Deletion of Drosophila insulin-like peptides causes growth defects and metabolic abnormalities.

Insulin/Insulin-like growth factor signaling regulates homeostasis and growth in mammals, and is implicated in diseases from diabetes to cancer. In Drosophila melanogaster, as in other invertebrates, multiple Insulin-Like Peptides (DILPs) are encoded by a family of related genes. To assess DILPs' physiological roles, we generated small deficiencies that uncover single or multiple dilps, generating genetic loss-of-function mutations. Deletion of dilps1-5 generated homozygotes that are small, severely growth-delayed, and poorly viable and fertile. These animals display reduced metabolic activity, decreased triglyceride levels and prematurely activate autophagy, indicative of "starvation in the midst of plenty," a hallmark of Type I diabetes. Furthermore, circulating sugar levels are elevated in Df [dilp1-5] homozygotes during eating and fasting. In contrast, Df[dilp6] or Df[dilp7] animals showed no major metabolic defects. We discuss physiological differences between mammals and insects that may explain the unexpected survival of lean, 'diabetic' flies.

The B' protein phosphatase 2A regulatory subunit well-rounded regulates synaptic growth and cytoskeletal stability at the Drosophila neuromuscular junction.

Synaptic growth is essential for the development and plasticity of neural circuits. To identify molecular mechanisms regulating synaptic growth, we performed a gain-of-function screen for synapse morphology mutants at the Drosophila neuromuscular junction (NMJ). We isolated a B' regulatory subunit of protein phosphatase 2A (PP2A) that we have named well-rounded (wrd). Neuronal overexpression of wrd leads to overgrowth of the synaptic terminal. Endogenous Wrd protein is present in the larval nervous system and muscle and is enriched at central and neuromuscular synapses. wrd is required for normal synaptic development; in its absence, there are fewer synaptic boutons and there is a decrease in synaptic strength. wrd functions presynaptically to promote normal synaptic growth and postsynaptically to maintain normal levels of evoked transmitter release. In the absence of wrd, the presynaptic cytoskeleton is abnormal, with an increased proportion of unbundled microtubules. Reducing PP2A enzymatic activity also leads to an increase in unbundled microtubules, an effect enhanced by reducing wrd levels. Hence, wrd promotes the function of PP2A and is required for normal cytoskeletal organization, synaptic growth, and synaptic function at the Drosophila NMJ.

Independent signaling by Drosophila insulin receptor for axon guidance and growth.

The Drosophila insulin receptor (DInR) regulates a diverse array of biological processes including growth, axon guidance, and sugar homeostasis. Growth regulation by DInR is mediated by Chico, the Drosophila homolog of vertebrate insulin receptor substrate proteins IRS1-4. In contrast, DInR regulation of photoreceptor axon guidance in the developing visual system is mediated by the SH2-SH3 domain adaptor protein Dreadlocks (Dock). In vitro studies by others identified five NPXY motifs, one in the juxtamembrane region and four in the signaling C-terminal tail (C-tail), important for interaction with Chico. Here we used yeast two-hybrid assays to identify regions in the DInR C-tail that interact with Dock. These Dock binding sites were in separate portions of the C-tail from the previously identified Chico binding sites. To test whether these sites are required for growth or axon guidance in whole animals, a panel of DInR proteins, in which the putative Chico and Dock interaction sites had been mutated individually or in combination, were tested for their ability to rescue viability, growth and axon guidance defects of dinr mutant flies. Sites required for viability were identified. Unexpectedly, mutation of both putative Dock binding sites, either individually or in combination, did not lead to defects in photoreceptor axon guidance. Thus, either sites also required for viability are necessary for DInR function in axon guidance and/or there is redundancy built into the DInR/Dock interaction such that Dock is able to interact with multiple regions of DInR. We also found that simultaneous mutation of all five NPXY motifs implicated in Chico interaction drastically decreased growth in both male and female adult flies. These animals resembled chico mutants, supporting the notion that DInR interacts directly with Chico in vivo to control body size. Mutation of these five NPXY motifs did not affect photoreceptor axon guidance, segregating the roles of DInR in the processes of growth and axon guidance.

Insulin stimulates translocation of human GLUT4 to the membrane in fat bodies of transgenic Drosophila melanogaster.

The fruit fly Drosophila melanogaster is an excellent model system for studies of genes controlling development and disease. However, its applicability to physiological systems is less clear because of metabolic differences between insects and mammals. Insulin signaling has been studied in mammals because of relevance to diabetes and other diseases but there are many parallels between mammalian and insect pathways. For example, deletion of Drosophila Insulin-Like Peptides resulted in 'diabetic' flies with elevated circulating sugar levels. Whether this situation reflects failure of sugar uptake into peripheral tissues as seen in mammals is unclear and depends upon whether flies harbor the machinery to mount mammalian-like insulin-dependent sugar uptake responses. Here we asked whether Drosophila fat cells are competent to respond to insulin with mammalian-like regulated trafficking of sugar transporters. Transgenic Drosophila expressing human glucose transporter-4 (GLUT4), the sugar transporter expressed primarily in insulin-responsive tissues, were generated. After expression in fat bodies, GLUT4 intracellular trafficking and localization were monitored by confocal and total internal reflection fluorescence microscopy (TIRFM). We found that fat body cells responded to insulin with increased GLUT4 trafficking and translocation to the plasma membrane. While the amplitude of these responses was relatively weak in animals reared on a standard diet, it was greatly enhanced in animals reared on sugar-restricted diets, suggesting that flies fed standard diets are insulin resistant. Our findings demonstrate that flies are competent to mobilize translocation of sugar transporters to the cell surface in response to insulin. They suggest that Drosophila fat cells are primed for a response to insulin and that these pathways are down-regulated when animals are exposed to constant, high levels of sugar. Finally, these studies are the first to use TIRFM to monitor insulin-signaling pathways in Drosophila, demonstrating the utility of TIRFM of tagged sugar transporters to monitor signaling pathways in insects.

Genetic mosaic analysis indicates that the bulb region of coat hair follicles contains a resident population of several active multipotent epithelial lineage progenitors.

The hair follicle represents an excellent model system for exploring the properties of lineage-forming units in a dynamic epithelium containing multiple cell types. During its growth (anagen) phase, the proximal-distal axis of the mouse coat hair (pelage) follicle provides a historical record of all epithelial lineages generated from its resident stem cell population. An unresolved question in the field is whether the bulb region of anagen pelage follicles contains multipotential progenitors and whether their individual contribution to cellular census fluctuates over time. To address this issue, chimeric follicles were harvested in midanagen from three types of genetic mosaic mouse models. Analysis of the distribution of genotypic markers, including digital three-dimensional reconstruction of serially sectioned chimeric follicles, revealed that on average the bulb contains four or fewer active progenitors, each capable of giving rise to all six follicular epithelial fates. Moreover, analysis of mosaic pelage, as well as cultured whisker follicles provided evidence that bulb-associated progenitors can give rise to expanding descendant clones during midanagen, leading to the conclusion that the bulb contains dormant or symmetrically dividing stem cells. This latter feature resembles the behavior of hematopoietic stem cells after bone marrow transplantation, and raises the question of whether this property may be shared by stem cells in other self-renewing epithelia.