Extended Data Fig. 4: Identification of genes involved in crystal cell function.

(a) Plasmatocyte-specific knockdown of u-shaped (ush) or string (stg) decreased plasmatocyte numbers. An RNAi-based screen targeted the following plasmatocyte proliferation genes: ush (Hml^Δ-Gal4 UAS-EGFP; lz-LexA LexAop-mCherry, UAS-ush RNAi (BL32950, BL44041) or UAS-stg RNAi (BL34831)). Quantification of total Hml⁺ plasmatocytes (left) or lz⁺ crystal cells (right) in each genetic background. b–c. Decreased plasmatocyte numbers did not alter crystal cell localization at 5% O₂. (b) Quantification of circulating Hml⁺ plasmatocytes (left) or lz⁺ crystal cells (right) in Hml^Δ-Gal4, UAS-ush RNAi (BL44041). (c) The numbers and localization patterns of hemocytes (Hml⁺ plasmatocytes, green; lz⁺ crystal cells, magenta) in controls (Hml^Δ-Gal4 UAS-EGFP; lz-LexA LexAop-mCherry) or in ush RNAi (Hml^Δ-Gal4, UAS-EGFP UAS-ush RNAi; lz-LexA LexAop-mCherry) animals. d–e. Decreased plasmatocyte numbers did not mimic hypoxic animal phenotypes. (d) Expression of RNAi against ush (BL44041) or stg (BL34831) did not alter pupal volume. (e) These genetic backgrounds did not increase the TTB numbers. Quantification of TTB numbers is presented in Supplementary Tables 1 and 2. f–i. An RNAi-based screen targeted the top 20 crystal-cell-specific genes: PPO1, fok, CG10467, Men, CG15343, Pde1c, CG9119, CG7860, CG10469, mthl10, Gip, CG17109, Naxd, peb, tna (lz-Gal4 UAS-EGFP, UAS-PPO1 RNAi, UAS-fok RNAi, UAS-CG10467 RNAi, UAS-Men RNAi, UAS-CG15343 RNAi, UAS-Pde1c RNAi, UAS-CG9119 RNAi, UAS-CG7860 RNAi, UAS-CG10469 RNAi, UAS-mthl10 RNAi, UAS-Gip RNAi, UAS-CG17109 RNAi, UAS-Naxd RNAi, UAS-peb RNAi, and UAS-tna RNAi). Quantification of circulating Pxn⁺ plasmatocytes (f) or lz⁺ crystal cells (h) in each condition and genetic background. Red dotted bars indicate genes, including those of unknown function (fok and CG10467), that disrupted hemocyte movement to the hematopoietic pocket at 4 h in 5% O₂. PPO1^Δ or PPO2^Δ mutants recapitulated the PPO1 or PPO2 RNAi phenotypes. Quantification of circulating Pxn⁺ plasmatocytes (g) or lz⁺ crystal cells (i) in each condition and genetic background. j–o. RNA interference (RNAi) efficiency of Prophenoloxidase 2 (PPO2, Hml^{LineageTracing(LT)}-Gal4 UAS-PPO2 RNAi) (j), Carbonic anhydrase 2 (CAH2, Hml^Δ-Gal4 UAS-CAH2 RNAi) (k), Metallothionein A (MtnA, Hml^LT-Gal4 UAS-MtnA RNAi) (l), Antioxidant 1 copper chaperone (Atox1, Hml^LT-Gal4 UAS-Atox1 RNAi) (m), Copper transporter 1 A (Ctr1A, Hml^LT-Gal4 UAS-Ctr1A RNAi) (n), or Prophenoloxidase 1 (PPO1, Hml^LT-Gal4 UAS-PPO1 RNAi) (o). White bars indicate 21% O₂, and green or magenta bars indicate 5% O₂. Scale bar: black, 500 µm; white, 50 µm. n.s (not significant), *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Extended Data Fig. 4a, d, f, g, h, and i were analysed using the Mann–Whitney test. Extended Data Fig. 4b was analysed by one-way ANOVA followed by Tukey’s post hoc test. Extended Data Fig. 4j–o were analysed by Student’s t test. Box and whiskers plots in 4a, 4b, 4d, and 4f–i denote maximum, 25%, median, 75%, and minimum values, respectively. Error bars in 4j–o represent standard deviations (SDs).

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