Summary Neurogenesis continues throughout adulthood in the brains of many animals, including some insects[1–3], and relies on the presence of mitotic neural stem cells. However, in mammals, unlike the liver or skin, the regenerative capacity of the adult nervous system is extremely limited. Using the Drosophila brain as a model, we investigate the mechanisms that restrict adult neurogenesis, with the eventual goal of devising ways to replace neurons lost by injury or disease. We find that in Drosophila, adult neurogenesis is absent due to the elimination of neural stem cells (neuroblasts) during development by cell death. Prior to their elimination, neuroblasts undergo a developmentally regulated reduction in their growth and proliferation rates due to downregulation of insulin/PI3 kinase signaling, resulting in increased levels of nuclear Foxo. These small, growth-impaired neuroblasts are often eliminated by caspase-dependent cell death and not exclusively by terminal differentiation as has been previously proposed[4]. Eliminating Foxo, together with inhibition of pro-apoptotic proteins promotes long-term survival of neuroblasts and sustained neurogenesis in the adult mushroom body (mb), the center for learning and memory in Drosophila. Foxo likely activates an autophagic cell death response, since simultaneous inhibition of ATG1 (autophagy-specific gene 1) and apoptosis also promotes long-term mb neuroblast survival. mb neurons generated in the adult incorporate into the existing mb neuropil suggesting that both neuroblast identity and neuronal pathfinding cues are intact. Thus neurogenesis can be induced in adults by antagonizing pathways that normally function to eliminate neural stem cells during development.
A comprehensive understanding of the brain requires analysis of individual neurons. Here we describe twin-spot MARCM for high-resolution lineage tracing by independent labeling of paired sister clones. We determine patterns of neurogenesis and the influences of lineage on neuron-type specification. Notably, neural progenitors may yield intermediate precursors that make one, two, or more neurons. Furthermore, neurons acquire stereotyped projections according to their temporal position within various brain sublineages. Twin-spot MARCM also permits birth dating of mutant clones, enabling the detection of a single temporal fate that requires chinmo in a sublineage of six Drosophila central complex neurons. In sum, twin-spot MARCM can reveal the developmental origins of neurons and the mechanisms that underlie cell fate.
Caspase family proteases play important roles in the regulation of apoptotic cell death. Initiator caspases are activated in response to death stimuli, and they transduce and amplify these signals by cleaving and thereby activating effector caspases. In Drosophila, the initiator caspase Nc (previously Dronc) cleaves and activates two short-prodomain caspases, Dcp-1 and Ice (previously Drice), suggesting these as candidate effectors of Nc killing activity. dcp-1-null mutants are healthy and possess few defects in normally occurring cell death. To explore roles for Ice in cell death, we generated and characterized an Ice null mutant. Animals lacking Ice show a number of defects in cell death, including those that occur during embryonic development, as well as during formation of adult eyes, arista and wings. Ice mutants exhibit subtle defects in the destruction of larval tissues, and do not prevent destruction of salivary glands during metamorphosis. Cells from Ice animals are also markedly resistant to several stresses, including X-irradiation and inhibition of protein synthesis. Mutations in Ice also suppress cell death that is induced by expression of Rpr, Wrinkled (previously Hid) and Grim. These observations demonstrate that Ice plays an important non-redundant role as a cell death effector. Finally, we demonstrate that Ice participates in, but is not absolutely required for, the non-apoptotic process of spermatid differentiation.
Insects act as vectors for diseases of plants, animals and humans. Replacement of wild insect populations with genetically modified individuals unable to transmit disease provides a potentially self-perpetuating method of disease prevention. Population replacement requires a gene drive mechanism in order to spread linked genes mediating disease refractoriness through wild populations. We previously reported the creation of synthetic Medea selfish genetic elements able to drive population replacement in Drosophila. These elements use microRNA-mediated silencing of myd88, a maternally expressed gene required for embryonic dorso-ventral pattern formation, coupled with early zygotic expression of a rescuing transgene, to bring about gene drive. Medea elements that work through additional mechanisms are needed in order to be able to carry out cycles of population replacement and/or remove existing transgenes from the population, using second-generation elements that spread while driving first-generation elements out of the population. Here we report the synthesis and population genetic behavior of two new synthetic Medea elements that drive population replacement through manipulation of signaling pathways involved in cellular blastoderm formation or Notch signaling, demonstrating that in Drosophila Medea elements can be generated through manipulation of diverse signaling pathways. We also describe the mRNA and small RNA changes in ovaries and early embryos associated from Medea-bearing females. Finally, we use modeling to illustrate how Medea elements carrying genes that result in diapause-dependent female lethality could be used to bring about population suppression.
Sterile inflammation triggered by endogenous factors is thought to contribute to the pathogenesis of acute and chronic inflammatory diseases. Here, we demonstrate that apoptosis-deficient mutants spontaneously develop a necrosis-driven systemic immune response in Drosophila and provide an in vivo model for studying the organismal response to sterile inflammation. Metabolomic analysis of hemolymph from apoptosis-deficient mutants revealed increased sarcosine and reduced S-adenosyl-methionine (SAM) levels due to glycine N-methyltransferase (Gnmt) upregulation. We showed that Gnmt was elevated in response to Toll activation induced by the local necrosis of wing epidermal cells. Necrosis-driven inflammatory conditions induced dFoxO hyperactivation, leading to an energy-wasting phenotype. Gnmt was cell-autonomously upregulated by dFoxO in the fat body as a possible rheostat for controlling energy loss, which functioned during fasting as well as inflammatory conditions. We propose that the dFoxO-Gnmt axis is essential for the maintenance of organismal SAM metabolism and energy homeostasis.
Many inhibitor of apoptosis (IAP) family proteins inhibit apoptosis. IAPs contain N-terminal baculovirus IAP repeatdomains and a C-terminal RING ubiquitin ligase domain. Drosophila IAP DIAP1 is essential for the survival of many cells, protecting them from apoptosis by inhibiting active caspases. Apoptosis initiates when proteins such as Reaper, Hid, and Grim bind a surface groove in DIAP1 baculovirus IAP repeat domains via an N-terminal IAP-binding motif. This evolutionarily conserved interaction disrupts DIAP1-caspase interactions, unleashing apoptosis-inducing caspase activity. A second Drosophila IAP, DIAP2, also binds Rpr and Hid and inhibits apoptosis in multiple contexts when overexpressed. However, due to a lack of mutants, little is known about the normal functions of DIAP2. We report the generation of diap2 null mutants. These flies are viable and show no defects in developmental or stress-induced apoptosis. Instead, DIAP2 is required for the innate immune response to Gram-negative bacterial infection. DIAP2 promotes cytoplasmic cleavage and nuclear translocation of the NF-B homolog Relish, and this requires the DIAP2 RING domain. Increasing the genetic dose of diap2 results in an increased immune response, whereas expression of Rpr or Hid results in down-regulation of DIAP2 protein levels. Together these observations suggest that DIAP2 can regulate immune signaling in a dose-dependent manner, and this can be regulated by IBM-containing proteins. Therefore, diap2 may identify a point of convergence between apoptosis and immune signaling pathways. The inhibitor of apoptosis (IAP)2 family proteins contain one or more repeats of an ϳ70-amino acid motif known as a baculovirus IAP repeat (BIR), which mediates interactions with multiple death activators and plays an essential role in the ability of these proteins to inhibit cell death. IAPs also contain a C-terminal RING E3 ubiquitin ligase domain that can target bound proteins, as well as the IAP itself, for ubiquitination and in some cases degradation (1). The Drosophila genome encodes two BIR and RING domain-containing IAP family members, DIAP1 and DIAP2, and ectopic expression of either protein inhibits apoptosis (2-4). DIAP1 is required continuously in many cells to inhibit the apical caspase Dronc and effector caspases activated by Dronc, such as Drice (5, 6). Critical interactions between caspases and DIAP1 are mediated by a surface groove within each DIAP1 BIR domain and short IAP-binding motifs (IBM) present in Dronc or Drice (7-9). Apoptosis in the fly can be induced by expression of proteins such as Reaper, Hid, Grim, Sickle, and Jafrac2 (the RHG proteins). Each of these proteins contains an N-terminal IBM that mediates competitive binding to DIAP1 through the same BIR surface grooves that are required for DIAP1-caspase interactions (8, 10). RHG proteins can also promote ubiquitin-dependent degradation of DIAP1. Both activities have the effect of liberating active caspases, resulting in apoptosis (6). IAPs that inhibit apoptosis, as well as inhibi...
Recent Zika virus (ZIKV) outbreaks have highlighted the necessity for development of novel vector control strategies to combat arboviral transmission, including genetic versions of the sterile insect technique, artificial infection with Wolbachia to reduce population size and/or vectoring competency, and gene drive-based methods. Here, we describe the development of mosquitoes synthetically engineered to impede vector competence to ZIKV. We demonstrate that a polycistronic cluster of engineered synthetic small RNAs targeting ZIKV is expressed and fully processed in Aedes aegypti, ensuring the formation of mature synthetic small RNAs in the midgut where ZIKV resides in the early stages of infection. Critically, we demonstrate that engineered Ae. aegypti mosquitoes harboring the anti-ZIKV transgene have significantly reduced viral infection, dissemination, and transmission rates of ZIKV. Taken together, these compelling results provide a promising path forward for development of effective genetic-based ZIKV control strategies, which could potentially be extended to curtail other arboviruses.
In this study, we uncover a role for microRNAs in Drosophila germline stem cell (GSC) maintenance. Disruption of Dicer-1 function in GSCs during adult life results in GSC loss. Surprisingly, however, loss of Dicer-1 during development does not result in a GSC maintenance defect, although a defect is seen if both Dicer-1 and Dicer-2 function are disrupted. Loss of the bantam microRNA mimics the Dicer-1 maintenance defect when induced in adult GSCs, suggesting that bantam plays a key role in GSC self-renewal. Mad, a component of the TGF-beta pathway, behaves similarly to Dicer-1: adult GSC maintenance requires Mad if it is lost during adult life, but not if it is lost during pupal development. Overall, these results show stage-specific differential sensitivity of GSC maintenance to certain perturbations, and suggest that there may be Dcr-2 dependent redundancy of GSC maintenance mechanisms during development that is lost in later life.
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