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

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 (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

Steroid Hormones in *Drosophila*:

**2.2 PTTH regulates of ecdysone levels** 

How Ecdysone Coordinates Developmental Signalling with Cell Growth and Division 147

the timing of the larval-pupal transition and metamorphosis. As the adult fly size is determined by the size of the larvae at the pupal molt, the timing of ecdysone release plays a vital role in the growth of the fly (reviewed in (King-Jones and Thummel 2005)). Studies in 2005 demonstrated the importance of the size of the PG and its effect on ecdysone production and, therefore, determination of the final adult fly size (Caldwell, Walkiewicz, and Stern 2005; Colombani et al. 2005; Mirth, Truman, and Riddiford 2005). Specifically, these groups reported a role for insulin signalling in the PG, and also characterised a sizeassessing feature of the PG (Figure 3). As a size-assessment tissue, inhibiting the growth of the PG causes an underestimation of body size and results in pupation at a larger size, whereas promoting this tissue's growth results in smaller flies (Mirth, Truman, and Riddiford 2005). Consistent with this, overexpression of activated *PI3K* or *Ras* (*RasV12*), both key components of growth control pathways in flies and mammals, specifically in the PG resulted in a larger PG but reduced the pupal and adult size (Caldwell, Walkiewicz, and Stern 2005; Colombani et al. 2005; Mirth, Truman, and Riddiford 2005), which we have recapitulated as shown in Figure 3 (compare 3B with 3A). Conversely, overexpression of a dominant negative isoform of *PI3K* (*Dp110DN*) reduced the PG size but resulted in larger pupae and adults, due to an extended larval growth period (Figure 3, compare C with A). Furthermore, through measurements of the ecdysone target *E74B* or through an enzyme immunoassay for ecdysteroid titres, it was shown that the extended larval growth period was due to reduced ecdysone levels, which was most likely a result of a smaller PG (Caldwell, Walkiewicz, and Stern 2005; Colombani et al. 2005; Mirth, Truman, and Riddiford 2005).

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).

**2.3 Juvenile hormone controls PTTH release and ecdysone production** 

The signals required for metamorphosis have been extensively studied in the tobacco hornworm *Manduca sexta*. In this insect, the pulse of ecdysone in the last larval instar is inhibited by another hormone, the Juvenile Hormone (JH). In the case of JH, levels need to

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 for controlling cell growth and division in *Drosophila*.
