Title: Body-Size Regulation: Combining Genetics and Physiology
Abstract: New research has revealed that the activity of the insulin-signaling pathway in the prothoracic gland of Drosophila modulates ecdysone release and thereby influences both the duration and rate of larval growth. New research has revealed that the activity of the insulin-signaling pathway in the prothoracic gland of Drosophila modulates ecdysone release and thereby influences both the duration and rate of larval growth. At its simplest, body size in multicellular organisms is determined by two factors: the duration of growth and the rate of growth. In insects, however, each of these two factors has historically been studied by a different school of biology. Mechanisms controlling the duration of growth have been studied principally by physiologists; but mechanisms controlling the rate of growth have been elucidated largely by geneticists. Perhaps because of this, it is unclear how, or even if, these two factors interact. Now, important research by Caldwell et al. [1Caldwell P.E. Walkiewicz M. Stern M. Ras activity in the Drosophila prothoracic gland regulates body size and developmental rate via ecdysone release.Curr. Biol. 2005; 15 (this issue)Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar] and Mirth et al. [2Mirth C. Truman J.W. Riddiford L.M. The role of the prothoracic gland in determining critical weight for metamorphosis in Drosophila melanogaster.Curr. Biol. 2005; 15 (this issue)Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar] reported in this issue of Current Biology, and Colombani et al. [3Colombani, J., Bianchini, L., Layalle, S., Pondeville, E., Dauphin-Villemant, C., Antoniewski, C., Carré, C., Noselli, S., and Léopold, P. (2005). Antagonistic actions of Ecdysone and Insulins determine final size in Drosophila. Science, 10.1126/science.1119432.Google Scholar] in Science, begins to show how the genetic and physiological controls of body size are related. Their studies show that the activity of the insulin-signaling pathway in the prothoracic gland modulates ecdysone release and influences both the duration and rate of larval growth. The duration of growth in insects is controlled by fluctuating levels of hormones [4Gilbert L.I. Rybczynski R. Warren J.T. Control and biochemical nature of the ecdysteroidogenic pathway.Annu. Rev. Entomol. 2002; 47: 883-916Crossref PubMed Scopus (395) Google Scholar]. Holometabolous insects, such as Drosophila, molt through a series of larval instars before entering a pupal stage, undergoing metamorphosis and emerging as an adult. The larval–pupal transition is initiated by the larvae reaching a 'critical size' in its final instar. By an unknown mechanism, attainment of critical size causes levels of juvenile hormone to fall. This allows prothoracicotropic hormone to be released, which stimulates the prothoracic gland to release ecdysone. It is the subsequent rise in ecdysone levels that triggers metamorphosis. Body growth is restricted to the larval instars and so adult size is fixed at the larval–pupal transition. The rate of growth in insects is controlled largely by the insulin-signaling pathway [5Brogiolo W. Stocker H. Ikeya T. Rintelen F. Fernandez R. Hafen E. An evolutionarily conserved function of the Drosophila insulin receptor and insulin-like peptides in growth control.Curr. Biol. 2001; 11: 213-221Abstract Full Text Full Text PDF PubMed Scopus (914) Google Scholar]. In Drosophila, insulin-like peptides bind to the insulin receptor and activate a signal transduction cascade that regulates the rate of cell growth and division. This cascade includes such enzymes as phosphatidyl inositol 3-kinase (PI 3-kinase, dp110 in Drosophila), Akt and the 'target of rapamycin' (TOR). Activation of this pathway within a particular tissue promotes autonomous growth, whilst non-autonomous growth is regulated by circulating insulin-like peptides. Recent studies have shown that this pathway is crucial in coordinating growth with nutritional conditions, not only in Drosophila but in metazoans in general. The critical size is a key determinant of the final adult size, as it ultimately specifies when a larva stops growing and begins metamorphosis. Almost nothing is known of how the critical size is measured in Drosophila, or how its attainment initiates metamorphosis. It is known that suppression of the insulin-signaling pathway delays attainment of critical size, but does not affect the critical size itself [6Shingleton A.W. Das J. Vinicius L. Stern D.L. The temporal requirements for insulin signaling during development in Drosophila.PLoS Biol. 2005; 3: e289Crossref PubMed Scopus (217) Google Scholar]. One hypothesis, therefore, is that critical size is monitored by some size-assessing organ that grows in response to the insulin-signaling pathway. The organ then initiates metamorphosis when it reaches a certain size. If this were correct, stimulating the insulin-signaling pathway in this organ alone should accelerate the organ's growth and cause it to initiate metamorphosis prematurely. Because the rest of the body is growing at a normal rate, metamorphosis would therefore be initiated in undersized larvae producing small adults. Both Mirth et al. [2Mirth C. Truman J.W. Riddiford L.M. The role of the prothoracic gland in determining critical weight for metamorphosis in Drosophila melanogaster.Curr. Biol. 2005; 15 (this issue)Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar] and Caldwell et al. [1Caldwell P.E. Walkiewicz M. Stern M. Ras activity in the Drosophila prothoracic gland regulates body size and developmental rate via ecdysone release.Curr. Biol. 2005; 15 (this issue)Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar] found that up-regulating PI 3-kinase in the prothoracic gland accelerates its growth and is sufficient to promote early metamorphosis, shortening the larval growth period and reducing adult size. Measurement of ecdysone levels revealed that precocious ecdysone release is responsible for these effects. Conversely, retarding the growth of the prothoracic gland by expressing either dominant negative PI 3-kinase or PTEN, a phosphatase suppressor of the insulin-signaling pathway, has the opposite effect, delaying ecdysone release, lengthening the larval growth period and increasing adult size. These data therefore suggest that the prothoracic gland is the putative critical size organ, and that it initiates metamorphosis when it alone, rather than the rest of the body, has grown to a certain size. As is often the case, however, the story is somewhat more complicated. In addition to altering the size of the prothoracic gland by manipulating PI 3-kinase, Caldwell et al. [1Caldwell P.E. Walkiewicz M. Stern M. Ras activity in the Drosophila prothoracic gland regulates body size and developmental rate via ecdysone release.Curr. Biol. 2005; 15 (this issue)Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar] also up-regulated and down-regulated the Ras–Raf–ErK pathway. This pathway is involved in an array of processes, such as cell proliferation, differentiation and apoptosis, and has a complex relationship with the insulin-signaling pathway; it is partially regulated by the insulin receptor and there appears to be considerable cross-talk between Ras/Raf/ErK and PI 3-kinase/Akt [7Jun T. Gjoerup O. Roberts T.M. Tangled webs: evidence of cross talk between c-Raf-1 and Akt.Sci. STKE. 1999; 13: pe1Google Scholar]. Caldwell et al. [1Caldwell P.E. Walkiewicz M. Stern M. Ras activity in the Drosophila prothoracic gland regulates body size and developmental rate via ecdysone release.Curr. Biol. 2005; 15 (this issue)Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar] found that over-expression of Raf in the prothoracic gland, like that of PI 3-kinase, also initiates premature metamorphosis and reduces final adult size, whilst suppression of Raf has the opposite effect. These phenotypic effects are, however, achieved without any increase or decrease in growth of the prothoracic gland, suggesting that the initiation of metamorphosis by ecdysone is not simply dependent on the size of the prothoracic gland. Other evidence supports this hypothesis. Changing growth of the prothoracic gland using the growth regulators dMyc or cyclinD–Cdk4 was found to have no effect on adult body size [3Colombani, J., Bianchini, L., Layalle, S., Pondeville, E., Dauphin-Villemant, C., Antoniewski, C., Carré, C., Noselli, S., and Léopold, P. (2005). Antagonistic actions of Ecdysone and Insulins determine final size in Drosophila. Science, 10.1126/science.1119432.Google Scholar]. Furthermore, mutations in DHR4, a gene that is expressed in the prothoracic gland and that represses ecdysone-response genes there, also induce premature metamorphosis and a reduction in adult size, apparently without influencing prothoracic gland growth [8King-Jones K. Charles J.P. Lam G. Thummel C.S. The ecdysone-induced DHR4 orphan nuclear receptor coordinates growth and maturation in Drosophila.Cell. 2005; 121: 773-784Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar]. There are several possible interpretations of these data. One possibility is that ecdysone release is regulated in two ways, firstly through insulin-dependent growth of the prothoracic gland and secondly through a Raf-dependent step which, along with DH4R, affects ecdysone synthesis directly. Alternatively, insulin-dependent growth of the prothoracic gland may be unimportant. There is evidence that PI 3-kinase regulates downstream components of the Ras–Raf–ErK pathway in vertebrates [9Chaudhary A. King W.G. Mattaliano M.D. Frost J.A. Diaz B. Morrison D.K. Cobb M.H. Marshall M.S. Brugge J.S. Phosphatidylinositol 3-kinase regulates Raf1 through Pak phosphorylation of serine 338.Curr. Biol. 2000; 10: 551-554Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar]. Expression of PI 3-kinase in the prothoracic gland may therefore regulate the same downstream effectors as expression of Raf, whilst coincidentally promoting growth. The notion that Raf and PI 3-kinase are directly involved in ecdysone synthesis is appealing. Both are regulated by insulin-like peptides, which have been shown to have prothoracicotropic activity in moths [10Nagata K. Maruyama K. Kojima K. Yamamoto M. Tanaka M. Kataoka H. Nagasawa H. Isogai A. Ishizaki H. Suzuki A. Prothoracicotropic activity of SBRPs, the insulin-like peptides of the saturniid silkworm Samia cynthia ricini.Biochem. Biophys. Res. Commun. 1999; 266: 575-578Crossref PubMed Scopus (22) Google Scholar] and which can initiate ecdysone production in the ovaries of mosquitoes [11Riehle M.A. Brown M.R. Insulin stimulates ecdysteroid production through a conserved signaling cascade in the mosquito Aedes aegypti.Insect Biochem. Mol. Biol. 1999; 29: 855-860Crossref PubMed Scopus (129) Google Scholar]. It is not known whether the manipulations by Mirth et al. [2Mirth C. Truman J.W. Riddiford L.M. The role of the prothoracic gland in determining critical weight for metamorphosis in Drosophila melanogaster.Curr. Biol. 2005; 15 (this issue)Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar] and Caldwell et al. [1Caldwell P.E. Walkiewicz M. Stern M. Ras activity in the Drosophila prothoracic gland regulates body size and developmental rate via ecdysone release.Curr. Biol. 2005; 15 (this issue)Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar] stimulate the changes in either juvenile hormone or prothoracicotropic hormone that normally precede the release of ecdysone. The manipulations may side-step the upstream regulatory mechanisms that typically control the timing of metamorphosis by influencing ecdysone synthesis directly. At least one regulatory mechanism appears to be being circumscribed by the manipulations: under-developed or continuously growing imaginal discs (developing adult appendages) can inhibit ecdysone release and delay metamorphosis [12Sehnal F. Bryant P.J. Delayed pupariation in Drosophila imaginal disc overgrowth mutants Is associated with reduced ecdysteroid titer.J. Insect Physiol. 1993; 39: 1051-1059Crossref Scopus (27) Google Scholar]. This does not appear to be the case in larvae in which PI 3-kinase or Raf is upregulated in the prothoracic gland. There remains the possibility, therefore, that there are other critical size organs also responsible for regulating the timing of metamorphosis but which lie upstream of the prothoracic gland. Surprisingly, manipulations of the prothoracic gland were also found to affect the rate of growth in developing larvae. Larvae in which either Raf or PI 3-kinase is up-regulated in the prothoracic gland show a significant decrease in their rate of growth during the third larval instar [1Caldwell P.E. Walkiewicz M. Stern M. Ras activity in the Drosophila prothoracic gland regulates body size and developmental rate via ecdysone release.Curr. Biol. 2005; 15 (this issue)Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 2Mirth C. Truman J.W. Riddiford L.M. The role of the prothoracic gland in determining critical weight for metamorphosis in Drosophila melanogaster.Curr. Biol. 2005; 15 (this issue)Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar, 3Colombani, J., Bianchini, L., Layalle, S., Pondeville, E., Dauphin-Villemant, C., Antoniewski, C., Carré, C., Noselli, S., and Léopold, P. (2005). Antagonistic actions of Ecdysone and Insulins determine final size in Drosophila. Science, 10.1126/science.1119432.Google Scholar]. This growth suppression is, however, rescued by nutritionally supplementing the diet of developing larvae, at least when PI 3-kinase is expressed in the prothoracic gland [2Mirth C. Truman J.W. Riddiford L.M. The role of the prothoracic gland in determining critical weight for metamorphosis in Drosophila melanogaster.Curr. Biol. 2005; 15 (this issue)Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar]. In contrast, down-regulation of PI 3-kinase in the prothoracic gland suppresses larval growth [3Colombani, J., Bianchini, L., Layalle, S., Pondeville, E., Dauphin-Villemant, C., Antoniewski, C., Carré, C., Noselli, S., and Léopold, P. (2005). Antagonistic actions of Ecdysone and Insulins determine final size in Drosophila. Science, 10.1126/science.1119432.Google Scholar]. How might expressing Raf or PI3 kinase in the prothoracic gland influence overall growth rate? It appears that driving PI 3-kinase or Raf expression in the prothoracic gland not only accelerates the peak of ecdysone that initiates metamorphosis, but also raises basal levels of ecdysone [3Colombani, J., Bianchini, L., Layalle, S., Pondeville, E., Dauphin-Villemant, C., Antoniewski, C., Carré, C., Noselli, S., and Léopold, P. (2005). Antagonistic actions of Ecdysone and Insulins determine final size in Drosophila. Science, 10.1126/science.1119432.Google Scholar]. Recent work has shown that ecdysone down-regulates PI 3-kinase signaling in the fat body [13Rusten T.E. Lindmo K. Juhasz G. Sass M. Seglen P.O. Brech A. Stenmark H. Programmed autophagy in the Drosophila fat body is induced by ecdysone through regulation of the PI3K pathway.Dev. Cell. 2004; 7: 179-192Abstract Full Text Full Text PDF PubMed Scopus (380) Google Scholar]. The fat body functions as a nutrient sensor and is involved in the coordination of organismal growth. Importantly, amino acid starvation of the fat body alone not only causes a slight decrease in PI 3-kinase activity there, but also shuts down PI 3-kinase activity in other tissues and reduces larval growth, through an unknown humoral mechanism [14Colombani J. Raisin S. Pantalacci S. Radimerski T. Montagne J. Leopold P. A nutrient sensor mechanism controls Drosophila growth.Cell. 2003; 114: 739-749Abstract Full Text Full Text PDF PubMed Scopus (597) Google Scholar]. Ecdysone-mediated suppression of the PI3 kinase signaling in the fat body may therefore have a similar effect. Evidence reported by Colombani et al. [3Colombani, J., Bianchini, L., Layalle, S., Pondeville, E., Dauphin-Villemant, C., Antoniewski, C., Carré, C., Noselli, S., and Léopold, P. (2005). Antagonistic actions of Ecdysone and Insulins determine final size in Drosophila. Science, 10.1126/science.1119432.Google Scholar] suggests that this is the case. They found that ecdysone-fed larvae show a decrease in PI 3-kinase activity in their fat body; inhibiting the effects of ecdsyone on the fat body, by silencing the ecdysone receptor there, removes these effects and leads to an increase in larval growth rate. The possibility remains, however, that ecdysone also acts directly on developing tissues and regulates their PI 3-kinase activity independent of the fat body. The effect of increased ecdysone release on a larva's growth rate appears to be countered by enhancing the nutrient content of its diet [2Mirth C. Truman J.W. Riddiford L.M. The role of the prothoracic gland in determining critical weight for metamorphosis in Drosophila melanogaster.Curr. Biol. 2005; 15 (this issue)Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar]. Increased nutrition up-regulates the insulin-signaling pathway, both cell autonomously and via the nutrient-dependent release of insulin-like peptides. Further, the ecdysone suppression of growth rate is lost in dFOXO mutants [3Colombani, J., Bianchini, L., Layalle, S., Pondeville, E., Dauphin-Villemant, C., Antoniewski, C., Carré, C., Noselli, S., and Léopold, P. (2005). Antagonistic actions of Ecdysone and Insulins determine final size in Drosophila. Science, 10.1126/science.1119432.Google Scholar]. dFOXO is a negative growth regulator itself inhibited by PI 3- kinase, so dFOXO mutants effectively have constitutively activated insulin signaling. Ecdysone and insulin appear, therefore, to counteract each other's effects on growth in Drosophila larvae [3Colombani, J., Bianchini, L., Layalle, S., Pondeville, E., Dauphin-Villemant, C., Antoniewski, C., Carré, C., Noselli, S., and Léopold, P. (2005). Antagonistic actions of Ecdysone and Insulins determine final size in Drosophila. Science, 10.1126/science.1119432.Google Scholar]. These new studies [1Caldwell P.E. Walkiewicz M. Stern M. Ras activity in the Drosophila prothoracic gland regulates body size and developmental rate via ecdysone release.Curr. Biol. 2005; 15 (this issue)Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 2Mirth C. Truman J.W. Riddiford L.M. The role of the prothoracic gland in determining critical weight for metamorphosis in Drosophila melanogaster.Curr. Biol. 2005; 15 (this issue)Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar, 3Colombani, J., Bianchini, L., Layalle, S., Pondeville, E., Dauphin-Villemant, C., Antoniewski, C., Carré, C., Noselli, S., and Léopold, P. (2005). Antagonistic actions of Ecdysone and Insulins determine final size in Drosophila. Science, 10.1126/science.1119432.Google Scholar] provide important insights into the mechanisms regulating body size in insects, and in metazoans in general. Of course, they also raise many questions. Nevertheless, such research begins the essential process of unifying our genetic and physiological understanding of growth and development. Only through such integration will we understand why flies are the size that they are, and why we are the size that we are.