**3.3 EcR is required for** *Wingless* **repression**

A key signalling molecule in the morphogenesis of the wing is the Wingless (Wg) protein, a member of the Wnt family of secreted morphogens. Wg is secreted in a band across the dorsal-ventral (D/V) boundary in the wing pouch (Figure 5; (Williams, Paddock, and Carroll 1993)) and is essential for cell cycle arrest in a region of the wing disc called the "Zone of Non-Proliferating Cells", or ZNC, at the end of larval development. The Wg pathway acts to downregulate key cell cycle genes (eg. *dmyc*, *cycE*, *dE2F1* and *stg*) to link the Wg patterning signal to the cell cycle delay preceding the onset of differentiation at the wing margin (Johnston and Edgar 1998; Johnston et al. 1999; Johnston and Sanders 2003; Duman-Scheel, Johnston, and Du 2004). Indeed, the cell cycle arrest in the ZNC mediated by Wg is required for these cells to differentiate and develop into the adult wing blade (Figure 5; (Johnston and Edgar 1998; Johnston et al. 1999)).

In the wing pouch EcR signalling is required for repression of *wg* transcription (Mitchell et al. 2008; Cranna and Quinn 2009), which together with the data above showing EcR is required for cell division, suggests the ecdysone signal might normally control cell cycle via Wg (Figure 6). Consistent with EcR normally being required to repress *wg* transcription, expansion of the *wg* expression domain occurs in *UAS-EcRAdN* (Mitchell et al. 2008) and *UAS-EcRBdN* (Cranna and Quinn 2009) "flip-out" clones generated in a *wg-lacZ* enhancer trap background (Kassis et al. 1992). These results suggest repression of *wg* transcription in the wing pouch is dependent on the ecdysone pathway. Given that increased Wg protein causes reduction of cell cycle regulators such as *dmyc* and *stg*, leading to decreased cells in Sphase and mitosis in the pouch (Figure 5; (Johnston and Edgar 1998; Johnston et al. 1999)), this finding is consistent with the reduced cell cycles observed in *EcR* loss-of-function clones.

Steroid Hormones in *Drosophila*:

wing pouch, which is required to form the adult wing.

binding the *wg* promoter to down-regulate *wg* transcription.

**3.4 Ecdysone couples growth and division in larval histoblasts** 

How Ecdysone Coordinates Developmental Signalling with Cell Growth and Division 159

Cross-talk between the Wg pathway and other signalling pathways is required to coordinate proliferation and patterning of the wing imaginal disc. Dpp is expressed in a band of cells in the anterior compartment along the anterior-posterior boundary (Lecuit et al. 1996) and is required for cell cycle progression and tissue growth (Martin-Castellanos and Edgar 2002). Proliferation is dependent on careful regulation of the relative levels of the Dpp and Wg signalling pathways (Edgar and Lehner 1996). The Hedgehog (Hh) (Strigini and Cohen 2000) and Notch (N) (de Celis, Garcia-Bellido, and Bray 1996) pathways are key upstream regulators of Wg in the wing disc. Notch activity also plays a role in cell cycle arrest during wing development (Herranz et al. 2008; Johnston and Edgar 1998). Notch is activated in cells along the dorso-ventral (D/V) boundary (ZNC) of the wing disc, where it is required for Wg expression (de Celis, Garcia-Bellido, and Bray 1996). The activation of Wg target genes *achaete* (*ac*) and *scute* (*sc*) specifically within the anterior compartment of the cells flanking the D/V boundary results in downregulation of the mitotic inducer, Cdc25c/Stg, to arrest these cells in G2 (Johnston and Edgar 1998). The expression of Notch within the D/V boundary prevents the G2 arrest, allowing Wg to mediate G1 arrest within the anterior cells comprising the D/V boundary and all cells comprising the posterior compartment ZNC (Figure 5); (Johnston and Edgar 1998; Johnston et al. 1999). More recent reports have demonstrated that Notch also acts downstream of Wg to control G1 to S phase progression in the ZNC (Herranz et al. 2008). Together these studies suggest that a Wg and N "doublerepression mechanism" controls cell cycle exit in the ZNC through controlling levels of *dmyc*  expression, which drives growth and regulates the S phase transcription factor, E2F1 (Johnston and Edgar 1998; Johnston et al. 1999; Herranz et al. 2008). Thus, interplay between these signalling pathways is essential for cell cycle patterning and differentiation of the

The Hh pathway is critical for regulating *wg* transcription during wing development (Murone, Rosenthal, and de Sauvage 1999), but as ectopic levels of the Hh pathway activator, Ci, were not detected in *crol* mutant clones, Crol is unlikely to affect *wg* transcription indirectly via the Hh pathway (Mitchell et al. 2008). Notch is required for Wg expression (de Celis, Garcia-Bellido, and Bray 1996) and plays a critical role in cell cycle arrest during wing development (Herranz et al. 2008; Johnston and Edgar 1998). The Notch target, En(spl)m7 was not however decreased in *crol* over-expressing cells, suggesting Notch signalling is not downregulated by Crol (Mitchell et al. 2008). The effects of Crol on cell cycle in the wing via downregulation of *wg* transcription are therefore unlikely to be due to indirect effects on either the Notch or Hh pathways. Future studies are therefore aimed to determine whether Crol mediates ecdysone signalling via repression of Wg by directly

Another *Drosophila* tissue where ecdysone has been connected with control of growth and/or cell division is the developing histoblast, which gives rise to the abdominal epithelium (Ninov, Chiarelli, and Martin-Blanco 2007). In the canonical cell division cycles of the eye and wing imaginal discs DNA synthesis is coupled with cell division; cells grow in G1, initiate DNA replication and enter S phase, which is separated from mitosis by the G2 phase. In these cells G1 progression is stimulated by growth factors, which trigger cell growth and activate the G1-S cell cycle machinery (see Introduction 1.4), including the cyclin/Cdk complexes and E2F activity. The progression from G2 to mitosis is coupled to S

Fig. 7. **S phase progression in UAS-EcRAdN clones is sensitive to the dose of crol.** (A,B) Representative images of the wing pouch with control clones in heterozygous crol mutant (crolk05205) background; (C,D) UAS-EcRdN-A clones and (E,F) UAS-EcRdN-A clones in heterozygous crol mutant background, (A, C, E) S-phase is shown using BrdU (red), (B, D, F) GFP (green) marks clonal tissue. Scale bars indicate 50μm. (G) Quantification of S-phases for each of the genotypes; heterozygous *crol* mutant, UAS-EcRAdN alone and EcRAdN in the heterozygous *crol* mutant background. A significant reduction in the number of S-phase cells was found for the UAS-EcRdN-A alone compared to the control (p=0.0055) and for the UAS-EcRdN-A in the *crol* mutant background compared to UAS-EcRdN-A alone (p=0.0011). (H) Mean number of BrdU (S-phase) cells + SEM in control (clones in tissue heterozygous for the crol mutant); UAS-EcRdN-A alone and UAS-EcRdN-A in the *crol* mutant background. n=sample size.

Together this data suggests that EcR activity and the ecdysone-responsive transcription factor Crol are required for cell cycle progression in the wing imaginal disc (Mitchell et al. 2008). First Crol affects the Wg pathway by downregulating *wg* transcription and driving cells through the Wg-mediated cell cycle arrest (Mitchell et al. 2008). In support of ecdysone acting upstream of Crol to regulate the Wg pathway, blocking EcR activity in the wing results in increased *wg* transcription and reduced cell cycle progression, which is further impaired by halving the dose of *crol* (Figure 7). As Wg is one of the key developmental signals required for inhibition of cell cycle progression in the wing pouch (Duman-Scheel, Johnston, and Du 2004; Johnston and Edgar 1998; Johnston et al. 1999; Johnston and Sanders 2003; Milan 1998), this would be consistent with EcR regulating cell cycle by acting to increase levels of *crol* transcription, which will in turn decrease levels of Wg signalling. Thus we would predict that ecdysone/EcR/USP would normally act to upregulate Crol and drive cell cycle progression in the wing pouch via inhibition of Wg (Figure 6).

Fig. 7. **S phase progression in UAS-EcRAdN clones is sensitive to the dose of crol.** (A,B) Representative images of the wing pouch with control clones in heterozygous crol mutant (crolk05205) background; (C,D) UAS-EcRdN-A clones and (E,F) UAS-EcRdN-A clones in heterozygous crol mutant background, (A, C, E) S-phase is shown using BrdU (red), (B, D, F) GFP (green) marks clonal tissue. Scale bars indicate 50μm. (G) Quantification of S-phases for each of the genotypes; heterozygous *crol* mutant, UAS-EcRAdN alone and EcRAdN in the heterozygous *crol* mutant background. A significant reduction in the number of S-phase cells was found for the UAS-EcRdN-A alone compared to the control (p=0.0055) and for the UAS-EcRdN-A in the *crol* mutant background compared to UAS-EcRdN-A alone (p=0.0011). (H) Mean number of BrdU (S-phase) cells + SEM in control (clones in tissue heterozygous

for the crol mutant); UAS-EcRdN-A alone and UAS-EcRdN-A in the *crol* mutant

cell cycle progression in the wing pouch via inhibition of Wg (Figure 6).

Together this data suggests that EcR activity and the ecdysone-responsive transcription factor Crol are required for cell cycle progression in the wing imaginal disc (Mitchell et al. 2008). First Crol affects the Wg pathway by downregulating *wg* transcription and driving cells through the Wg-mediated cell cycle arrest (Mitchell et al. 2008). In support of ecdysone acting upstream of Crol to regulate the Wg pathway, blocking EcR activity in the wing results in increased *wg* transcription and reduced cell cycle progression, which is further impaired by halving the dose of *crol* (Figure 7). As Wg is one of the key developmental signals required for inhibition of cell cycle progression in the wing pouch (Duman-Scheel, Johnston, and Du 2004; Johnston and Edgar 1998; Johnston et al. 1999; Johnston and Sanders 2003; Milan 1998), this would be consistent with EcR regulating cell cycle by acting to increase levels of *crol* transcription, which will in turn decrease levels of Wg signalling. Thus we would predict that ecdysone/EcR/USP would normally act to upregulate Crol and drive

background. n=sample size.

Cross-talk between the Wg pathway and other signalling pathways is required to coordinate proliferation and patterning of the wing imaginal disc. Dpp is expressed in a band of cells in the anterior compartment along the anterior-posterior boundary (Lecuit et al. 1996) and is required for cell cycle progression and tissue growth (Martin-Castellanos and Edgar 2002). Proliferation is dependent on careful regulation of the relative levels of the Dpp and Wg signalling pathways (Edgar and Lehner 1996). The Hedgehog (Hh) (Strigini and Cohen 2000) and Notch (N) (de Celis, Garcia-Bellido, and Bray 1996) pathways are key upstream regulators of Wg in the wing disc. Notch activity also plays a role in cell cycle arrest during wing development (Herranz et al. 2008; Johnston and Edgar 1998). Notch is activated in cells along the dorso-ventral (D/V) boundary (ZNC) of the wing disc, where it is required for Wg expression (de Celis, Garcia-Bellido, and Bray 1996). The activation of Wg target genes *achaete* (*ac*) and *scute* (*sc*) specifically within the anterior compartment of the cells flanking the D/V boundary results in downregulation of the mitotic inducer, Cdc25c/Stg, to arrest these cells in G2 (Johnston and Edgar 1998). The expression of Notch within the D/V boundary prevents the G2 arrest, allowing Wg to mediate G1 arrest within the anterior cells comprising the D/V boundary and all cells comprising the posterior compartment ZNC (Figure 5); (Johnston and Edgar 1998; Johnston et al. 1999). More recent reports have demonstrated that Notch also acts downstream of Wg to control G1 to S phase progression in the ZNC (Herranz et al. 2008). Together these studies suggest that a Wg and N "doublerepression mechanism" controls cell cycle exit in the ZNC through controlling levels of *dmyc*  expression, which drives growth and regulates the S phase transcription factor, E2F1 (Johnston and Edgar 1998; Johnston et al. 1999; Herranz et al. 2008). Thus, interplay between these signalling pathways is essential for cell cycle patterning and differentiation of the wing pouch, which is required to form the adult wing.

The Hh pathway is critical for regulating *wg* transcription during wing development (Murone, Rosenthal, and de Sauvage 1999), but as ectopic levels of the Hh pathway activator, Ci, were not detected in *crol* mutant clones, Crol is unlikely to affect *wg* transcription indirectly via the Hh pathway (Mitchell et al. 2008). Notch is required for Wg expression (de Celis, Garcia-Bellido, and Bray 1996) and plays a critical role in cell cycle arrest during wing development (Herranz et al. 2008; Johnston and Edgar 1998). The Notch target, En(spl)m7 was not however decreased in *crol* over-expressing cells, suggesting Notch signalling is not downregulated by Crol (Mitchell et al. 2008). The effects of Crol on cell cycle in the wing via downregulation of *wg* transcription are therefore unlikely to be due to indirect effects on either the Notch or Hh pathways. Future studies are therefore aimed to determine whether Crol mediates ecdysone signalling via repression of Wg by directly binding the *wg* promoter to down-regulate *wg* transcription.
