Figure 3

Imp-L2 binds Dilp2 and counteracts its activity. (a,b) Antibody staining of larval brains with an Imp-L2 antibody (green). (a) Specific neurons of both brain hemispheres, the subesophageal ganglion region (gray arrow) and the corpora cardiaca (white arrow) express Imp-L2 protein. The corpora allata are innervated by Imp-L2 expressing axons. White arrowheads mark the Dilp-producing m-NSCs. (b) In larvae carrying a dilp2-lacZ.nls transgene, co-staining with β-galactosidase and Imp-L2 antibodies reveals that the seven dilp-expressing m-NSCs also produce low levels of Imp-L2. (c) The size increase of arm-Gal4, UAS-dilp2 flies is dominantly enhanced by reducing Imp-L2 levels. In an Imp-L2^-/- background, dilp2 overexpression results in lethality, which can be rescued by a copy of the Imp-L2 genomic rescue construct (GR). (d) Overexpression of dilp2 as well as of Imp-L2 at high levels by ppl-Gal4 causes lethality, whereas concomitant overexpression of dilp2 and Imp-L2 yields flies of wild-type size. The lacZ transgene was introduced to rule out a dosage effect of the UAS/Gal4-system. (e) Imp-L2 binds Dilp2. In-vitro-translated, ³⁵S-labeled wild-type (ImpL2-IVT, about 32 kDa) or mutant (ImpL2^MG2-IVT, about 30 kDa) Imp-L2 (lane 1) was incubated with cell lysates of either non-transfected (lane 3) or stably transfected S2 cells expressing Flag-Dilp2 (lane 2). Imp-L2 could only be pulled down in the presence of Dilp2. The Imp-L2^MG2 mutation abolished Dilp2 binding. Genotypes in (c): 'Imp-L2^+/-' Imp-L2^Def42/+; 'Imp-L2^-/-' Imp-L2^Def42/Imp-L2^Def20; 'Imp-L2^-/-, GR' Imp-L2^Def42/Imp-L2^Def20, GR-57; 'control' (black bar) arm-Gal4, UAS-GFP. P-values were determined using unpaired Student's t-test (n = 40, except for bars 1–3 in (c): bar 1, n = 31; bars 2 and 3, n = 17). Error bars represent s.d.