**2.2 PTTH regulates of ecdysone levels**

146 Steroids – Basic Science

Thummel 1997; Yin and Thummel 2005), the relationship between ecdysone and cell cycle is a relatively unexplored field. Here we review the evidence that the ecdysone pulse is critical

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 (Johnston et al. 1999), the essential G1 to S-phase Cyclin complex, Cyclin E (CycE) and its Cyclin-dependent-kinase (Cdk) partner Cdk2, which triggers S-phase by promoting DNA replication (Knoblich et al. 1994; Neufeld et al. 1998; Richardson et al. 1995), 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 (Edgar and Datar 1996). 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 (Neufeld et al. 1998). dE2F1 responds to the relevant Cdk-Cyclin complex (CycE/Cdk2 for S-phase and CycB/Cdk1 for mitosis) to coordinate cell cycle progression

During metamorphosis, following removal of the obsolete larval structures, proliferation of the remaining tissue 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 the remaining tissue to form the adult midgut (Jiang, Baehrecke, and Thummel 1997). 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 (Li and White 2003). Analysis of *EcR* null mutants also 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 body of this chapter will discuss how the ecdysone pulse achieves changes to cell growth and cell cycle progression. First we will describe how ecdysone levels dictate body size cell extrinsically by controlling developmental timing. Then we will discuss how ecdysone works with its receptors, in a tissue autonomous manner to control transcription of cell cycle genes, which most likely occurs indirectly by modifying the

**2. Cell extrinsic effects of ecdysone on larval growth and body size** 

The ecdysone pulse can act indirectly to affect larval growth as a consequence of the link between the ecdysone titre and developmental timing. Here we will discuss how cell extrinsic effects of the ecdysone pathway control *Drosophila* larval growth and final body size non-autonomously, at least in part, through interactions between the ecdysone and insulin pathways (King-Jones and Thummel 2005; Shingleton 2005; Mirth and Riddiford

The prothoracic gland (PG) is tightly associated with the developmental timing of all holometabolous insects, including *Drosophila*, as it produces the ecdysone pulse that dictates

for controlling cell growth and division in *Drosophila*.

from G1 to S-phase and G2 into mitosis (Reis and Edgar 2004).

activity of developmental signalling pathways.

**2.1 The prothoracic gland directs body size** 

2007; Nijhout 2008).

**1.4 Linking the Ecdysone pulse to cell cycle** 

In insects, the production and release of ecdysone is responsive to the prothoracicotropic hormone (PTTH), a small, secreted peptide. PTTH is thought to induce the transcription of ecdysone biosynthetic genes that encode enzymes driving the series of dehydrogenation and hydroxylation reactions required to synthesise the active metabolite 20E from the cholesterol precursor (Marchal et al. 2010). In *Drosophila* PTTH is produced by a pair of bilateral neurosecretory cells in the brain, which innervate the prothoracic gland (PG) ((Figure 2; (McBrayer et al. 2007)). PTTH is expressed throughout 3rd instar in an 8 hour cyclic pattern, with upregulation noticed around 12 hours before pupariation (McBrayer et al. 2007). Ablation of the neurons that produce PTTH results in a 5-day developmental delay in the onset of pupariation, larger 3rd instar larvae and pupae, and adults with larger wings due to increased cell number. In line with the predicted role for PTTH in modulating ecdysone synthesis and release, larvae lacking PTTH producing neurons have reduced ecdysone titres. This suggests PTTH normally modulates ecdysone levels to coordinate larval growth with the onset of metamorphosis. However, as the ecdysone levels still eventually peak in larvae with ablated neurons, PTTH may not be the sole factor required for increasing ecdysone titres (McBrayer et al. 2007). Thus PTTH might be required in addition to the insulin-dependent growth pathways discussed above, to coordinate larval growth with ecdysone-induced moulting and metamorphosis (Figure 2-3).
