**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

Steroid Hormones in *Drosophila*:

body, but not in the wing disc during pupariation.

Arquier, and Leopold 2008; reviewed in (Nijhout 2008)).

imaginal tissues to the ecdysone titre.

(Herranz et al. 2008; Johnston et al. 1999).

How Ecdysone Coordinates Developmental Signalling with Cell Growth and Division 149

reduced levels of *dmyc* promoter activity (Cranna and Quinn 2009). Thus the effect of ecdysone on this key growth regulator appears to be both 1) developmental-stage specific; being required for *dmyc* activation in the wing at the earlier growth phase but not after pupariation and 2) tissue specific; resulting in downregulation of *dmyc* expression in the fat

The lack of consensus binding sites for EcR/Usp (EcREs) in the *dmyc* promoter region suggests that *dmyc* is not a direct target of EcR-mediated gene repression in the fat body or activation in the wing, but rather that EcR signalling indirectly controls *dmyc* transcription. Although the fat-specific target of EcR leading to altered *dmyc* expression is unknown, in the wing imaginal disc EcR has been shown to modulate levels of the Wingless morphogen (Mitchell et al. 2008), which in turn can lead to downregulation of *dmyc* transcription

**2.5 Interplay between insulin pathway and ecdysone determines final body size**  Taken together the above findings suggest that the insulin-signalling pathway acts in the prothoracic gland (PG) to regulate the release of ecdysone and determine the length of the larval growth period (Caldwell, Walkiewicz, and Stern 2005; Colombani et al. 2005; King-Jones et al. 2005; Mirth, Truman, and Riddiford 2005; Shingleton 2005; Prober and Edgar 2002). For instance, increased PG growth occurs when PI3-kinase (PI3K, a downstream regulator of the insulin pathway) is upregulated in the PG (Caldwell, Walkiewicz, and Stern 2005; Mirth, Truman, and Riddiford 2005). The PG overgrowth causes accelerated metamorphosis, which results in reduced adult size due to the rapid progression through the larval growth stage. Precocious ecdysone release, as measured by premature increase in levels of the early response ecdysone genes, correlates with this disruption to larval growth. Conversely, reducing growth of the PG, using a dominant negative form of PI3K, results in longer larval growth periods and larger adults due to slower ecdysone release and delayed onset of pupariation. More recently it has been shown that Target of Rapamycin (TOR) may link the ecdysone-regulated development to the PI3K mediated growth pathways (Layalle,

The levels of ecdysone release are therefore inversely proportional to larval growth and adult body size; with early onset of the ecdysone peak giving small flies and reduced ecdysone prolonging the growth period to give larger adults. Thus the time spent in the larval growth phase is a critical determinant of body size, with longer growth periods resulting in more cell division cycles and delayed onset of differentiation. In the next section we address the question of how the ecdysone pulse works to affect rates of cell growth and cell cycle progression within specific larval tissues. In particular we discuss the developmental signalling pathways implicated in linking cell cycle patterning of larval

**3. Cell intrinsic roles for ecdysone, EcR and USP in cell growth and division**  The *Drosophila* imaginal discs (see also Introduction 1.1), which form the adult head structures (eyes and antenna), appendages (wings and legs) and genitalia, have provided an excellent model for studying developmental signals controlling cell proliferation. The imaginal disc precursor cells arise early in embryonic development from invaginations of the embryonic epithelium (Alberts 2002). By the early larval stage each disc consists of a ball

drop below a threshold for metamorphosis to begin (Nijhout and Williams 1974, 1974; Dominick and Truman 1985). Whether the drop in JH abundance signals the attainment of critical weight, which defines the larval size response to starvation (Davidowitz, D'Amico, and Nijhout 2003), or reaching critical weight initiates the drop in JH levels is unclear. However, at least in *Manduca*, a drop of JH levels below a critical threshold is required for PTTH to be released and activate the production of ecdysone to start metamorphosis. As pupae do not receive any additional nutrition, the transition into pupation marks the termination of larval growth and establishes the final adult size.

For *Drosophila*, the role of JH in regulating PTTH is not as well defined. Studies suggests PTTH may operate upstream to set the critical weight as loss of PTTH results in an increase in critical weight and an extended developmental delay (Figure 3; (McBrayer et al. 2007)). Control of developmental timing is likely achieved by minor pulses of PTTH and subsequent ecdysone pulses, which occur prior to the major ecdysone peak. This is consistent with the observation that loss of PTTH impairs ecdysone release and leads to developmental delays and larger adult flies (McBrayer et al. 2007). As ecdysone levels determine the transition from each developmental stage the PG, therefore, plays a critical role in regulating *Drosophila* organ and tissue growth.

## **2.4 Ecdysone controls animal growth rate via the fat body**

In holometabolous insects, growth is mainly restricted to the larval period and maturation occurs during metamorphosis or pupal development. In all multicellular animals, tissue growth relies on the insulin-signalling pathway, which couples nutrition with growth (Edgar 2006; Britton et al. 2002). A recent study suggests an ecdysone-dependent control mechanism for restricting growth to the juvenile period, where ecdysone controls growth rate via effects on the growth regulator Myc in the fat body (Delanoue, Slaidina, and Leopold 2010). The fat body, which is functionally homologous to the vertebrate liver, appears to act as a relay tissue for the control of larval growth by circulating ecdysone. Loss of Ecdysone receptor (EcR) function in fat body increases dMyc expression and its ability to upregulate growth by increasing ribosome biogenesis and protein translation. Together with RNA profiling of dissected fat bodies, this suggests that EcR signalling represses dMyc and its downstream targets. Importantly, manipulation of dMyc levels in the fat body is sufficient to affect animal growth-rate. In addition, the downregulation of dMyc in fat cells is required for growth inhibition by ecdysone as the growth increase induced by silencing EcR in the fat body is suppressed by cosilencing dMyc. This work suggests a model where the rise of ecdysone levels at the end of the juvenile period represses dMyc expression in the fat body. This steroid hormone-dependent inhibition restricts ribosome biosynthesis and translation efficiency in fat cells via dMyc and, therefore, induces a general pause in the growth program that precedes entry into metamorphosis.

The ability of circulating ecdysone to control dMyc expression during the pupal stage was found to be specific to the fat body. For example, *dmyc* mRNA levels were elevated in fat body after reducing the level of circulating ecdysone via inhibition of PI3K pathway in the prothoracic gland, but at this stage *dmyc* levels are not altered in wing imaginal discs (Delanoue, Slaidina, and Leopold 2010). Interestingly, inhibition of ecdysone gene activation at the earlier 3rd larval instar stage revealed that EcR function is actually required for normal levels of *dmyc* transcription in some tissues. In these studies, blocking the ecdysone pathway in wing imaginal disc cells using EcR dominant negative (dN) transgenes results in

drop below a threshold for metamorphosis to begin (Nijhout and Williams 1974, 1974; Dominick and Truman 1985). Whether the drop in JH abundance signals the attainment of critical weight, which defines the larval size response to starvation (Davidowitz, D'Amico, and Nijhout 2003), or reaching critical weight initiates the drop in JH levels is unclear. However, at least in *Manduca*, a drop of JH levels below a critical threshold is required for PTTH to be released and activate the production of ecdysone to start metamorphosis. As pupae do not receive any additional nutrition, the transition into pupation marks the

For *Drosophila*, the role of JH in regulating PTTH is not as well defined. Studies suggests PTTH may operate upstream to set the critical weight as loss of PTTH results in an increase in critical weight and an extended developmental delay (Figure 3; (McBrayer et al. 2007)). Control of developmental timing is likely achieved by minor pulses of PTTH and subsequent ecdysone pulses, which occur prior to the major ecdysone peak. This is consistent with the observation that loss of PTTH impairs ecdysone release and leads to developmental delays and larger adult flies (McBrayer et al. 2007). As ecdysone levels determine the transition from each developmental stage the PG, therefore, plays a critical

In holometabolous insects, growth is mainly restricted to the larval period and maturation occurs during metamorphosis or pupal development. In all multicellular animals, tissue growth relies on the insulin-signalling pathway, which couples nutrition with growth (Edgar 2006; Britton et al. 2002). A recent study suggests an ecdysone-dependent control mechanism for restricting growth to the juvenile period, where ecdysone controls growth rate via effects on the growth regulator Myc in the fat body (Delanoue, Slaidina, and Leopold 2010). The fat body, which is functionally homologous to the vertebrate liver, appears to act as a relay tissue for the control of larval growth by circulating ecdysone. Loss of Ecdysone receptor (EcR) function in fat body increases dMyc expression and its ability to upregulate growth by increasing ribosome biogenesis and protein translation. Together with RNA profiling of dissected fat bodies, this suggests that EcR signalling represses dMyc and its downstream targets. Importantly, manipulation of dMyc levels in the fat body is sufficient to affect animal growth-rate. In addition, the downregulation of dMyc in fat cells is required for growth inhibition by ecdysone as the growth increase induced by silencing EcR in the fat body is suppressed by cosilencing dMyc. This work suggests a model where the rise of ecdysone levels at the end of the juvenile period represses dMyc expression in the fat body. This steroid hormone-dependent inhibition restricts ribosome biosynthesis and translation efficiency in fat cells via dMyc and, therefore, induces a general pause in the

The ability of circulating ecdysone to control dMyc expression during the pupal stage was found to be specific to the fat body. For example, *dmyc* mRNA levels were elevated in fat body after reducing the level of circulating ecdysone via inhibition of PI3K pathway in the prothoracic gland, but at this stage *dmyc* levels are not altered in wing imaginal discs (Delanoue, Slaidina, and Leopold 2010). Interestingly, inhibition of ecdysone gene activation at the earlier 3rd larval instar stage revealed that EcR function is actually required for normal levels of *dmyc* transcription in some tissues. In these studies, blocking the ecdysone pathway in wing imaginal disc cells using EcR dominant negative (dN) transgenes results in

termination of larval growth and establishes the final adult size.

role in regulating *Drosophila* organ and tissue growth.

**2.4 Ecdysone controls animal growth rate via the fat body** 

growth program that precedes entry into metamorphosis.

reduced levels of *dmyc* promoter activity (Cranna and Quinn 2009). Thus the effect of ecdysone on this key growth regulator appears to be both 1) developmental-stage specific; being required for *dmyc* activation in the wing at the earlier growth phase but not after pupariation and 2) tissue specific; resulting in downregulation of *dmyc* expression in the fat body, but not in the wing disc during pupariation.

The lack of consensus binding sites for EcR/Usp (EcREs) in the *dmyc* promoter region suggests that *dmyc* is not a direct target of EcR-mediated gene repression in the fat body or activation in the wing, but rather that EcR signalling indirectly controls *dmyc* transcription. Although the fat-specific target of EcR leading to altered *dmyc* expression is unknown, in the wing imaginal disc EcR has been shown to modulate levels of the Wingless morphogen (Mitchell et al. 2008), which in turn can lead to downregulation of *dmyc* transcription (Herranz et al. 2008; Johnston et al. 1999).
