A New Role for Neuropeptide F Signaling in Controlling Developmental Timing and Body Size in Drosophila melanogaster.

As juvenile animals grow, their behavior, physiology, and development need to be matched to environmental conditions to ensure they survive to adulthood. However, we know little about how behavior and physiology are integrated with development to achieve this outcome. Neuropeptides are prime candidates for achieving this due to their well-known signaling functions in controlling many aspects of behavior, physiology, and development in response to environmental cues. In the growing Drosophila larva, while several neuropeptides have been shown to regulate feeding behavior, and a handful to regulate growth, it is unclear if any of these play a global role in coordinating feeding behavior with developmental programs. Here, we demonstrate that Neuropeptide F Receptor (NPFR), best studied as a conserved regulator of feeding behavior from insects to mammals, also regulates development in Drosophila Knocking down NPFR in the prothoracic gland, which produces the steroid hormone ecdysone, generates developmental delay and an extended feeding period, resulting in increased body size. We show that these effects are due to decreased ecdysone production, as these animals have reduced expression of ecdysone biosynthesis genes and lower ecdysone titers. Moreover, these phenotypes can be rescued by feeding larvae food supplemented with ecdysone. Further, we show that NPFR negatively regulates the insulin signaling pathway in the prothoracic gland to achieve these effects. Taken together, our data demonstrate that NPFR signaling plays a key role in regulating animal development, and may, thus, play a global role in integrating feeding behavior and development in Drosophila.

Torso-Like Is a Component of the Hemolymph and Regulates the Insulin Signaling Pathway in Drosophila.

In Drosophila, key developmental transitions are governed by the steroid hormone ecdysone. A number of neuropeptide-activated signaling pathways control ecdysone production in response to environmental signals, including the insulin signaling pathway, which regulates ecdysone production in response to nutrition. Here, we find that the Membrane Attack Complex/Perforin-like protein Torso-like, best characterized for its role in activating the Torso receptor tyrosine kinase in early embryo patterning, also regulates the insulin signaling pathway in Drosophila We previously reported that the small body size and developmental delay phenotypes of torso-like null mutants resemble those observed when insulin signaling is reduced. Here we report that, in addition to growth defects, torso-like mutants also display metabolic and nutritional plasticity phenotypes characteristic of mutants with impaired insulin signaling. We further find that in the absence of torso-like, the expression of insulin-like peptides is increased, as is their accumulation in insulin-producing cells. Finally, we show that Torso-like is a component of the hemolymph and that it is required in the prothoracic gland to control developmental timing and body size. Taken together, our data suggest that the secretion of Torso-like from the prothoracic gland influences the activity of insulin signaling throughout the body in Drosophila.

Genome-Wide Screen for New Components of the Drosophila melanogaster Torso Receptor Tyrosine Kinase Pathway.

Patterning of the Drosophila embryonic termini by the Torso (Tor) receptor pathway has long served as a valuable paradigm for understanding how receptor tyrosine kinase signaling is controlled. However, the mechanisms that underpin the control of Tor signaling remain to be fully understood. In particular, it is unclear how the Perforin-like protein Torso-like (Tsl) localizes Tor activity to the embryonic termini. To shed light on this, together with other aspects of Tor pathway function, we conducted a genome-wide screen to identify new pathway components that operate downstream of Tsl. Using a set of molecularly defined chromosomal deficiencies, we screened for suppressors of ligand-dependent Tor signaling induced by unrestricted Tsl expression. This approach yielded 59 genomic suppressor regions, 11 of which we mapped to the causative gene, and a further 29 that were mapped to <15 genes. Of the identified genes, six represent previously unknown regulators of embryonic Tor signaling. These include twins (tws), which encodes an integral subunit of the protein phosphatase 2A complex, and α-tubulin at 84B (αTub84B), a major constituent of the microtubule network, suggesting that these may play an important part in terminal patterning. Together, these data comprise a valuable resource for the discovery of new Tor pathway components. Many of these may also be required for other roles of Tor in development, such as in the larval prothoracic gland where Tor signaling controls the initiation of metamorphosis.

MACPF/CDC proteins in development: Insights from Drosophila torso-like.

The Membrane Attack Complex Perforin-like/Cholesterol-Dependent Cytolysin (MACPF) superfamily is an ancient and biologically diverse group of proteins that are best known for pore-forming roles in mammalian immunity and bacterial pathogenesis. Intriguingly, however, some eukaryotic proteins which contain the MACPF domain that defines this family do not act in attack or defence, and instead have distinct developmental functions. It remains unclear whether these proteins function via pore formation or have a different mechanism of action. Of these, by far the best characterised is Torso-like (Tsl), the only MACPF member that has been identified in the fruit fly, Drosophila melanogaster. While it has long been known to have a role in embryonic patterning, recent studies have shown that Tsl in fact has multiple roles in development. As such, it presents an excellent opportunity to investigate how the MACPF domain functions in a developmental context. Here, we review what is known about Tsl in Drosophila and other insects, and discuss the potential molecular mechanism by which Tsl and thus other developmental MACPF proteins may function.

Torso-like mediates extracellular accumulation of Furin-cleaved Trunk to pattern the Drosophila embryo termini.

Patterning of the Drosophila embryonic termini is achieved by localized activation of the Torso receptor by the growth factor Trunk. Governing this event is the perforin-like protein Torso-like, which is localized to the extracellular space at the embryo poles and has long been proposed to control localized proteolytic activation of Trunk. However, a protease involved in terminal patterning remains to be identified, and the role of Torso-like remains unknown. Here we find that Trunk is cleaved intracellularly by Furin proteases. We further show that Trunk is secreted, and that levels of extracellular Trunk are greatly reduced in torso-like null mutants. On the basis of these and previous findings, we suggest that Torso-like functions to mediate secretion of Trunk, thus providing the mechanism for spatially restricted activation of Torso. Our data represent an alternative mechanism for the spatial control of receptor signalling, and define a different role for perforin-like proteins in eukaryotes.

High resolution structure of cleaved Serpin 42 Da from Drosophila melanogaster.

BACKGROUND: The Drosophila melanogaster Serpin 42 Da gene (previously Serpin 4) encodes a serine protease inhibitor that is capable of remarkable functional diversity through the alternative splicing of four different reactive centre loop exons. Eight protein isoforms of Serpin 42 Da have been identified to date, targeting the protease inhibitor to both different proteases and cellular locations. Biochemical and genetic studies suggest that Serpin 42 Da inhibits target proteases through the classical serpin 'suicide' inhibition mechanism, however the crystal structure of a representative Serpin 42 Da isoform remains to be determined. RESULTS: We report two high-resolution crystal structures of Serpin 42 Da representing the A/B isoforms in the cleaved conformation, belonging to two different space-groups and diffracting to 1.7 Å and 1.8 Å. Structural analysis reveals the archetypal serpin fold, with the major elements of secondary structure displaying significant homology to the vertebrate serpin, neuroserpin. Key residues known to have central roles in the serpin inhibitory mechanism are conserved in both the hinge and shutter regions of Serpin 42 Da. Furthermore, these structures identify important conserved interactions that appear to be of crucial importance in allowing the Serpin 42 Da fold to act as a versatile template for multiple reactive centre loops that have different sequences and protease specificities. CONCLUSIONS: In combination with previous biochemical and genetic studies, these structures confirm for the first time that the Serpin 42 Da isoforms are typical inhibitory serpin family members with the conserved serpin fold and inhibitory mechanism. Additionally, these data reveal the remarkable structural plasticity of serpins, whereby the basic fold is harnessed as a template for inhibition of a large spectrum of proteases by reactive centre loop exon 'switching'. This is the first structure of a Drosophila serpin reported to date, and will provide a platform for future mutational studies in Drosophila to ascertain the functional role of each of the Serpin 42 Da isoforms.

Trunk cleavage is essential for Drosophila terminal patterning and can occur independently of Torso-like.

Terminal patterning in Drosophila is governed by a localized interaction between the Torso kinase (Tor) and its ligand Trunk (Trk). Currently, it is proposed that Trk must be cleaved in order to bind Tor, and that these proteolytic events are controlled by secretion of Torso-like (Tsl) only at the embryo poles. However, controversy surrounds these ideas since neither cleaved Trk nor a protease that functions in terminal patterning have been identified. Here we show that Trk is cleaved multiple times in vivo and that these proteolytic events are essential for its function. Unexpectedly, however, the Trk cleavage patterns we observe are unaltered in tsl-null mutants. One explanation for these data is that the influence of Tsl on localized Trk cleavage at the embryo poles is subtle and cannot be readily detected. Alternatively, we favour a scenario where Tsl functions post proteolytic processing of Trk to control localized terminal patterning.

Torso-like functions independently of Torso to regulate Drosophila growth and developmental timing.

Activation of the Drosophila receptor tyrosine kinase Torso (Tor) only at the termini of the embryo is achieved by the localized expression of the maternal gene Torso-like (Tsl). Tor has a second function in the prothoracic gland as the receptor for prothoracicotropic hormone (PTTH) that initiates metamorphosis. Consistent with the function of Tor in this tissue, Tsl also localizes to the prothoracic gland and influences developmental timing. Despite these commonalities, in our studies of Tsl we unexpectedly found that tsl and tor have opposing effects on body size; tsl null mutants are smaller than normal, rather than larger as would be expected if the PTTH/Tor pathway was disrupted. We further found that whereas both genes regulate developmental timing, tsl does so independently of tor. Although tsl null mutants exhibit a similar length delay in time to pupariation to tor mutants, in tsl:tor double mutants this delay is strikingly enhanced. Thus, loss of tsl is additive rather than epistatic to loss of tor. We also find that phenotypes generated by ectopic PTTH expression are independent of tsl. Finally, we show that a modified form of tsl that can rescue developmental timing cannot rescue terminal patterning, indicating that Tsl can function via distinct mechanisms in different contexts. We conclude that Tsl is not just a specialized cue for Torso signaling but also acts independently of PTTH/Tor in the control of body size and the timing of developmental progression. These data highlight surprisingly diverse developmental functions for this sole Drosophila member of the perforin-like superfamily.