**3.1 Ecdysone mediates morphogenetic Furrow progression in the eye imaginal disc**

The *Drosophila* eye is composed of an ordered array of photoreceptor clusters or ommatidia, which develop from an epithelial monolayer known as the eye imaginal disc, via an organised pattern of proliferation and differentiation (Figure 4; (Ready, Hanson, and Benzer 1976; Wolff and Ready 1991)). Differentiation of the ommatidia occurs in a wave that moves from the posterior toward the anterior (Thomas and Zipursky 1994). The margin between the asynchronously dividing anterior cells and the differentiated posterior cells is marked by the morphogenetic furrow (MF) (Ready, Hanson, and Benzer 1976). Mitotic division cycles become synchronized in the MF where cells are delayed in G1 and a subset of photoreceptor cells are specified. The remaining retinal cells synchronously re-enter the cell cycle in the "Second Mitotic Wave" (SMW), which is composed of a tight band of DNA synthesis and mitosis (Figure 4). These final cell divisions provide the cells required for differentiation of the ommatidial structures that form the adult eye (Ready, Hanson, and Benzer 1976; Wolff and Ready 1991).

Studies in the eye primordium of the tobacco hornworm moth, *Manduca sexta*, suggest that progression of the MF, including proliferation and differentiation of ommatidial clusters, requires ecdysone. Eye primordia proliferation responds to a critical concentration of ecdysone and below this threshold cells arrest in the G2 phase of the cell cycle (Champlin and Truman 1998). Premature exposure to high levels of ecdysone will also result in MF arrest and precocious maturation of ommatida (Champlin and Truman 1998). These cell cycle responses to ecdysone are consistent with the moderate ecdysone pulse during the larval stage first stimulating eye proliferation and the high levels of ecdysone released after pupariation driving cell cycle exit and eye maturation.

The ecdysone pathway has also been implicated in regulation of MF progression in the *Drosophila* larval eye imaginal disc. The *ecdysoneless* mutation (*ecd-ts*) is a hypomorphic temperature-sensitive allele, which reduces ecdysone secretion from the ring gland (Henrich et al. 1987). Homozygous *ecd-ts* flies show eye defects when shifted to the restrictive temperature during the third instar larval stage (Brennan, Ashburner, and Moses 1998). Consistent with the MF moving much more slowly than normal in the *ecd-ts* mutant, delayed eye differentiation was shown using the neuronal marker Elav.

Microarray analysis has linked the ecdysone pulse during metamorphosis to transcriptional changes in mitogenic signalling molecules, which are essential for coordinating cell cycle and patterning of imaginal tissues. The observation that ecdysone signalling was essential for the activation of factors involved in regulatory signalling pathways such as Wg, Notch

of around 10-50 undifferentiated stem cells, which undergo extensive growth and proliferation to comprise up to 100,000 cells by the end of the third larval instar. The imaginal discs start differentiation at the end of third instar and complete the process by the end of pupariation, when all adult structures such as the wings, legs and eyes have developed (Fristrom and Fristrom 1993). The third instar larval stage is a critical stage of *Drosophila* development, containing the major growth and proliferation of all tissues required to form the adult fly (Church and Robertson 1966). Indeed the size of the adult fly is determined at the time when the pupal case is formed, as after this the animal cannot feed again until eclosion. Here we will discuss the developmental signals (including Wingless, Dpp, Hedgehog, Notch) controlling growth of the eye and wing imaginal discs, and how

**3.1 Ecdysone mediates morphogenetic Furrow progression in the eye imaginal disc**  The *Drosophila* eye is composed of an ordered array of photoreceptor clusters or ommatidia, which develop from an epithelial monolayer known as the eye imaginal disc, via an organised pattern of proliferation and differentiation (Figure 4; (Ready, Hanson, and Benzer 1976; Wolff and Ready 1991)). Differentiation of the ommatidia occurs in a wave that moves from the posterior toward the anterior (Thomas and Zipursky 1994). The margin between the asynchronously dividing anterior cells and the differentiated posterior cells is marked by the morphogenetic furrow (MF) (Ready, Hanson, and Benzer 1976). Mitotic division cycles become synchronized in the MF where cells are delayed in G1 and a subset of photoreceptor cells are specified. The remaining retinal cells synchronously re-enter the cell cycle in the "Second Mitotic Wave" (SMW), which is composed of a tight band of DNA synthesis and mitosis (Figure 4). These final cell divisions provide the cells required for differentiation of the ommatidial structures that form the adult eye (Ready, Hanson, and

Studies in the eye primordium of the tobacco hornworm moth, *Manduca sexta*, suggest that progression of the MF, including proliferation and differentiation of ommatidial clusters, requires ecdysone. Eye primordia proliferation responds to a critical concentration of ecdysone and below this threshold cells arrest in the G2 phase of the cell cycle (Champlin and Truman 1998). Premature exposure to high levels of ecdysone will also result in MF arrest and precocious maturation of ommatida (Champlin and Truman 1998). These cell cycle responses to ecdysone are consistent with the moderate ecdysone pulse during the larval stage first stimulating eye proliferation and the high levels of ecdysone released after

The ecdysone pathway has also been implicated in regulation of MF progression in the *Drosophila* larval eye imaginal disc. The *ecdysoneless* mutation (*ecd-ts*) is a hypomorphic temperature-sensitive allele, which reduces ecdysone secretion from the ring gland (Henrich et al. 1987). Homozygous *ecd-ts* flies show eye defects when shifted to the restrictive temperature during the third instar larval stage (Brennan, Ashburner, and Moses 1998). Consistent with the MF moving much more slowly than normal in the *ecd-ts* mutant,

Microarray analysis has linked the ecdysone pulse during metamorphosis to transcriptional changes in mitogenic signalling molecules, which are essential for coordinating cell cycle and patterning of imaginal tissues. The observation that ecdysone signalling was essential for the activation of factors involved in regulatory signalling pathways such as Wg, Notch

ecdysone impacts on these signalling pathways to control cell division.

Benzer 1976; Wolff and Ready 1991).

pupariation driving cell cycle exit and eye maturation.

delayed eye differentiation was shown using the neuronal marker Elav.

Fig. 4. **A - Eye imaginal disc differentiation occurs in a wave that moves from posterior (P) to anterior (A)**. The margin between the asynchronously dividing anterior cells and the differentiated posterior cells is marked by the morphogenetic furrow (MF), where cells are delayed in G1. Mitotic division cycles become synchronized in the "Second Mitotic Wave" (SMW), which is composed of a tight band of DNA synthesis (Marked by BrdU in red) and mitosis (marked by PH3 in green). **B - The Hedgehog (Hh) and Dpp pathways control cell division in the larval eye.** *Drosophila* eye development is dependent on *hedgehog* (*hh*) expression posterior to the MF and *decapentaplegic* (*dpp*) expression within the MF. Hh and Dpp regulate key cell cycle genes to coordinate cell cycle and differentiation. Dpp and Hh act redundantly to ensure G1 arrest, thus cells unable to respond to Dpp will arrest later in response to Hh. Dpp and Hh inhibit Cyclin E and dE2F1 in the cells comprising the MF. In the anterior of the MF, Hh acts to promote cell division in the SMW by upregulating Cyclin D to promote cell growth and Cyclin E to drive S-phase entry.

and Dpp, suggests there might be many connections between ecdysone, developmental pathways and cell cycle regulation during metamorphosis in *Drosophila* (Li and White 2003). The first evidence for these connections in the *Drosophila* larval eye imaginal disc came from studies implicating the ecdysone pathway in regulation of MF progression via effects on Hh and Dpp (Brennan, Ashburner, and Moses 1998; Brennan et al. 2001). In *Drosophila*, eye development is dependent on *hedgehog* (*hh*) expression posterior to the MF (Heberlein,

Steroid Hormones in *Drosophila*:

observed (Zelhof et al. 1997).

or *usp* mutants.

Ghbeish et al. 2001; Ghbeish and McKeown 2002).

SMW cell cycles and MF progression.

How Ecdysone Coordinates Developmental Signalling with Cell Growth and Division 153

cycles in *usp*-/- clones may be the underlying cause of the premature differentiation

Together these data show that reduction in either ecdysone or USP results in reduced cell cycles. Paradoxically, however, *usp* mutations increase the rate of MF movement (Zelhof et al. 1997; Ghbeish et al. 2001; Ghbeish and McKeown 2002) while loss of ecdysone stops the MF (Brennan, Ashburner, and Moses 1998; Brennan et al. 2001). One explanation for these observations is that in the absence of ligand, the EcR/USP heterodimer normally acts as a repressor at certain *EcRE*s. For these target genes ecdysone would be required to relieve the transcriptional repression caused by unliganded binding of the EcR/USP complex. This idea emerged from the finding that the *Broad-complex* (*BR-C*), which encodes the family of zincfinger transcription factors upregulated early in response to high ecdysone titres (Karim, Guild, and Thummel 1993), becomes ectopically expressed in loss-of-function wing imaginal disc cells for either *usp* (Schubiger and Truman 2000) or *EcR* (Schubiger et al. 2005). Although concrete evidence is lacking, the idea is that the early (pre-ecdysone pulse) repressive effect of the EcR/USP heterodimer at the *BR-C* promoter will be lost in either *EcR*

The apparently contradictory effects of USP and ecdysone in the eye might actually be a consequence of the differential effects of the pathway on *BR-C* transcription. The Z1 isoform of the *BR-C* (*BrC-Z1*) is normally expressed posterior to the MF but not anterior to the MF (Emery, Bedian, and Guild 1994; Bayer, Holley, and Fristrom 1996) and reduced induction of *BrC-Z1* occurs in *ecd-ts* eye discs (Brennan, Ashburner, and Moses 1998). Loss of USP function has the opposite effect, leading to high level BrC-Z1 protein expression both anterior and posterior to the MF, which might occur as a consequence of de-repression of *BR-C* transcription (Brennan et al. 2001). This high level of BrC-Z1 protein in *usp* mutant clones may explain the MF advancement phenotypes, as ectopic BrC-Z1 protein has been shown to induce premature differentiation of photoreceptor cells (Zelhof et al. 1997;

Yet even though *BrC-Z1* expression is downregulated in *ecd-ts* mutants (Brennan, Ashburner, and Moses 1998), *BrC-Z1* loss of function eye imaginal discs are phenotypically different (Ghbeish et al. 2001), suggesting that other downstream targets of ecdysone pathway transcription mediate the reported effects on eye development. Like *ecd-ts*, impaired *BrC-Z1* function results in decreased levels of Hh, defective MF progression and photoreceptor recruitment. However, unlike the findings for *ecd-ts*, reduced levels of Cyclin B were not detected in *BrC-Z1* loss of function clones (Ghbeish et al. 2001). Rather loss of *BrC-Z1* function results in defects in ommatidial assembly, suggesting a role for *BR-C* in post-MF differentiation rather than cell cycle regulation in the SMW (Brennan et al. 2001). This suggests that some ecdysone regulation in the eye is mediated by BrC-Z1, but that an alternate target(s) of the ecdysone pathway regulates the cell cycle activity required for

The *ecd-ts* and USP studies suggest a role for the ecdysone pathway and the USP receptor in furrow progression, however, analysis of *EcR* mutant clones led to the conclusion that EcR was not required for furrow progression (Brennan et al. 2001). This was surprising given the EcR isoforms are the major mediators of the ecdysone signal, combined with the *Manduca Sexta* (Champlin and Truman 1998, 1998) and *Drosophila* studies (Brennan, Ashburner, and Moses 1998) that have demonstrated a requirement for ecdysone in MF progression. This led the authors of this study to propose a novel hormone transduction pathway involving an uncharacterized receptor to explain USP functioning independent of EcR in the eye, which

Wolff, and Rubin 1993; Heberlein et al. 1995) and *decapentaplegic* (*dpp*) expression within the MF (Figure 4; (Blackman et al. 1991)). *Drosophila* Dpp is a member of the mammalian transforming growth factor-beta (TGF-beta) family of secreted proteins. TGF-beta can behave as a tumour-suppressor or oncogene depending on the tissue microenvironment, thus pathway inhibition or activation can result in cancer progression (Serra and Moses 1996; Derynck, Akhurst, and Balmain 2001; Wakefield and Roberts 2002; Bachman and Park 2005; Elliott and Blobe 2005; Jakowlew 2006; Massague 2008). Aberrant Hh signalling has also been associated with human cancer, with much literature linking activation of the pathway with increased tumour progression (Toftgard 2000; Vestergaard, Bak, and Larsen 2005; Evangelista, Tian, and de Sauvage 2006; Epstein 2008; Varjosalo and Taipale 2008). In the eye disc, Dpp and Hedgehog (Hh) act redundantly to ensure G1 arrest within the MF (Penton, Selleck, and Hoffmann 1997; Horsfield et al. 1998; Firth and Baker 2005) where both pathways can inhibit Cyclin E and dE2F1 (Escudero and Freeman 2007). In addition to this cell cycle inhibitory role of Hh in the MF, Hh promotes cell division in the SMW by upregulating Cyclin D to promote cell growth and Cyclin E to drive S-phase entry (Figure 4; (Duman-Scheel et al. 2002)).

In line with ecdysone regulating eye development via Hh, the phenotype observed in *ecdysoneless* (*ecd-ts*) mutant is similar to phenotypes resulting from *hh* loss of function (Heberlein et al. 1995). In addition, the decreased levels of Hh protein posterior to the MF in *ecd-ts* larval eye discs are consistent with *hh* being a downstream target of the ecdysone signal (Brennan, Ashburner, and Moses 1998). The delayed MF progression may be, therefore, a consequence of the requirement for Hh in activation of the S-phase genes *cyclin D* and *cyclin E* and, therefore, cell cycle re-entry in the SMW (Duman-Scheel et al. 2002). Indeed, the failure of MF movement in *ecd-ts* mutants is likely a result of impaired cell cycle progression as S-phase numbers were dramatically decreased in the SMW (Brennan, Ashburner, and Moses 1998). Consistent with reduced cell division within the SMW, levels of the mitotic cyclin, Cyclin B, were also reduced posterior to the MF (Brennan, Ashburner, and Moses 1998).

The USP receptor has also been implicated in regulation of cell cycle progression and differentiation in the developing eye imaginal disc. Loss-of-function *usp* clones spanning the morphogenetic furrow show an anterior shift in expression of the MF-specific marker Dpp, consistent with premature progression of the MF and a role for USP in repressing morphogenetic furrow movement (Zelhof et al. 1997). In addition, loss of USP results in ectopic activation of many genes involved in cell fate specification in the eye, including the differentiation markers Spalt and Atonal (Zelhof et al. 1997). Although expression of these differentiation markers occurs prematurely, specification of cells contributing to the ommatidia occurs normally. The cell cycle analysis of *usp* mutant clones suggested that although the MF was advanced, cell cycle progression was disrupted in the SMW. First staining for Cyclin A, as a marker for cells in either S or G2 phase, revealed fewer Cyclin Apositive cells in *usp*- clones posterior to the morphogenetic furrow (Ghbeish et al. 2001). Similarly, although the Cyclin B band was not shifted in *usp-* clones posterior to the MF, the numbers of cells expressing Cyclin B were reduced (Ghbeish and McKeown 2002). The reduction in cell cycle markers posterior of the MF suggests that USP is required for cell cycle progression in the SMW. In support of cell cycle induction in the SMW depending on the presence of USP protein, *usp* overexpression using the *GMR-*promoter, which is only expressed posterior of the furrow, can rescue the loss of Cyclin B in the *usp* mutant clone. As progression through the SMW and differentiation are tightly coupled, the reduced cell

Wolff, and Rubin 1993; Heberlein et al. 1995) and *decapentaplegic* (*dpp*) expression within the MF (Figure 4; (Blackman et al. 1991)). *Drosophila* Dpp is a member of the mammalian transforming growth factor-beta (TGF-beta) family of secreted proteins. TGF-beta can behave as a tumour-suppressor or oncogene depending on the tissue microenvironment, thus pathway inhibition or activation can result in cancer progression (Serra and Moses 1996; Derynck, Akhurst, and Balmain 2001; Wakefield and Roberts 2002; Bachman and Park 2005; Elliott and Blobe 2005; Jakowlew 2006; Massague 2008). Aberrant Hh signalling has also been associated with human cancer, with much literature linking activation of the pathway with increased tumour progression (Toftgard 2000; Vestergaard, Bak, and Larsen 2005; Evangelista, Tian, and de Sauvage 2006; Epstein 2008; Varjosalo and Taipale 2008). In the eye disc, Dpp and Hedgehog (Hh) act redundantly to ensure G1 arrest within the MF (Penton, Selleck, and Hoffmann 1997; Horsfield et al. 1998; Firth and Baker 2005) where both pathways can inhibit Cyclin E and dE2F1 (Escudero and Freeman 2007). In addition to this cell cycle inhibitory role of Hh in the MF, Hh promotes cell division in the SMW by upregulating Cyclin D to promote cell growth and Cyclin E to drive S-phase entry (Figure 4;

In line with ecdysone regulating eye development via Hh, the phenotype observed in *ecdysoneless* (*ecd-ts*) mutant is similar to phenotypes resulting from *hh* loss of function (Heberlein et al. 1995). In addition, the decreased levels of Hh protein posterior to the MF in *ecd-ts* larval eye discs are consistent with *hh* being a downstream target of the ecdysone signal (Brennan, Ashburner, and Moses 1998). The delayed MF progression may be, therefore, a consequence of the requirement for Hh in activation of the S-phase genes *cyclin D* and *cyclin E* and, therefore, cell cycle re-entry in the SMW (Duman-Scheel et al. 2002). Indeed, the failure of MF movement in *ecd-ts* mutants is likely a result of impaired cell cycle progression as S-phase numbers were dramatically decreased in the SMW (Brennan, Ashburner, and Moses 1998). Consistent with reduced cell division within the SMW, levels of the mitotic cyclin, Cyclin B, were also reduced posterior to the MF (Brennan, Ashburner,

The USP receptor has also been implicated in regulation of cell cycle progression and differentiation in the developing eye imaginal disc. Loss-of-function *usp* clones spanning the morphogenetic furrow show an anterior shift in expression of the MF-specific marker Dpp, consistent with premature progression of the MF and a role for USP in repressing morphogenetic furrow movement (Zelhof et al. 1997). In addition, loss of USP results in ectopic activation of many genes involved in cell fate specification in the eye, including the differentiation markers Spalt and Atonal (Zelhof et al. 1997). Although expression of these differentiation markers occurs prematurely, specification of cells contributing to the ommatidia occurs normally. The cell cycle analysis of *usp* mutant clones suggested that although the MF was advanced, cell cycle progression was disrupted in the SMW. First staining for Cyclin A, as a marker for cells in either S or G2 phase, revealed fewer Cyclin Apositive cells in *usp*- clones posterior to the morphogenetic furrow (Ghbeish et al. 2001). Similarly, although the Cyclin B band was not shifted in *usp-* clones posterior to the MF, the numbers of cells expressing Cyclin B were reduced (Ghbeish and McKeown 2002). The reduction in cell cycle markers posterior of the MF suggests that USP is required for cell cycle progression in the SMW. In support of cell cycle induction in the SMW depending on the presence of USP protein, *usp* overexpression using the *GMR-*promoter, which is only expressed posterior of the furrow, can rescue the loss of Cyclin B in the *usp* mutant clone. As progression through the SMW and differentiation are tightly coupled, the reduced cell

(Duman-Scheel et al. 2002)).

and Moses 1998).

cycles in *usp*-/- clones may be the underlying cause of the premature differentiation observed (Zelhof et al. 1997).

Together these data show that reduction in either ecdysone or USP results in reduced cell cycles. Paradoxically, however, *usp* mutations increase the rate of MF movement (Zelhof et al. 1997; Ghbeish et al. 2001; Ghbeish and McKeown 2002) while loss of ecdysone stops the MF (Brennan, Ashburner, and Moses 1998; Brennan et al. 2001). One explanation for these observations is that in the absence of ligand, the EcR/USP heterodimer normally acts as a repressor at certain *EcRE*s. For these target genes ecdysone would be required to relieve the transcriptional repression caused by unliganded binding of the EcR/USP complex. This idea emerged from the finding that the *Broad-complex* (*BR-C*), which encodes the family of zincfinger transcription factors upregulated early in response to high ecdysone titres (Karim, Guild, and Thummel 1993), becomes ectopically expressed in loss-of-function wing imaginal disc cells for either *usp* (Schubiger and Truman 2000) or *EcR* (Schubiger et al. 2005). Although concrete evidence is lacking, the idea is that the early (pre-ecdysone pulse) repressive effect of the EcR/USP heterodimer at the *BR-C* promoter will be lost in either *EcR* or *usp* mutants.

The apparently contradictory effects of USP and ecdysone in the eye might actually be a consequence of the differential effects of the pathway on *BR-C* transcription. The Z1 isoform of the *BR-C* (*BrC-Z1*) is normally expressed posterior to the MF but not anterior to the MF (Emery, Bedian, and Guild 1994; Bayer, Holley, and Fristrom 1996) and reduced induction of *BrC-Z1* occurs in *ecd-ts* eye discs (Brennan, Ashburner, and Moses 1998). Loss of USP function has the opposite effect, leading to high level BrC-Z1 protein expression both anterior and posterior to the MF, which might occur as a consequence of de-repression of *BR-C* transcription (Brennan et al. 2001). This high level of BrC-Z1 protein in *usp* mutant clones may explain the MF advancement phenotypes, as ectopic BrC-Z1 protein has been shown to induce premature differentiation of photoreceptor cells (Zelhof et al. 1997; Ghbeish et al. 2001; Ghbeish and McKeown 2002).

Yet even though *BrC-Z1* expression is downregulated in *ecd-ts* mutants (Brennan, Ashburner, and Moses 1998), *BrC-Z1* loss of function eye imaginal discs are phenotypically different (Ghbeish et al. 2001), suggesting that other downstream targets of ecdysone pathway transcription mediate the reported effects on eye development. Like *ecd-ts*, impaired *BrC-Z1* function results in decreased levels of Hh, defective MF progression and photoreceptor recruitment. However, unlike the findings for *ecd-ts*, reduced levels of Cyclin B were not detected in *BrC-Z1* loss of function clones (Ghbeish et al. 2001). Rather loss of *BrC-Z1* function results in defects in ommatidial assembly, suggesting a role for *BR-C* in post-MF differentiation rather than cell cycle regulation in the SMW (Brennan et al. 2001). This suggests that some ecdysone regulation in the eye is mediated by BrC-Z1, but that an alternate target(s) of the ecdysone pathway regulates the cell cycle activity required for SMW cell cycles and MF progression.

The *ecd-ts* and USP studies suggest a role for the ecdysone pathway and the USP receptor in furrow progression, however, analysis of *EcR* mutant clones led to the conclusion that EcR was not required for furrow progression (Brennan et al. 2001). This was surprising given the EcR isoforms are the major mediators of the ecdysone signal, combined with the *Manduca Sexta* (Champlin and Truman 1998, 1998) and *Drosophila* studies (Brennan, Ashburner, and Moses 1998) that have demonstrated a requirement for ecdysone in MF progression. This led the authors of this study to propose a novel hormone transduction pathway involving an uncharacterized receptor to explain USP functioning independent of EcR in the eye, which

Steroid Hormones in *Drosophila*:

How Ecdysone Coordinates Developmental Signalling with Cell Growth and Division 155

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* 

**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

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*

reduced BrdU staining (red) for S-phase.

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 required for eye development (Figure 4).
