Fig. 4.

Upd3 secreted from the brain triggers a systemic signal. c–f, r–s indicate CC numbers in a single lymph gland. n.s: not significant (p > 0.01). *p < 0.01; **p < 0.001; ***p < 0.0001. Error bars in a–b, l–q: standard deviation. Bars in c–f, r–s: the median. Scale Bar: 50μm. CO₂SN inhibited conditions: –CO₂R with shaded yellow in e, m, o–r. Soda-lime generated low CO₂ conditions: shaded blue in b, f, s. a-b upd3 upregulation in the brain is linked to CC differentiation. Loss of Gr21a (Gr63a-gal4; UAS-Gr21a^RNAi) in the CO₂SN (a) or scavenging of CO₂ (b) results in increased upd3 mRNA in the brain. c-m. Upd3 functions downstream of Sima. upd3 in the brain causes increased CC formation (Elav-gal4; UAS-upd3) (c). Loss of neuronal upd3 (Elav-gal4; UAS-upd3^RNAi) alone does not alter CC number (d). Silencing neuronal upd3 suppresses the CC phenotype caused by inhibition of CO₂SN (Gr63a-LexA, LexAop-Shi^ts1; Elav-gal4, UAS-upd3^RNAi) (e) or by soda-lime treatment (Elav-gal4; UAS-upd3^RNAi) (f). Compared with control, upd3 is elevated in the posterior VNC of Gr63a¹/ + mutants (Upd3-gal4, UAS-GFP; Gr63a¹/ + ) (g). No overlap is observed between Sima⁺ and upd3⁺ neurons (h). HypSN seen in the posterior VNC are occasionally upd3-positive (white arrow heads), but the majority of upd3⁺ cells are not HypSNs (Upd3-gal4, UAS-GFP; Gyc89da-LexA; LexAop-mCherry) (i). Syb::GRASP expression of the HypSNs and Upd3⁺ neurons in the VNC (Magnified images in the following panels) (Upd3, red; GRASP, green) (j). Gyc89da-LexA alone (Gyc89da-LexA; UAS-CD4-spGFP₁₁, LexAop-nSyb-spGFP_1-10) or Upd3-gal4 alone (Upd3-gal4 UAS-mCherry; UAS-CD4-spGFP₁₁, LexAop-nSyb-spGFP_1-10) does not give rise to a GRASP signal (k). Overexpression of sima in HypSNs increases neuronal upd3 mRNA (Gyc89da-gal4; UAS-sima) (l). sima RNAi or inhibition of HypSNs suppresses the elevated upd3 levels (Gr63a-LexA, LexAop-Shi^ts1; Gyc89da-gal4, UAS-sima^RNAi or Gr63a-LexA, LexAop-Shi^ts1; Gyc89da-gal4, UAS-Shi^ts1) (m). n–s Brain-secreted Upd3 functions in the fat body. Socs36e is induced in the fat body upon loss of CO₂SN (Gr63a-LexA; LexAop-Shi^ts1) (n). This expression is suppressed upon: loss of neuronal upd3 (Gr63a-LexA, LexAop-Shi^ts1; Elav-gal4, UAS-upd3^RNAi) (o), by simultaneous inhibition of HypSNs (Gr63a-LexA, LexAop-Shi^ts1; Gyc89da-gal4, UAS-Shi^ts1) (p), by sima RNAi in the HypSNs (Gr63a-LexA, LexAop-Shi^ts1; Gyc89da-gal4, UAS-sima^RNAi) (p), or upon loss of dome in the fat body (Gr63a-LexA, LexAop-Shi^ts1; ppl-gal4, UAS-dome^RNAi) (q). dome RNAi in the fat body reverts the CC numbers in the CO₂SN mutant (Gr63a-LexA, LexAop-Shi^ts1; ppl-gal4, UAS-dome^RNAi) (r) or in soda-lime conditions (ppl-gal4; UAS-dome^RNAi) (s) to wild-type