The major developmental hormone in Drosophila, the steroid hormone 20-hydroxyecdysone, commonly known as ecdysone, is secreted from the prothoracic gland (PG) and plays a major role in regulating imaginal disc development. Ecdysone release is controlled by a complex combination of upstream factors, including peptide hormones and neuropeptide signals 3. For example, Prothoracicotropic hormone (PTTH) from the central nervous system (CNS) is required to regulate the synthesis and release of ecdysone from the PG 4. Ecdysone pulses are required for all aspects of morphogenesis, starting with the formation of the body plan during late embryogenesis required to develop to first instar larvae and for the cuticle moulting at the end of the first and second instars. A large titre of ecdysone is released at the end of the third instar, in the wandering larvae in preparation for pupation, which marks the beginning of adult tissue metamorphosis 5678. The range of larval structures that respond to the ecdysone pulse at this transformation stage all elicit diverse cellular responses to achieve conversion from the larval tissue to the adult structures 9.

During metamorphosis, a cascade of gene transcription is triggered by ecdysone, which activates the ecdysone receptor (EcR), a member of the nuclear receptor family, and its receptor binding partner Ultraspiricle (USP) 5678. The EcR gene spans 77 kb in length, and through the use of two promoters and as a result of alternate splicing, encodes three major protein isoforms EcR-A, EcR-B1, EcR-B2. The EcR-A, EcR-B1, EcR-B2 isoforms have conserved DNA binding domains and ligand binding domains but differ in their N-terminal regions, with variable N terminal domains of 197, 226 and 17 amino acid residues, respectively 810. Although EcR can bind ecdysone alone, optimal binding to the ecdysone response elements (EcRE) and activation of transcriptional targets requires the addition of USP 1112.

USP exhibits a strong structural and functional similarity to the orthologous vertebrate retinoid × receptor (RXR) 1314. Like RXR, which forms heterodimers with non-steroid receptors for thyroid hormone, retinoic acid and vitamin D, and thereby activates them for DNA-binding 15, USP interacts with each of the EcR isoforms to form DNA-binding heterodimers 913. Drosophila EcRs are therefore analogous to the vertebrate family of RXR heterodimeric receptors rather than the vertebrate family of steroid hormone receptors, which bind DNA as homodimers 16. In the presence of the ecdysone ligand, the appropriate EcR nuclear receptor isoform dimerizes with Ultraspiricle (USP), and the complex is stabilised by the ecdysone ligand to allow efficient binding to the ecdysone response element (EcRE) 1718 and transcriptional activation of ecdysone-responsive genes (such as BR-C, E74, E75) 1920212223. Early genes encode transcription factors that activate late genes and this hierarchy of gene activation is required for subsequent development 5. To increase the output of the ecdysone pulse, EcR provides an autoregulatory loop by increasing the level of it's own transcription in order to further increase receptor levels in response to the ecdysone ligand 8.

An essential process driven by the ecdysone pulse is the removal of larval tissues no longer required in the adult 24. The process of steroid hormone driven apoptosis is an important part of tissue remodelling, whereby selective death removes unwanted cells towards generating the mature structure 25. For example, the histolysis of the larval salivary gland and midgut at the end of metamorphosis is stage-specific, ecdysone triggered, programmed cell death, which results in the removal of the component of these larval structures no longer required in the adult fly. Consistent with this, previous studies have shown that cell death activators are upregulated in the third instar larval tissues, including the salivary glands and midgut in response to ecdysone (reviewed 242627).

The ecdysone is pulse is also essential for differentiation and patterning of the larval imaginal tissues required for development of adult structures 17192829. As cell division and patterning are tightly linked in Drosophila imaginal tissues the process of metamorphosis controlled by ecdysone will therefore involve coordination of the developmental signals that regulate proliferation and differentiation. Although much work has focused on the downstream targets linking the ecdysone pathway to programmed cell death and cell differentiation 242627, the relationship between ecdysone and cell cycle is less clear. Here we integrate evidence providing a link between the ecdysone pulse and cell cycle progression in Drosophila.

In Drosophila, cell growth and cell cycle progression are regulated by a number of key genes, which have been shown to control the cell cycle in an analogous manner in all multicellular organisms. These include the Drosophila orthologue of the mammalian c-myc transcription factor and oncogene, dMyc, which drives growth and progression through G1 to S-phase 30, the essential G1 to S-phase Cyclin complex, Cyclin E (CycE) and it's Cyclin-dependent-kinase (Cdk) partner Cdk2, which triggers S-phase by promoting DNA replication 313233, and the Drosophila orthologue of the Cdc25 phosphatase, String (Stg), which is required for G2/M progression and promotes mitotic entry by activating the Cdk1/Cyclin B complex 34. CycE and Stg are the rate limiting factors for S-phase and mitosis, respectively, and both are activated by the Drosophila orthologue of human E2F1 protein, dE2F1 32. dE2F1 responds to the relevant Cdk-Cyclin complex (CycE/Cdk2 for S-phase and CycB/Cdk1 for mitosis) to coordinate cell cycle progression from G1 to S-phase and G2 into mitosis 35.

During metamorphosis, growth of larval tissues occurs in an ecdysone-dependent manner to produce adult structures. For example, during pupal development the larval midgut is removed by apoptosis and is replaced through proliferation of imaginal tissues to form the adult midgut 2636. Microarray analysis has revealed that the ecdysone signal is associated with the activation of key cell cycle genes, including Cyclin B, Cdc2 and Cyclin D, during the initiation of midgut metamorphosis 37. Analysis of EcR null mutants revealed that EcR function was necessary for the cell cycle and growth genes to be activated in the larval midgut, suggesting that the ecdysone pathway is required for cell division control.

The ecdysone pulse has been shown to act non-autonomously to affect larval growth. These cell extrinsic effects of the ecdysone pathway are reviewed elsewhere 38394041 and therefore only mentioned briefly here. This control of Drosophila larval growth and final body size occurs non-autonomously, at least in part through interactions between the ecdysone and insulin pathways. The insulin signaling pathway acts in the prothoracic gland (PG) to regulate the release of ecdysone, therefore influencing the rate and duration of larval growth 383942434445. For instance, increased PG growth occurs when PI3-kinase (PI3K, a downstream regulator of the insulin pathway) is upregulated in the PG 4244. The PG overgrowth causes accelerated metamorphosis, which results in reduced adult size due to the rapid progression through the larval growth stage. Precocious ecdysone release, as measured by premature increase in levels of the early response ecdysone genes, correlates with this disruption to larval growth. Conversely, reducing growth of the PG, using a dominant negative form of PI3K, resulted in a longer larval growth period and larger adults due to slower ecdysone release and delayed onset of pupariation. More recently it has been shown that Target of Rapamycin (TOR) may link the ecdysone regulated development to the PI3K mediated growth pathways 46 (reviewed 41).

Correct timing of the critical peak in ecdysone is therefore essential for controlling larval growth and adult body size, but how does the ecdysone pulse achieve these changes in cell growth and cell cycle progression within the larval tissues? In particular, how does ecdysone connect with the major developmental signaling pathways that regulate cell cycle patterning in Drosophila?

Drosophila eye development is dependent on hedgehog (hh) expression posterior to the MF 5253 and decapentaplegic (dpp) expression within the MF 54. 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 55565758596061. Aberrant Hh signaling has also been associated with numerous human cancers, with much literature linking activation of the pathway with increased tumour progression 6263646566.

In the eye disc, the Hh and Dpp signaling pathways regulate key cell cycle genes to mediate cell cycle arrest or progression. Cell cycle arrest occurs in G1 phase within and anterior to the MF in response to Dpp 6768. Dpp and Hedgehog (Hh) act redundantly to ensure G1 arrest, thus cells unable to respond to Dpp will arrest later in response to Hh 69. Dpp and Hh are both required to ensure Cyclin E and dE2F1 are inhibited and the cells comprising the MF undergo a coordinated G1 arrest 70. In addition to the cell cycle inhibitory role of Hh 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 71. These contrasting roles of Hh in different cells indicate that levels of Hh may be important to dictate cell cycle outcome or that other factors are involved. Indeed Notch signaling has been shown to be critical to drive S-phase entry in the SMW 7273.

Studies in the eye primordium of the 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 74. Premature exposure to high levels of ecdysone will also result in MF arrest and precocious maturation of ommatida 75. These cell cycle responses to ecdysone are consistent with the moderate ecdysone pulse during the larval stage stimulating eye proliferation, whilst the high levels of ecdysone released after pupariation would be required for 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 76. The ecdysoneless mutation (ecd-ts) is a hypomorphic temperature-sensitive allele, which reduces ecdysone secretion from the ring gland 77. Homozygous ecd-ts flies show eye defects when shifted to the restrictive temperature during the third instar larval stage 76. Consistent with the MF moving much more slowly than normal in the ecd-ts mutant, delayed eye differentiation was shown using the Elav marker. This phenotype is similar to phenotypes resulting from hh loss of function 53. Consistent with hh being a downstream target of the ecdysone signal, decreased levels of Hh protein were detected posterior to the MF in ecd-ts larval eye discs 76. Delayed MF progression would be consistent with a requirement for Hh in activation of the S-phase genes Cyclin D and Cyclin E and therefore cell cycle re-entry in the SMW 71. The failure of MF movement in ecd-ts mutants was attributed to impaired cell cycle progression as S-phase numbers were dramatically decreased in the SMW 76. Consistent with reduction of cell division within the SMW, levels of the mitotic cyclin, Cyclin B, were also reduced posterior to the MF 76.

USP also regulates cell cycle and differentiation in developing larval imaginal discs. Loss-of-function usp clones spanning the morphogenetic furrow in larval eye imaginal discs 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 78. Loss of functional USP affects many genes involved in cell fate specification in the eye, including the differentiation markers Spalt and Atonal 78. 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 A-positive cells in usp- clones posterior to the morphogenetic furrow 79. 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 80. 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 cycles in usp-/- clones may be associated with the premature differentiation observed 78.

Thus reduction in either ecdysone or USP results in reduced cell cycles, yet usp mutations increase the rate of MF movement 787980 and loss of ecdysone stops the MF 7681. One explanation for these observations is that in the absence of ligand, the EcR/USP heterodimer normally acts as a repressor at certain EcREs. 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 a family of zinc-finger transcription factors upregulated in response to high ecdysone titres 82, becomes ectopically expressed in wing imaginal disc cells loss-of-function for either usp 83 or EcR 84. 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 therefore 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 8586 and reduced induction of BrC-Z1 occurs in ecd-ts eye discs 76. 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 81. 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 787980.

Yet even though BrC-Z1 expression is downregulated in ecd-ts mutants 76, BrC-Z1 loss of functioneye imaginal discs are phenotypically different 79, suggesting that other downstream transcriptional targets of the ecdysone pathway 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 79. 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 81. 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 cell cycles in the second mitotic wave.

Although a direct cell cycle role has not been demonstrated in the eye, the ecdysone-responsive BR-C has been implicated in regulating DNA synthesis in the adult ovary during oogenesis. Ectopic BR-C expression leads to ectopic G1 to S-phase endoreplication cycles during oogenesis, consistent with the ecdysone pathway promoting DNA replication 87. These studies suggest ecdysone is required for endocycles, which are cycles of DNA replication (S-phase progression) without cell division required to amplify specific regions of the genome (the chorion genes) required for formation of the egg shell [reviewed in 88. BR-C loss of function causes premature arrest of chorion gene amplification, whereas overexpression of BR-C isoforms lead to the formation of additional foci of BrdU incorporation in follicle cells 89. BR-C most likely promotes endoreplication in the Drosophila ovary via the key cell growth and S-phase regulators, dMyc 90 and Cyclin E 319192.

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. The wing pouch, which ultimately forms the adult wing blade, has been a focus for studying signals impacting upon the cell cycle. The morphogenesis of the wing blade involves patterned cell cycles that are tightly regulated and the signaling pathways affecting these cell cycles are well characterised 3093949596. A key signaling 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 297 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 the key cell cycle genes; including dmyc, cycE, dE2F1 and stg, to link the Wg patterning signal to proliferation 30949598. 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 2) 3094.

Cross-talk between the Wg pathway and other signaling 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 99 and is required for cell cycle progression and tissue growth 100. Proliferation is dependent on careful regulation of the relative levels of the DPP and Wg signaling pathways [reviewed in 101. The Hedgehog (Hh) 102 and Notch (N) 103 pathways are key upstream regulators of Wg in the wing disc. Notch activity also plays a role in cell cycle arrest during wing development 94104. Notch is activated in cells along the dorso-ventral (D/V) boundary (ZNC) of the wing disc, where it is required for Wg expression 103. 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 94. The expression of Notch specifically 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 2) 3094. The interplay of these signaling pathways therefore achieves the pattern of cell cycles within the wing pouch required for wing disc differentiation.

Our recent work revealed a link between the ecdysone pulse, the Wg pathway and developmental cell cycle regulation in the wing 105. Previous studies have shown that Crol, which is a zinc finger transcription factor, is activated in late larval imaginal discs by the steroid hormone ecdysone 19. Pupal lethal, hypomorphic crol mutants (crol⁴⁴¹⁸) have defects in ecdysone-induced gene expression 19. We demonstrated that Crol is required for cell cycle progression in the wing imaginal disc 105. First, crol mutant clones in the wing pouch fail to proliferate, whilst overexpression of crol results in ectopic proliferation in the larval wing disc ZNC, suggesting Crol is both necessary and sufficient for third instar larval wing cell cycles. Crol is also required to downregulate the Wg pathway, which normally acts in the ZNC to drive cell cycle exit and differentiation (Figure 2). Therefore, by inhibiting the Wg pathway, crol positively drives cell cycle and potentially provides a link between the ecdysone pathway and the developmental signals that regulate cell cycle (Figure 3).

To determine whether ecdysone signaling via the EcR normally plays a role in cell cycle regulation we used two independent dominant negative lines to inactivate signaling through the EcR/USP/ecdysone complex; first the EcRA dominant negative (dN) receptor (EcRAdN), which still binds ecdysone, USP and the EcRE, but is completely defective in the activation of target-gene transcription due to a mutation in the ligand binding domain (LBD) 106; and second the EcR-B2 dominant negative receptor, which dimerizes with USP and binds the EcRE, but cannot bind ecdysone, thus preventing optimal activation of ecdysone responsive genes 106107.

We have previously shown that blocking the EcR signal via overexpression of EcRAdN in third instar wing imaginal disc flip-out clones 108 results in cell cycle inhibition 105. These results suggested that the EcR pathway was required for the normal pattern of wing imaginal disc cell cycles. Here we show the result of blocking the pathway with EcRBdN in wing imaginal disc clones (Figure 4A'–D'). EcRBdN overexpression results in wing disc clones with an overall decrease in BrdU positive cells. Quantification of BrdU revealed a significant decrease in S-phase progression in the UAS-EcRBdN clones compared with control clones (p = 0.0038, Figure 4E'–F'). Consistent with signaling through the EcR also being required to positively regulate progression through mitosis, clonal tissue also exhibited reduced numbers of PH3 positive cells (Figure 4A"–D") compared with control (p = 0.0047, Figure 4 E"–F"). Cell cycle analysis was carried out as described previously 105.

Therefore, blocking ecdysone pathway signaling using UAS-EcR dominant negative transgenes significantly reduces the number of cells progressing through the cell cycle. As both the UAS-EcRAdN and UAS-EcRBdN block the activation of ecdysone responsive genes, these findings suggest that targets of the ecdysone pathway are required for cell cycle progression in the Drosophila wing imaginal disc.

dMyc is a key mediator of growth and S-phase progression in the wing imaginal disc 30. To test for changes to dmyc transcription UAS-EcRAdN flip-out clones were generated in the dmyc-lacZ enhancer trap background 109 (Figure 5). The control in Figure 5 (A–D) shows the expected pattern of dmyc transcription throughout the cycling cells of the wing pouch, with reduced staining within the cell cycle arrested cells of the ZNC 30. Reduced β-gal staining in the GFP-positive UAS-EcRAdN clones (Figure 5 E–H), suggests that dmyc transcription is downregulated as a consequence of blocking EcR signaling.

These results are consistent with ecdysone signaling through EcR/USP normally being required for dmyc transcription. As increased dMyc leads to up-regulation of its cell cycle targets cycE, cycD, and cdk4, resulting in inactivation of Rbf and increased activity of the S-phase transcription factor E2f1 98, this suggests EcR signaling might normally regulate S-phase progression by modulating dmyc levels (Figure 3).

We recently demonstrated that EcR signaling is required for repression of wg transcription. Consistent with the EcR pathway normally being required to repress wg transcription, expansion of the wg expression domain occurs in UAS-EcRAdN 105 and UAS-EcRBdN (Figure 6) flip-out clones generated in a wg-lacZ enhancer trap background 110. These results suggest suppression of wg transcription in the wing pouch is dependent on the EcR pathway. Given that increased Wg protein causes reduction of cell cycle regulators such as dmyc and stg, leading to decreased cells in S-phase and mitosis in the pouch 3094, this finding is consistent with the reduced cell cycles observed in EcR loss-of-function clones. Given that Crol is ecdysone responsive 19 and capable of repressing wg transcription 105, the proposed mechanism is that the ecdysone signal normally upregulates Crol to repress wg transcription and drive cell cycle progression in the pouch (Figure 3).