The Ecdysone receptor constrains wingless expression to pattern cell cycle across the Drosophila wing margin in a Cyclin B-dependent manner

Abstract

Background: Ecdysone triggers transcriptional changes via the ecdysone receptor (EcR) to coordinate developmental programs of apoptosis, cell cycle and differentiation. Data suggests EcR affects cell cycle gene expression indirectly and here we identify Wingless as an intermediary factor linking EcR to cell cycle.

Results: We demonstrate EcR patterns cell cycle across the presumptive Drosophila wing margin by constraining wg transcription to modulate CycB expression, but not the previously identified Wg-targets dMyc or Stg. Furthermore co-knockdown of Wg restores CycB patterning in EcR knockdown clones. Wg is not a direct target of EcR, rather we demonstrate that repression of Wg by EcR is likely mediated by direct interaction between the EcR-responsive zinc finger transcription factor Crol and the wg promoter.

Conclusions: Thus we elucidate a critical mechanism potentially connecting ecdysone with patterning signals to ensure correct timing of cell cycle exit and differentiation during margin wing development.

Figure 1

Patterning cell cycle across the wing imaginal disc. (A) - Schematic of the 3rd instar wing disc. The red and blue region develops to form the notum and hinge while the purple region forms the wing blade. The green line marks the anterior-posterior (A/P) boundary while the red line defines the dorsal-ventral (D/V) boundary. (B) - Across the margin Notch (N) expression triggers the activation of Wingless (Wg). In the margin Wg induces G1 delay via repression of dE2F1 activity in a narrow domain within the anterior compartment and also in a broader region of the posterior compartment. In the anterior compartment, Wg also induces G2 delay in the cells flanking the G1 band by down regulating expression of stg.

Figure 2

EcR is required for normal patterning of E2F1 activity in the wing margin. (A-D) - PCNA-GFP/+ wing imaginal discs stained with the EcR antibody. PCNA-GFP (A) is normally detected in cycling cells of the wing pouch and in the G1 band within the anterior and posterior of the wing margin. EcR staining in red (B) overlaps with PCNA-GFP throughout the pouch and the G1 cells of the margin (C and D, DNA in blue). (E-H) - EcR RNAi flip out clones generated in the PCNA-GFP background are marked with CD8-RFP. White arrows mark ectopic G1 cells in the G2 region. The DNA stain in H is for the equivalent confocal section and white arrows show the position of ectopic PCNA-GFP corresponding to clones in the anterior, while the red arrow corresponds to the clone with reduced PCNA-GFP in the posterior.

Figure 3

EcR pathway is required for Wg repression. (A-C) - EcR antibody staining (green) in the wg-lacZ background, detected with β-gal (red). (D-F) - wg-lacZ activity (red) in EcR RNAi clones marked with GFP. (G) - Control pattern of Wg antibody staining (red). (G-I) - shows wg RNAi and EcR RNAi double knockdown (marked with CD8-RFP) with PCNA-GFP patterning across the margin, while (J-L) shows a second example of wg RNAi and EcR RNAi co-knockdown focusing on the wing margin.

Figure 4

EcR is essential for CycB expression at the wing margin. (A-D) - Pattern of overlap between the Cyclin B-GFP protein trap (CycB-PT) in green (A) and anti-EcR antibody in red (B). The merge is shown in (C) and overlap with DNA in (D). (E-H) - EcR RNAi clones marked with β-gal (red) with CycB-PT (green). (E) - disruption to the Cyc B pattern across the margin marked with white arrows corresponds to the EcR RNAi clones shown with β-gal in F. The merge is shown in G and the presence of cells across the margin lacking CycB shown with DNA in H. (I-L) - Disruption to both CycB-PT activity in green (I) and the G2-marker Geminin in purple (J) for large EcR RNAi clones detected with β-gal in red (K). In (I-K) - the position of two sets of non-EcR knockdown cells is show across the margin to highlight non-autonomous affects of EcR knockdown. The presence of cells across the margin shown with DNA in (L).

Figure 5

Overexpression of CycB or knockdown of Wg partially restores cell cycle patterning in EcR RNAi clones. (A-D) - UAS-CycB clones (marked with CD8-RFP in A) do not disrupt PCNA-GFP patterning across the anterior margin (B). The merge is shown in (C) and overlap with DNA in D. (E-H) - Overexpression of CycB partially restores PCNA-GFP patterning (F, see arrow for remaining G1 cells) in EcR RNAi clones marked with CD8-RFP in (E). The merge is shown in (G) and overlap with DNA in (H). (I-L)- wg RNAi clones marked with CD8-RFP (I) disrupt CycB-PT patterning (J, see arrow). The merge is shown in (K) and overlap with DNA in (L). (M-P) - The double wg RNAi and EcR RNAi clones marked with CD8-RFP (M) result in restoration of CycB-PT patterning (N). The merge is shown in (O) and overlap with DNA in (P).

Figure 6

Crol restores repression of wg expression in EcR knockdown. (A-B) - Crol-GFP protein trap activity (A) is reduced in EcR RNAi clones marked with β-gal in red for the merge (B). (C-D) - wg-lacZ activity marked with β-gal in red (C) is reduced in UAS-crol clones marked with GFP in the merge (D). (E-F) - UAS-crol restores repression of wg-lacZ activity in the EcR RNAi clones.

Figure 7

Crol is enriched across consensus zinc finger binding sites in the wg promoter. (A) - 6 overlapping primer sets spanning 5 kb of the wg promoter, wg transcription start site +1. (B) - region of wg promoter containing overlapping amplicons for wg (Wg primers 1–6) used for ChIP with anti-Crol or Ci, the boxed region highlights amplicons positive for Crol enrichment (see gel in C) and the dashed grey line shows the region of the wg promoter containing the zinc finger and Ci consensus sites shown in 7B. (B) - Position of primers for ChIP-qPCR (Wg2.1-Wg2.4) are marked with arrows. The in vitro defined Ci binding site and consensus zinc finger binding sites are boxed and labelled. (C) – DNA gel electrophoresis of ChIP-PCR across the Wg promoter using Wg primers (as marked in 7) for Input DNA; Ci ChIP or Crol ChIP as marked. (D-E) Fine mapping of Crol and Ci enrichment using ChIP-qPCR for small amplicons - Wg2.1-2.4 shown in (B). (D) - ChIP for Crol - percentage enrichment and error for percentage enrichment for Crol are as follows; Wg 2.1 (0.28 ± 0.0045); Wg 2.2 (0.453 ± 0.0083); Wg 2.3 (0.055 ± 0.0082) and Wg 2.4 (0.00116 ± 0.0095). (E) - ChIP for Ci - percentage enrichment and Error for percentage enrichment for Ci are as follows; Wg 2.1 (0.482 ± 0.0066); Wg 2.2 (1.031 ± 0.0141); Wg 2.3 (0.24 ± 0.0103) and Wg 2.4 (0.137 ± 0.019). (F) - Model for regulation of cell cycle patterning across the anterior wing margin by EcR.