**3.2 The ecdysone pathway regulates cell cycle progression in the larval wing disc**

Like the eye disc, the larval wing disc is also comprised of an epithelial sheet, which can be divided into distinct domains based on cell fate in the adult wing; the notum, hinge and pouch (Figure 5). The wing pouch, which ultimately forms the adult wing blade, has been a focus for studying signals impacting upon cell cycle, as wing morphogenesis involves patterned cell cycles that are tightly linked with developmental signalling (Johnson, Grenier, and Scott 1995; Johnston and Edgar 1998; Johnston et al. 1999; Johnston and Sanders 2003; Baker 2007).

Early studies demonstrated that Crol*,* which is a zinc finger transcription factor, is activated in late larval imaginal discs by the steroid hormone ecdysone (D'Avino and Thummel 1998). Pupal lethal, hypomorphic *crol* mutants (*crol4418*) have defects in ecdysone-induced gene expression (D'Avino and Thummel 1998). Crol is both necessary and sufficient for cell cycle progression in the wing imaginal disc as *crol* mutant clones in the wing pouch fail to proliferate, whilst overexpression of *crol* results in ectopic proliferation (Mitchell et al. 2008). Crol is also required to downregulate the Wingless (Wg) pathway, which normally acts to drive cell cycle exit and differentiation (Figure 5). Therefore, by inhibiting the Wg pathway, *crol* drives wing disc cell division and potentially provides a link between the ecdysone pathway and the developmental signals that regulate cell cycle patterning (discussed in more detail section 3.3; Figure 6).

could occur via heterodimerisation of USP with one of the 16 orphan nuclear receptors identified in *Drosophila* (Sullivan and Thummel 2003). For example USP has been found to heterodimerize with the orphan nuclear receptor, DHR38, to regulate cuticle formation (Kozlova et al. 1998; Sutherland et al. 1995). The USP/DHR38 complex responds to a different class of ecdysteroids in larval fat body and epidermis in an EcR independent manner, which does not involve direct binding of the ecdysone ligand to either DHR38 or USP (Baker et al. 2003). However, as *DHR38* expression does not appear to be induced by ecdysteroids in the larval eye (Baker et al. 2003), it is unlikely that DHR38 partners USP during eye development. We believe it is premature to rule out a function for EcR in MF progression as the absence of a furrow progression phenotype reported (Brennan et al. 2001) may be a consequence of perdurance of EcR protein after clone induction. As studies using dominant negative EcR transgenes have shown that EcR is required for normal signalling and cell cycle progression in the wing (discussed in section 3.2; (Mitchell et al. 2008; Cranna and Quinn 2009)), similar methods should be used to inhibit EcR activity before any definitive conclusions about whether EcR is required for eye proliferation can be made. Together the evidence suggests that larval ecdysone signalling is essential for cell cycle progression in the eye imaginal disc. The effect of ecdysone on cell division may, in part, be mediated by increasing Hedgehog (Hh) signalling (Brennan, Ashburner, and Moses 1998) posterior to the MF to drive S-phase gene activity and cell cycle progression in the second mitotic wave (SMW) (Duman-Scheel et al. 2002). In addition to this cell cycle promoting role of ecdysone via Hh activity in the SMW, the shift in the Dpp band of expression in *usp*clones suggests the ecdysone pathway might also act on Dpp to coordinate G1 arrest in the furrow with division in the SMW (Escudero and Freeman 2007; Firth and Baker 2005). Further work is required to understand how ecdysone might coordinate these developmental signals with the G1 arrest/MF formation and stimulation of the SMW

**3.2 The ecdysone pathway regulates cell cycle progression in the larval wing disc**  Like the eye disc, the larval wing disc is also comprised of an epithelial sheet, which can be divided into distinct domains based on cell fate in the adult wing; the notum, hinge and pouch (Figure 5). The wing pouch, which ultimately forms the adult wing blade, has been a focus for studying signals impacting upon cell cycle, as wing morphogenesis involves patterned cell cycles that are tightly linked with developmental signalling (Johnson, Grenier, and Scott 1995; Johnston and Edgar 1998; Johnston et al. 1999; Johnston and Sanders 2003;

Early studies demonstrated that Crol*,* which is a zinc finger transcription factor, is activated in late larval imaginal discs by the steroid hormone ecdysone (D'Avino and Thummel 1998). Pupal lethal, hypomorphic *crol* mutants (*crol4418*) have defects in ecdysone-induced gene expression (D'Avino and Thummel 1998). Crol is both necessary and sufficient for cell cycle progression in the wing imaginal disc as *crol* mutant clones in the wing pouch fail to proliferate, whilst overexpression of *crol* results in ectopic proliferation (Mitchell et al. 2008). Crol is also required to downregulate the Wingless (Wg) pathway, which normally acts to drive cell cycle exit and differentiation (Figure 5). Therefore, by inhibiting the Wg pathway, *crol* drives wing disc cell division and potentially provides a link between the ecdysone pathway and the developmental signals that regulate cell cycle patterning (discussed in

required for eye development (Figure 4).

more detail section 3.3; Figure 6).

Baker 2007).

Fig. 5. **(A) -** *Drosophila* **wing imaginal disc patterning.** (A) The orange domain forms the notum, the blue region gives the hinge and the purple region (the pouch) 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) Within D/V boundary of the pouch, Notch (N) expression activates Wingless (Wg) in the central domain. In the anterior compartment, *Wg*  induces G2 arrest via *string* (*stg)* through *Achaete* (*ac)* and *Scute* (*sc)*. In the posterior compartment, *Wg* induces G1 arrest via repression of dE2F. (**B) - Wg protein,** *dmyc* **expression and cell cycle patterning in the** *Drosophila* **wing pouch.** (A) Wg protein (red) is strongly expressed along the dorsal-ventral boundary of the wing pouch. (B) β-gal antibody staining (pink) of *dmyc-lacZ* discs shows a pattern consistent with *dmyc* transcription throughout the cycling cells of the pouch and downregulation of *dmyc* within the G1 arrested cells of the zone of non-proliferating cells (ZNC). (C) The ZNC can be seen by the reduced BrdU staining (red) for S-phase.

Steroid Hormones in *Drosophila*:

(Cranna and Quinn 2009).

regulates cell cycle in a Crol-dependent manner.

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

(Johnston and Edgar 1998; Johnston et al. 1999)).

clones.

How Ecdysone Coordinates Developmental Signalling with Cell Growth and Division 157

the *EcRE*, but cannot bind ecdysone, thus preventing optimal activation of ecdysone responsive genes (Cherbas et al. 2003; Hu, Cherbas, and Cherbas 2003). Blocking the EcR signal via overexpression of either *EcRAdN* or *EcRB2dN* in third instar wing imaginal disc flip-out clones (Pignoni and Zipursky 1997) results in a significant decrease in S-phase progression and mitosis, as measured by BrdU incorporation (Figure 7) and staining for anti-phosphohistone-H3, respectively (Cranna and Quinn 2009; Mitchell et al. 2008). Consistent with ecdysone signalling through EcR/USP normally being required for *dmyc*  transcription, reduced *dmyc* promoter activity was observed in *EcRAdN* clones generated in the *dmyc-lacZ* enhancer trap background (Cranna and Quinn 2009). Thus ecdysone signalling through EcR/USP might normally control cell cycle progression in the wing imaginal disc by upregulating dMyc to drive growth by increasing ribosome biogenesis and protein translation (Johnston et al. 1999) and S phase via increased activity of the G1 cyclins (Duman-Scheel, Johnston, and Du 2004). Together this suggests EcR signalling might modulate cell growth and division of the wing imaginal disc by modulating *dmyc* levels

In support of the reduced cell division in loss-of-function EcR cells being mediated by Crol, EcRAdN clones generated in the heterozygous *crol* mutant background show a further, significant reduction in cell cycle progression, when compared with either EcRdN cells alone or *crol* heterozygotes (Figure 7). This suggests that the reduction in cell cycle resulting from loss of EcR is sensitive to the level of Crol and that the ecdysone pathway normally

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;

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

Fig. 6. **Working model connecting Crol to steroid hormone signalling and cell cycle progression in the wing pouch.** Crol is up-regulated in response to ecdysone signalling and increased Crol results in decreased *wg* mRNA expression. Reduced Wg signalling leads to increased *dmyc* expression to drive S-phase and mitosis via increased Stg.

In addition, EcR function is required for wing imaginal disc cell cycles as inactivation of signalling through the EcR/USP/ecdysone complex results in reduced cell division (Cranna and Quinn 2009; Mitchell et al. 2008). In this work the pathway was inhibited using either of 2 dominant negative EcR isoforms; 1) the *EcRA* dominant negative (dN) receptor (EcRAdN), which still binds ecdysone, USP and the *EcRE*, but is defective in the activation of targetgene transcription due to a mutation in the ligand binding domain (LBD) (Cherbas et al. 2003); or 2) the EcR-B2 dominant negative receptor, which dimerizes with USP and binds

Fig. 6. **Working model connecting Crol to steroid hormone signalling and cell cycle progression in the wing pouch.** Crol is up-regulated in response to ecdysone signalling and increased Crol results in decreased *wg* mRNA expression. Reduced Wg signalling leads to

In addition, EcR function is required for wing imaginal disc cell cycles as inactivation of signalling through the EcR/USP/ecdysone complex results in reduced cell division (Cranna and Quinn 2009; Mitchell et al. 2008). In this work the pathway was inhibited using either of 2 dominant negative EcR isoforms; 1) the *EcRA* dominant negative (dN) receptor (EcRAdN), which still binds ecdysone, USP and the *EcRE*, but is defective in the activation of targetgene transcription due to a mutation in the ligand binding domain (LBD) (Cherbas et al. 2003); or 2) the EcR-B2 dominant negative receptor, which dimerizes with USP and binds

increased *dmyc* expression to drive S-phase and mitosis via increased Stg.

the *EcRE*, but cannot bind ecdysone, thus preventing optimal activation of ecdysone responsive genes (Cherbas et al. 2003; Hu, Cherbas, and Cherbas 2003). Blocking the EcR signal via overexpression of either *EcRAdN* or *EcRB2dN* in third instar wing imaginal disc flip-out clones (Pignoni and Zipursky 1997) results in a significant decrease in S-phase progression and mitosis, as measured by BrdU incorporation (Figure 7) and staining for anti-phosphohistone-H3, respectively (Cranna and Quinn 2009; Mitchell et al. 2008). Consistent with ecdysone signalling through EcR/USP normally being required for *dmyc*  transcription, reduced *dmyc* promoter activity was observed in *EcRAdN* clones generated in the *dmyc-lacZ* enhancer trap background (Cranna and Quinn 2009). Thus ecdysone signalling through EcR/USP might normally control cell cycle progression in the wing imaginal disc by upregulating dMyc to drive growth by increasing ribosome biogenesis and protein translation (Johnston et al. 1999) and S phase via increased activity of the G1 cyclins (Duman-Scheel, Johnston, and Du 2004). Together this suggests EcR signalling might modulate cell growth and division of the wing imaginal disc by modulating *dmyc* levels (Cranna and Quinn 2009).

In support of the reduced cell division in loss-of-function EcR cells being mediated by Crol, EcRAdN clones generated in the heterozygous *crol* mutant background show a further, significant reduction in cell cycle progression, when compared with either EcRdN cells alone or *crol* heterozygotes (Figure 7). This suggests that the reduction in cell cycle resulting from loss of EcR is sensitive to the level of Crol and that the ecdysone pathway normally regulates cell cycle in a Crol-dependent manner.
