Extended Data Fig. 2: Hemocyte responses until 24-h hypoxia or under various oxygen concentrations in the circulation.
From: Drosophila immune cells transport oxygen through PPO2 protein phase transition
a–b. Larvae were synchronized and reared in 21% O2 until moved hourly to 5% O2 over 1 to 24 h before dissection and observation at 120-h AEL. Quantification of (a) circulating Hml+ plasmatocytes (left) or (b) lz+crystal cells (right) in each oxygen condition. c–d. Hematopoiesis is not altered in 5% O2 up to 12 h. Total hemocyte numbers of larvae carrying HmlΔ-Gal4 UAS-EGFP; lz-LexA LexAop-mCherry exposed to 5% O2 for the indicated lengths of time up to 24 h. Quantification of total plasmatocytes (Hml+) (c) or crystal cells (lz+) (d) grown in each condition. e–f. Oxygen levels determine hemocyte dynamics between the hematopoietic pocket (sessile) and the haemolymph (circulation). Synchronized larvae carrying HmlΔ-Gal4 UAS-EGFP; lz-LexA LexAop-mCherry were reared in 21% O2 and shifted to 0.1% (anoxia) or 60% (hyperoxia) O2 for the indicated amounts of time, and the phenotypes were observed at 120-h AEL. (e) Schematics of anoxia experiments; a vertical grey bar indicates the time of the shift between oxygen conditions (top). Quantitation of circulating plasmatocytes (Hml+) (left) or crystal cells (lz+) (right) exposed to 0.1% O2 for the indicated numbers of hours. (f) Schematics of hyperoxia experiments; a vertical grey bar indicates the time of the shift between oxygen conditions (top). Quantification of circulating plasmatocytes (Hml+) (left) or crystal cells (lz+) (right) exposed to 60% O2 for the indicated hours. (g) Lateral view of segment A6 is magnified to highlight components of the hematopoietic pocket (neuron, green; trachea, magenta; oenocyte, blue). Schematic or confocal images of segment A6 labelled with btl-LexA LexAop-mCherry; 21-7-Gal4 UAS-mCD8::GFP (bottom). Reproduced with permission from ref. 10, Elsevier. h–i. Increased numbers of hemocytes were closely associated with PNS neurons or the trachea during hypoxia. (h) The proximity of hemocytes to tissues in the hematopoietic pocket marked by trachea-Hml+ plasmatocytes (btl-Gal4 UAS-GFP; HmlΔ-dsRed), trachea-lz+crystal cells (btl-Gal4 UAS-GFP; lz-LexA LexAop-mCherry), PNS neuron-Hml+ plasmatocytes (HmlΔ-LexA LexAop-mCherry; 21-7-Gal4 UAS-mCD8::GFP), or PNS neuron-lz+ crystal cells (21-7-Gal4 UAS-mCD8::GFP; lz-LexA LexAop-mCherry) in normoxia or 4-h hypoxia. (i) Quantification of the proximity index (a.u.) of the trachea and hemocytes (left) or PNS neurons and hemocytes (right). Scale bars: blue, 20 µm. n.s. (not significant), *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Extended Data Fig. 2a–f were analysed by one-way ANOVA followed by Tukey’s post hoc test. Box and whiskers plots in 2a–f and 2i denote maximum, 25%, median, 75%, and minimum values, respectively.