**2.1 The prothoracic gland directs body size**

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 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).
