3, 3 fat bodies (I). J, K Adult female flies of the indicated genotype were starved for about 30 min and then glued alive on a glass slide with the ventral abdomen imaged. Ca²⁺ activities indicated by fat body-specific expression of GCaMP5G were monitored and quantified. N = 3, 3, 3 adults (K). L, M Effects of gap junction disruption in fat bodies during the larval and adult stages on TAG metabolism. N = 7, 7, 11, 13 independent biological replicates (L). N = 4, 4, 3, 3 independent biological replicates (M). Ca²⁺ intensity (a.u.) was presented using a linear colour scale (minimum = 0, maximum = 255) (B, D, F, H, J). Unpaired two-tailed Student’s t-test was used in (C, G, I). Ordinary two-way ANOVA with Sidak’s multiple comparisons was used in (E, L). Ordinary one-way ANOVA with Dunnett’s multiple comparisons test was used in (K). Ordinary two-way ANOVA with uncorrected Fisher’s LSD was used in (M). Data were plotted as mean ± SD. Scale bars, 200 μm (B, H), 500 μm (D, J), 1 mm (F). Source data are provided as a Source Data file.