The ecdysone receptor controls the post-critical weight switch to nutrition-independent differentiation in Drosophila wing imaginal discs

Abstract

In holometabolous insects, a species-specific size, known as critical weight, needs to be reached for metamorphosis to be initiated in the absence of further nutritional input. Previously, we found that reaching critical weight depends on the insulin-dependent growth of the prothoracic glands (PGs) in Drosophila larvae. Because the PGs produce the molting hormone ecdysone, we hypothesized that ecdysone signaling switches the larva to a nutrition-independent mode of development post-critical weight. Wing discs from pre-critical weight larvae [5 hours after third instar ecdysis (AL3E)] fed on sucrose alone showed suppressed Wingless (WG), Cut (CT) and Senseless (SENS) expression. Post-critical weight, a sucrose-only diet no longer suppressed the expression of these proteins. Feeding larvae that exhibit enhanced insulin signaling in their PGs at 5 hours AL3E on sucrose alone produced wing discs with precocious WG, CT and SENS expression. In addition, knocking down the Ecdysone receptor (EcR) selectively in the discs also promoted premature WG, CUT and SENS expression in the wing discs of sucrose-fed pre-critical weight larvae. EcR is involved in gene activation when ecdysone is present, and gene repression in its absence. Thus, knocking down EcR derepresses genes that are normally repressed by unliganded EcR, thereby allowing wing patterning to progress. In addition, knocking down EcR in the wing discs caused precocious expression of the ecdysone-responsive gene broad. These results suggest that post-critical weight, EcR signaling switches wing discs to a nutrition-independent mode of development via derepression.

Fig. 1.

The development of the Wingless, Cut and Senseless expression patterns in wing discs of early third instar w¹¹¹⁸Drosophila larvae. Expression of (A-H) Wingless (WG), (I-P) Cut (CT) and (Q-X) Senseless (SENS) in wing discs at 0 (A,I,Q), 5 (B,J,R), 10 (C,K,S), 15 (D,L,T), 20 (E,M,U), 25 (F,N,V) 30 (G,O,W) and 35 (H,P,X) hours (h) after third instar ecdysis (AL3E). In C, the arrow points to the ring of WG expression around the wing pouch. The asterisks in U-X mark the precursor of the wing hinge chordotonal organ, and the small arrowheads indicate sensory bristle precursors. The double arrowhead in W and X points to the sensory bristle precursors in the triple row of the wing margin. Scale bar: 100 μm.

Fig. 2.

Patterning in the imaginal discs differs in pre- and post-critical weight w¹¹¹⁸ larvae fed on sucrose alone. The expression of (A,D,G,J,M,P) WG, (B,E,H,K,N,Q) SENS and (C,F,I,L,O,R) CT in w¹¹¹⁸ larvae. Wing discs are from (A-C) larvae at 5 hours (h) after third instar ecdysis (AL3E), (D-F) larvae fed on sucrose alone between 5 and 30 hours AL3E, (G-I) larvae at 30 hours AL3E, (J,K) larvae at 14 hours AL3E, (L) larvae dissected at 15 hours AL3E, (M,N) larvae fed on sucrose alone from 14-38 hours AL3E, (O) larvae fed on sucrose alone from 15-39 hours AL3E, (P,Q) larvae staged to 38 hours AL3E, and (R) larvae at 39 hours AL3E. The asterisks in H, N and Q are the sensory organ precursors (SOPs) of the wing hinge chordotonal organ. The double arrowheads in H, N and Q mark the SOPs in the triple row at the wing margin. Other SOPs in the wing pouch and notum are marked by arrowheads (H,N,Q). In R, the arrows mark CT expression in some of the SOPs of the notum and wing pouch. Scale bar: 100 μm.

Fig. 3.

Enhancing insulin signaling in the ring gland, using the P0206 ring gland GAL4 driver, changes the developmental response of wing discs to starvation. Wing discs from (A-I) P0206>GFP control larvae and (J-R) P0206>InR larvae. Wing discs are from (A-C,J-L) larvae staged to 5 hours (h) after third instar ecdysis (AL3E), (D-F,M-O) larvae fed on sucrose alone from 5-29 hours AL3E, and (G-I,P-R) larvae at 29 hours AL3E. Shown is expression of (A,D,G,J,M,P) WG, (B,E,H,K,N,Q) CT and (C,F,I,L,O,R) SENS. The asterisks in I, O and R mark the SOPs of the wing chordotonal organ. The double arrowheads (I,O,R) point to the SOPs of the triple row. SOPs in the wing pouch and notum are indicated with arrowheads (I,O,R). Scale bar: 100 μm.

Fig. 4.

Suppressing ecdysone receptor expression in the wing discs of starved pre-critical weight larvae promotes premature differentiation of the Wingless and Senseless expression patterns. Wing discs (A-H) dissected at 5 hours AL3E, (I-P) from larvae fed on sucrose alone from 5-29 hours AL3E, and (Q-X) from larvae fed on standard food and dissected at 29 hours AL3E. Four different genotypes were examined: C765 (A,B,I,J,Q,R), C765>EcR^DN (C,D,K,L,S,T), C765>EcRi (E,F,M,N,U,V) and MS1096-EcRi (G,H,O,P,W,X). Shown is expression of WG and SENS as labeled. The SOPs of the triple row are marked by double arrowheads (N,P,R,T,V,X). Asterisks mark the SOPs of the hinge chordotonal organ (N,P,R,T,V,X). Arrowheads indicate the SOPs of the wing pouch and notum (N,P,R,T,V,X). (P′) This inset shows the expression of SENS in a single row of sensory organ precursors at the margin. Scale bar: 100 μm.

Fig. 5.

Larvae in which EcR is suppressed in the wing discs reach critical weight at normal times. (A) The growth rate over time of C765 and C765>EcRi larvae. (B) The time to pupariation in C765 and C765>EcRi larvae of different size classes when either fed on standard food or starved on 2% agar. Error bars represent s.e.m.

Fig. 6.

The ecdysone response gene broad is upregulated in discs of w¹¹¹⁸ larvae post-critical weight. (A,D) Broad (BR) was expressed at low levels throughout the wing discs at 5 and 15 hours (h) after third instar ecdysis (AL3E). (B) BR levels remained low when larvae were fed on sucrose alone from 5-30 hours AL3E. (E) Larvae fed on sucrose alone from 15-39 hours AL3E, or (C,F) larvae staged to 30 and 39 hours AL3E, expressed BR at considerably higher levels than at 5 or 15 hours AL3E. All images were scanned at the same laser power. Scale bar: 100 μm.

Fig. 7.

The regulation of Broad in discs with suppressed EcR signaling. (A-L) Expression of Broad in wing imaginal discs from C765 (A-C), C765>EcRi (D-F), C765>EcR^DN (G-I), and MS1096>EcRi (J-L) larvae. (M-O) GFP expression pattern driven by the MS1096 GAL4 driver. Discs are from (A,D,G,J,M) larvae staged to 5 hours (h) after third instar ecdysis (AL3E), (B,E,H,K,N) larvae fed on sucrose alone from 5-29 hours AL3E, and (C,F,I,L,O) larvae at 29 hours AL3E. Images A-L were scanned at the same laser power. Scale bar: 100 μm.