Figure 4. vvl, kni and mld have a specific role in regulating ecdysone biosynthesis in the L3 stage.

(A) Expression of steroidogenic genes in ring glands from wild type (phm-GFP) larvae increases little from early (72 hours AEL) to mid (96 hours AEL) third instar, but rises dramatically in the late (120 hours AEL) third instar. vvl expression exhibits a minor increase in the late third instar, while mld, kni and especially Br-Z4 show a strong increase (n = 4). (B) Expression in the ring gland from larvae with knock down of vvl, kni or mld during the L3 stage two days after temperature induced activation of the RNAi with the Gal80^ts;phm> driver 96 hours AEL. Expression of all the steroidogenic genes were significantly reduced in animals with reduces vvl or kni expression. Knock down of mld results in a dramatic reduction in expression of nvd and spok that mediate two early steps in the biosynthesis of ecdysone (n = 4). (C) Quantified level of Phm protein in brain-ring gland complexes (BRGCs) from L3 larvae two days after temperature induced RNAi (96 hours AEL) normalized to Tubulin levels determined by immunoblotting (top panel) (n = 3). (D) Ecdysteroid levels determined by ELISA in L3 larvae with reduced PG expression of vvl, kni or mld two days after temperature induced activation of the RNAi effect (96 hours AEL) (n = 4). Error bars indicate s.e.m. *P<0.05, **P<0.01, ***P<0.001, versus the Gal80^ts;phm>+ control.