**1.1 The ecdysone pathway directs** *Drosophila* **development**

Ecdysone is the major steroid hormone in all holometabolous insects responsible for driving the metamorphosis of larval tissues into adult structures. During metamorphosis, ecdysone is essential for upregulating the genes required to control apoptosis and differentiation, essential processes for removal of larval structures which have become obsolete and for tissue remodelling. In addition, ecdysone directs cell growth and division in many tissues throughout the larval to pupal transition. This chapter will discuss the many diverse mechanisms reported for connecting the ecdysone pulse to the developmentally regulated cell growth and cycle progression required for tissue growth and for insects to reach their target body size.

Like all other holometabolous insects, the size of *Drosophila* adult flies is set by the size of the larvae prior to metamorphosis, at the time of pupariation when feeding has ceased and growth can no longer occur. The major developmental hormone in *Drosophila*, the steroid hormone 20-hydroxyecdysone (20E), commonly known as ecdysone, is required for all the developmental transitions needed for metamorphosis (Figure 1-3; (Thummel 1995, 1996, 2001)). Ecdysone is produced in and released by the prothoracic gland (PG), a component of the ring gland, which also contains the corpora allata (CA) and corpora cardiaca (CC) (Figure 1; (Zitnan et al. 2007; McBrayer et al. 2007)). Ecdysone release is controlled by a complex combination of upstream factors, including peptide hormones and neuropeptide signals (see section 2.2). For example, Prothoracicotropic hormone (PTTH) from the central nervous system (CNS) is required to regulate the synthesis and release of ecdysone from the PG (McBrayer et al. 2007).

Ecdysone pulses from the PG are required for all aspects of morphogenesis, starting with the formation of the body plan during late embryogenesis, hatching and development of the first larval instar, and for cuticle moulting at the end of the first and second instars. A large

Steroid Hormones in *Drosophila*:

How Ecdysone Coordinates Developmental Signalling with Cell Growth and Division 143

Fig. 2. **The ecdysone pathway.** Abbreviations: Juvenile hormone (JH), prothoracicotropic hormone (PTTH), Target of Rapamycin (TOR), 20-hydroxyecdysone (20E), ecdysone receptor (EcR), Ultraspiracle (USP), prothoracic gland (PG). References are in blue.

(Alberts 2002). Ecdysone triggers the imaginal disc eversion and elongation, which is accomplished through cell shape changes, rather than by additional cell division (Condic, Fristrom, and Fristrom 1991). As the new appendages emerge from the imaginal discs, the larval tissues undergo programmed cell death and are eventually replaced by the adult structures (Ward et al. 2003). About 12 hours after puparium formation, another major ecdysone pulse initiates the prepupal-pupal transition, and forms the basic body plan of the adult fly. This pulse causes the head to evert from the anterior end of the puparium, the final growth of leg and wings, as well as the removal of most of the remaining larval tissues (Ward et al. 2003). The following 4 days of pupal development involves terminal differentiation of the

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

remaining tissues to form the adult fly (Figure 3; reviewed in (Thummel 2001)).

residues, respectively (Koelle et al. 1991; Talbot, Swyryd, and Hogness 1993).

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

Fig. 1. **The ring gland.** An important organ in *Drosophila* is the ring gland, which is situated in between the two brain lobes in the larvae. (A) Diagram of the components of the ring gland: the prothoracic gland (PG), the corpora allata (CA) and corpora cardiaca (CC). Ecdysone is produced by the PG, whilst the CA is thought to synthesise the Juvenile hormone. (B) A confocal image of a 3rd instar prothoracic gland overexpressing GFP.

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 (Figure 3; (Thummel 1995, 1996, 2001)).

Insect metamorphosis is characterized by vast changes in tissue morphology, where larval tissues are replaced by adult structures. In *Drosophila*, the pulse of ecdysone at the end of the third larval instar initiates metamorphosis (Riddiford 1993). During metamorphosis, an extensive range of larval structures respond to the ecdysone pulse, which triggers the complex array of cellular responses required to achieve conversion from the larval tissue to give the adult (Bender et al. 1997). This begins with the secretion of glue proteins for the larvae to attach itself onto a surface for puparium development. Subsequently ecdysone drives the larval body to shorten, and promotes the subsequent cuticle darkening and hardening required to form the pupal case. During the late larval to early pupal stages the ecdysone pulse drives removal of obsolete larval tissues, such as the larval gut and salivary glands (Thummel 2001).

The major morphological changes for metamorphosis involve development of adult structures from the imaginal discs, which are epithelial structures formed from invaginations of the ectoderm during embryogenesis (Gates and Thummel 2000). The imaginal discs include 2 pairs for the eye/antenna, wing, and halteres, 3 pairs for the legs and a single disc for the gonads, which evert, elongate, and differentiate during metamorphosis

Fig. 1. **The ring gland.** An important organ in *Drosophila* is the ring gland, which is situated in between the two brain lobes in the larvae. (A) Diagram of the components of the ring gland: the prothoracic gland (PG), the corpora allata (CA) and corpora cardiaca (CC). Ecdysone is produced by the PG, whilst the CA is thought to synthesise the Juvenile hormone. (B) A confocal image of a 3rd instar prothoracic gland overexpressing GFP.

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 (Figure

Insect metamorphosis is characterized by vast changes in tissue morphology, where larval tissues are replaced by adult structures. In *Drosophila*, the pulse of ecdysone at the end of the third larval instar initiates metamorphosis (Riddiford 1993). During metamorphosis, an extensive range of larval structures respond to the ecdysone pulse, which triggers the complex array of cellular responses required to achieve conversion from the larval tissue to give the adult (Bender et al. 1997). This begins with the secretion of glue proteins for the larvae to attach itself onto a surface for puparium development. Subsequently ecdysone drives the larval body to shorten, and promotes the subsequent cuticle darkening and hardening required to form the pupal case. During the late larval to early pupal stages the ecdysone pulse drives removal of obsolete larval tissues, such as the larval gut and salivary

The major morphological changes for metamorphosis involve development of adult structures from the imaginal discs, which are epithelial structures formed from invaginations of the ectoderm during embryogenesis (Gates and Thummel 2000). The imaginal discs include 2 pairs for the eye/antenna, wing, and halteres, 3 pairs for the legs and a single disc for the gonads, which evert, elongate, and differentiate during metamorphosis

3; (Thummel 1995, 1996, 2001)).

glands (Thummel 2001).

Fig. 2. **The ecdysone pathway.** Abbreviations: Juvenile hormone (JH), prothoracicotropic hormone (PTTH), Target of Rapamycin (TOR), 20-hydroxyecdysone (20E), ecdysone receptor (EcR), Ultraspiracle (USP), prothoracic gland (PG). References are in blue.

(Alberts 2002). Ecdysone triggers the imaginal disc eversion and elongation, which is accomplished through cell shape changes, rather than by additional cell division (Condic, Fristrom, and Fristrom 1991). As the new appendages emerge from the imaginal discs, the larval tissues undergo programmed cell death and are eventually replaced by the adult structures (Ward et al. 2003). About 12 hours after puparium formation, another major ecdysone pulse initiates the prepupal-pupal transition, and forms the basic body plan of the adult fly. This pulse causes the head to evert from the anterior end of the puparium, the final growth of leg and wings, as well as the removal of most of the remaining larval tissues (Ward et al. 2003). The following 4 days of pupal development involves terminal differentiation of the remaining tissues to form the adult fly (Figure 3; reviewed in (Thummel 2001)).
