**1.2 Ecdysone, EcR and USP structure and function**

Insect metamorphosis is achieved by the cascade of gene transcription triggered by ecdysone, which activates the ecdysone receptor (EcR), a member of the nuclear receptor family, and its receptor binding partner Ultraspiricle (USP) (Thummel 1996, 1990, 1995; Koelle et al. 1991) (Figure 2). The *EcR* gene spans 77kb 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. All three 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 (Koelle et al. 1991; Talbot, Swyryd, and Hogness 1993).

Steroid Hormones in *Drosophila*:

as homodimers (Beato and Klug 2000).

response to the ecdysone ligand (Koelle et al. 1991).

**1.3 Ecdysone signalling coordinates proliferation, death and differentiation** 

(Jiang, Baehrecke, and Thummel 1997; Baehrecke 2000; Yin and Thummel 2005)).

The ecdysone pulse is also essential for differentiation and patterning of the larval imaginal tissues required for development of adult structures (Hall and Thummel 1998; D'Avino and Thummel 2000, 1998; Zheng et al. 2003). As cell division and patterning are tightly linked in *Drosophila* imaginal tissues, the process of metamorphosis controlled by ecdysone involves 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 (Baehrecke 2000; Jiang, Baehrecke, and

Metamorphosis of *Drosophila* requires co-ordination of proliferation (cell growth and division), differentiation and death in order to form an adult fly of the appropriate size and with correctly differentiated structures. An essential process driven by the ecdysone pulse is the removal of larval tissues no longer required in the adult (Baehrecke 2000). 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 (Rusconi, Hays, and Cagan 2000; Thummel 2001). 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. In line with an apoptotic mechanism, 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 in

How Ecdysone Coordinates Developmental Signalling with Cell Growth and Division 145

*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

Therefore, similar to vertebrate nuclear receptors, the EcR/USP heterodimer functions as a ligand-dependent transcription factor. In the presence of the ecdysone ligand, the appropriate EcR nuclear receptor isoform dimerizes with USP, and the complex is stabilised by the active form of ecdysone, 20-hydroxyecdysone (20E) to allow efficient binding to the ecdysone response element (*EcRE*) (Hall and Thummel 1998; Yao et al. 1993) and transcriptional activation of ecdysone-responsive genes (D'Avino and Thummel 1998; Kozlova and Thummel 2002; Thummel 2002; Thummel, Burtis, and Hogness 1990; Urness and Thummel 1995). Genes that are directly activated by the 20E/EcR/USP complex include three "early" ecdysone pathway genes; *E74*, *E75* and the *Broad-Complex* (*BR-C*), which all encode transcription factors. The E74, E75 and BR-C transcription factors control the late genes in order to elicit the biological changes associated with each ecdysone pulse. This hierarchy of gene activation is required for modulating expression of the many cell death, cell cycle and differentiation genes required for metamorphosis (Thummel 1996, 2001). *E74*  encodes two proteins with an identical ETS DNA binding domain, designated E74A and E74B (Burtis et al. 1990; Thummel, Burtis, and Hogness 1990). *E75* encodes three members of the nuclear receptor superfamily (designated E75A, E75B, and E75C), which are often referred to as orphan nuclear receptors due to their unidentified ligand (Segraves and Hogness 1990). The *BR-C* is a multigene locus, which encodes several zinc finger proteins (DiBello et al. 1991). To increase the output of the ecdysone pulse, EcR provides an autoregulatory loop to activate its own transcription and further increase receptor levels in

Fig. 3**. Manipulation of growth pathways in the prothoracic gland alters body size.** Light micrographs of female adult flies raised at 25°C bearing the genotypes: (A) control (*AmnC651>+*) (B) overexpressing activated *Ras* (*AmnC651>RasV12*) in the PG (C) overexpressing dominant negative *PI3K* (*AmnC651>Dp110DN*) in the PG. Red arrows indicate length of larval growth period. (D) Graph of ecdysone titres during *Drosophila* development (modified from (Thummel, 2001)).

Although EcR can bind ecdysone alone, optimal binding to the ecdysone response elements (*EcRE*) and activation of transcriptional targets requires the addition of USP (Grad et al. 2001; Grebe, Fauth, and Spindler-Barth 2004). USP exhibits a strong structural and functional similarity to the orthologous vertebrate retinoid X receptor (RXR) (Yao et al. 1992; Oro, McKeown, and Evans 1990). Like RXR, which forms heterodimers with non-steroid receptors for thyroid hormone, retinoic acid and vitamin D, and thereby activates them for DNA-binding (Mangelsdorf and Evans 1995), USP interacts with each of the EcR isoforms to form DNA-binding heterodimers (Yao et al. 1992; Bender et al. 1997). In this respect

Fig. 3**. Manipulation of growth pathways in the prothoracic gland alters body size.** Light

(*AmnC651>+*) (B) overexpressing activated *Ras* (*AmnC651>RasV12*) in the PG (C) overexpressing dominant negative *PI3K* (*AmnC651>Dp110DN*) in the PG. Red arrows indicate length of larval growth period. (D) Graph of ecdysone titres during *Drosophila* development (modified

Although EcR can bind ecdysone alone, optimal binding to the ecdysone response elements (*EcRE*) and activation of transcriptional targets requires the addition of USP (Grad et al. 2001; Grebe, Fauth, and Spindler-Barth 2004). USP exhibits a strong structural and functional similarity to the orthologous vertebrate retinoid X receptor (RXR) (Yao et al. 1992; Oro, McKeown, and Evans 1990). Like RXR, which forms heterodimers with non-steroid receptors for thyroid hormone, retinoic acid and vitamin D, and thereby activates them for DNA-binding (Mangelsdorf and Evans 1995), USP interacts with each of the EcR isoforms to form DNA-binding heterodimers (Yao et al. 1992; Bender et al. 1997). In this respect

micrographs of female adult flies raised at 25°C bearing the genotypes: (A) control

from (Thummel, 2001)).

*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 (Beato and Klug 2000).

Therefore, similar to vertebrate nuclear receptors, the EcR/USP heterodimer functions as a ligand-dependent transcription factor. In the presence of the ecdysone ligand, the appropriate EcR nuclear receptor isoform dimerizes with USP, and the complex is stabilised by the active form of ecdysone, 20-hydroxyecdysone (20E) to allow efficient binding to the ecdysone response element (*EcRE*) (Hall and Thummel 1998; Yao et al. 1993) and transcriptional activation of ecdysone-responsive genes (D'Avino and Thummel 1998; Kozlova and Thummel 2002; Thummel 2002; Thummel, Burtis, and Hogness 1990; Urness and Thummel 1995). Genes that are directly activated by the 20E/EcR/USP complex include three "early" ecdysone pathway genes; *E74*, *E75* and the *Broad-Complex* (*BR-C*), which all encode transcription factors. The E74, E75 and BR-C transcription factors control the late genes in order to elicit the biological changes associated with each ecdysone pulse. This hierarchy of gene activation is required for modulating expression of the many cell death, cell cycle and differentiation genes required for metamorphosis (Thummel 1996, 2001). *E74*  encodes two proteins with an identical ETS DNA binding domain, designated E74A and E74B (Burtis et al. 1990; Thummel, Burtis, and Hogness 1990). *E75* encodes three members of the nuclear receptor superfamily (designated E75A, E75B, and E75C), which are often referred to as orphan nuclear receptors due to their unidentified ligand (Segraves and Hogness 1990). The *BR-C* is a multigene locus, which encodes several zinc finger proteins (DiBello et al. 1991). To increase the output of the ecdysone pulse, EcR provides an autoregulatory loop to activate its own transcription and further increase receptor levels in response to the ecdysone ligand (Koelle et al. 1991).
