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Current papers in developmental biology and gene function


Friday, September 28th, 2018 - Cytoskeleton and Junctions

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Cristo, I., Carvalho, L., Ponte, S. and Jacinto, A. (2018). Novel role for Grainy head in the regulation of cytoskeletal and junctional dynamics during epithelial repair. J Cell Sci 131(17). PubMed ID: 30131442
Tissue repair is critical for the maintenance of epithelial integrity and permeability. Simple epithelial repair relies on a combination of collective cell movements and the action of a contractile actomyosin cable at the wound edge that together promote the fast and efficient closure of tissue discontinuities. The Grainy head family of transcription factors (Grh in flies; GRHL1-GRHL3 in mammals) are essential proteins that have been implicated both in the development and repair of epithelia. However, the genes and the molecular mechanisms that it controls remain poorly understood. This study shows that Grh knockdown disrupts actomyosin dynamics upon injury of the Drosophila pupa epithelial tissue. This leads to the formation of an ectopic actomyosin cable away from the wound edge and impaired wound closure. It was also uncovered that E-Cadherin is downregulated in the Grh-depleted tissue around the wound, likely as a consequence of Dorsal (an NF-kappaB protein) misregulation, which also affects actomyosin cable formation. This work highlights the importance of Grh as a stress response factor and its central role in the maintenance of epithelial characteristics necessary for tissue repair through regulating cytoskeleton and E-Cadherin dynamics.
Dunn, B. S., Rush, L., Lu, J. Y. and Xu, T. (2018). Mutations in the Drosophila tricellular junction protein M6 synergize with Ras(V12) to induce apical cell delamination and invasion. Proc Natl Acad Sci U S A 115(33): 8358-8363. PubMed ID: 30061406
Complications from metastasis are responsible for the majority of cancer-related deaths. Despite the outsized medical impact of metastasis, remarkably little is known about one of the key early steps of metastasis: departure of a tumor cell from its originating tissue. It is well documented that cellular delamination in the basal direction can induce invasive behaviors, but it remains unknown if apical cell delamination can induce migration and invasion in a cancer context. To explore this feature of cancer progression, a genetic screen was performed in Drosophila, and mutations in the protein M6 were found to synergize with oncogenic Ras to drive invasion following apical delamination without crossing a basement membrane. Mechanistically, it was observed that M6-deficient Ras(V12) clones delaminate as a result of alterations in a Canoe-RhoA-myosin II axis that is necessary for both the delamination and invasion phenotypes. To uncover the cellular roles of M6, this study showed that it localizes to tricellular junctions in epithelial tissues where it is necessary for the structural integrity of multicellular contacts. This work provides evidence that apical delamination can precede invasion and highlights the important role that tricellular junction integrity can play in this process.
Greenan, G. A., Keszthelyi, B., Vale, R. D. and Agard, D. A. (2018). Insights into centriole geometry revealed by cryotomography of doublet and triplet centrioles. Elife 7. PubMed ID: 30080137
Centrioles are cylindrical assemblies comprised of 9 singlet, doublet, or triplet microtubules, essential for the formation of motile and sensory cilia. While the structure of the cilium is being defined at increasing resolution, centriolar structure remains poorly understood. This study used electron cryo-tomography to determine the structure of mammalian (triplet) and Drosophila (doublet) centrioles. Mammalian centrioles have two distinct domains: a 200 nm proximal core region connected by A-C linkers, and a distal domain where the C-tubule is incomplete and a pair of novel linkages stabilize the assembly producing a geometry more closely resembling the ciliary axoneme. Drosophila centrioles resemble the mammalian core, but with their doublet microtubules linked through the A tubules. The commonality of core-region length, and the abrupt transition in mammalian centrioles, suggests a conserved length-setting mechanism. The unexpected linker diversity suggests how unique centriolar architectures arise in different tissues and organisms.
Gottardo, M., Persico, V., Callaini, G. and Riparbelli, M. G. (2018). The "transition zone" of the cilium-like regions in the Drosophila spermatocytes and the role of the C-tubule in axoneme assembly. Exp Cell Res 371(1): 262-268. PubMed ID: 30130520
The fruit-fly Drosophila melanogaster harbours different types of ciliary structures: ciliary projections associated with neurons of type I and cilium-like regions (CLRs) found during male gametogenesis. The latter deserve particular attention since they are morphologically similar to vertebrate primary cilia and transform into the sperm axonemes during spermiogenesis. Although, all the centrioles are able to organize the CLRs, this study found that the mother centriole docks first to the plasma membrane suggesting a new intrinsic functional asymmetry between the parent centrioles. The CLRs lack the Y-links that connect the axoneme doublets with the plasma membrane in conventional primary cilia. Moreover, the C-tubules, that are lacking in the axoneme of the primary cilia, persisted along the CLRs albeit modified into longitudinal blades. Remarkably, mutant flies in which the CLRs are devoid of the C-tubules or their number is reduced lack sperm axonemes or have incomplete axonemes. Therefore, the C-tubules are dispensable for the assembly of the CLRs but are essential for sperm axoneme elongation and maintenance in Drosophila.

Thursday, September 27th - Natural Immunity

Weavers, H., Franz, A., Wood, W. and Martin, P. (2018). Long-term in vivo tracking of inflammatory cell dynamics within Drosophila pupae. J Vis Exp(136). PubMed ID: 29985351
During the rapid inflammatory response to tissue damage, cells of the innate immune system are quickly recruited to the injury site. Once at the wound, innate immune cells perform a number of essential functions, such as fighting infection, clearing necrotic debris, and stimulating matrix deposition. In order to fully understand the diverse signaling events that regulate this immune response, it is crucial to observe the complex behaviors of (and interactions that occur between) multiple cell lineages in vivo, and in real-time, with the high spatio-temporal resolution. The optical translucency and the genetic tractability of Drosophila embryos have established Drosophila as an invaluable model to live-image and dissect fundamental aspects of inflammatory cell behavior, including mechanisms of developmental dispersal, clearance of apoptotic corpses and/or microbial pathogens, and recruitment to wounds. However, more recent work has now demonstrated that employing a much later stage in the Drosophila lifecycle - the Drosophila pupa - offers a number of distinct advantages, including improved RNAi efficiency, longer imaging periods, and significantly greater immune cell numbers. This study describes a protocol for imaging wound repair and the associated inflammatory response at the high spatio-temporal resolution in live Drosophila pupae. To follow the dynamics of both re-epithelialization and inflammation, a number of specific in vivo fluorescent markers is used for both the epithelium and innate immune cells. The effectiveness is demonstrated of photo-convertible fluorophores, such as Kaede, for following the specific immune cell subsets, to track their behavior as they migrate to, and resolve from, the injury site.
Benguettat, O., Jneid, R., Soltys, J., Loudhaief, R., Brun-Barale, A., Osman, D. and Gallet, A. (2018). The DH31/CGRP enteroendocrine peptide triggers intestinal contractions favoring the elimination of opportunistic bacteria. PLoS Pathog 14(9): e1007279. PubMed ID: 30180210
The digestive tract is the first organ affected by the ingestion of foodborne bacteria. While commensal bacteria become resident, opportunistic or virulent bacteria are eliminated from the gut by the local innate immune system. This study characterized a new mechanism of defense, independent of the immune system, in Drosophila melanogaster. Strong contractions of longitudinal visceral muscle fibers were observed for the first 2 hours following bacterial ingestion. These visceral muscle contractions are induced by immune reactive oxygen species (ROS) that accumulate in the lumen and depend on the ROS-sensing TRPA1 receptor. It was then demonstrated that both ROS and TRPA1 are required in a subset of anterior enteroendocrine cells for the release of the DH31 neuropeptide which activates its receptor in the neighboring visceral muscles. The resulting contractions of the visceral muscles favors quick expulsion of the bacteria, limiting their presence in the gut. These results unveil a precocious mechanism of defense against ingested opportunistic bacteria, whether they are Gram-positive like Bacillus thuringiensis or Gram-negative like Erwinia carotovora carotovora. Finally, it was found that the human homolog of DH31, CGRP, has a conserved function in Drosophila.
Goto, A., Okado, K., Martins, N., Cai, H., Barbier, V., Lamiable, O., Troxler, L., Santiago, E., Kuhn, L., Paik, D., Silverman, N., Holleufer, A., Hartmann, R., Liu, J., Peng, T., Hoffmann, J. A., Meignin, C., Daeffler, L. and Imler, J. L. (2018). The kinase IKKbeta regulates a STING- and NF-kappaB-dependent antiviral response pathway in Drosophila. Immunity 49(2): 225-234.e224. PubMed ID: 30119996
Antiviral immunity in Drosophila involves RNA interference and poorly characterized inducible responses. This study showed that two components of the IMD pathway, the kinase dIKKbeta and the transcription factor Relish, were required to control infection by two picorna-like viruses. A set of genes was identified that was induced by viral infection and regulated by dIKKbeta and Relish, which included an ortholog of STING. This study showed that dSTING participated in the control of infection by picorna-like viruses, acting upstream of dIKKbeta to regulate expression of Nazo, an antiviral factor. These data reveal an antiviral function for STING in an animal model devoid of interferons and suggest an evolutionarily ancient role for this molecule in antiviral immunity.
Yang, S., Yu, J., Fan, Z., Gong, S. T., Tang, H. and Pan, L. (2018). Bub1 facilitates virus entry through endocytosis in a model of Drosophila pathogenesis. J Virol. PubMed ID: 29976667
In order to establish productive infection and dissemination, viruses usually evolve a number of strategies to hijack and/or subvert the host defense systems. However, host factors utilized by the virus to facilitate infection remain poorly characterized. Drosophila melanogaster deficient in budding uninhibited by benzimidazoles 1 (bub1), a highly conserved subunit of kinetochores complex regulating chromosome congression, became resistant to Drosophila C virus (DCV) infection evidenced in increased survival rates and reduced viral loads, compared to the wild type control. Mechanistic analysis further showed that Bub1 also functioned in the cytoplasm and was essentially involved in clathrin-dependent endocytosis of DCV and other pathogens, thus limiting pathogen entry. DCV infection potentially had strengthened the interaction between Bub1 and the clathrin adaptor on the cell membrane. Furthermore, the conserved function of Bub1 was as well verified in a mammalian cell line. Thus, these data demonstrated a previously unknown function of Bub1 that could be hijacked by pathogens to facilitate their infection and spread.

Wednesday, September 27th - Adult Physiology

Farkas, R., Benova-Liszekova, D., Mentelova, L., Beno, M., Babisova, K., Trusinova-Pecenova, L., Raska, O., Chase, B. A. and Raska, I. (2018). Endosomal vacuoles of the prepupal salivary glands of Drosophila play an essential role in the metabolic reallocation of iron. Dev Growth Differ. PubMed ID: 30123964
After fruit flies release their glycoprotein-rich secretory glue during pupariation, early-to-mid prepupal salivary glands undergo extensive endocytosis with widespread vacuolation of the cytoplasm followed by massive apocrine secretion. This study describes additional novel properties of these endosomes. The use of vital pH-sensitive probes provided confirmatory evidence that these endosomes have acidic contents and that there are two types of endocytosis seen in the prepupal glands. The salivary glands simultaneously generate mildly acidic, small, basally-derived endosomes and strongly acidic, large and apical endosomes. Multiple lines of evidence were obtained that the small basally-derived endosomes are chiefly involved in the uptake of dietary Fe(3+) iron. The fusion of basal endosomes with the larger and strongly acidic apical endosomes appears to facilitate optimal conditions for ferrireductase activity inside the vacuoles to release metabolic Fe(2+) iron. This study found increasing fluorescence of a glutathione-sensitive probe in large vacuoles, which appeared to depend on the amount of iron released by ferrireductase. Moreover, labeled mammalian iron-bound transferrin is actively taken up, providing direct evidence for active iron uptake by basal endocytosis. In addition, it was serendipitously found that small (basal) endosomes were uniquely recognized by PNA lectin, whereas large (apical) vacuoles bound DBA lectin.
Bednarova, A., Tomcala, A., Mochanova, M., Kodrik, D. and Krishnan, N. (2018). Disruption of Adipokinetic Hormone mediated energy homeostasis has subtle effects on physiology, behavior and lipid status during aging in Drosophila. Front Physiol 9: 949. PubMed ID: 30079029
The impact of disruption of adipokinetic hormone (AKH) signaling was studied during aging in Drosophila in a sexually dimorphic manner. A mutant (Akh1) producing a non-functional AKH peptide was compared with isogenized wild-type controls (w1118), and Akh-rescue line where AKH was ectopically expressed in the mutant background (EE-Akh). Longevity, fecundity, and locomotor activity rhythms remained unaffected by lack of AKH signaling. While the strength of rhythms declined in general with age across all fly lines tested this was more so in case of Akh1 flies. Negative geotaxis was significantly impaired in Akh1 flies. Only young Akh1 flies of both sexes and old Akh1 females showed significantly higher body weight compared to age-matched iso-control flies (except in case of EE-Akh). Expression of genes involved in energy homeostasis and aging indicated that dTOR and Akt expression were elevated in Akh1 flies compared to other genotypes, whereas AMPK and dFoxO expression levels were significantly reduced. Multivariate analysis of the distribution of lipid species revealed a significant accumulation of specific diglyceride (DG) and triglyceride (TG) lipid species, irrespective of sex, attributable in part due to lack of AKH. Moreover, irrespective of lack of AKH, older flies of all genotypes accumulated TGs. Taken together, the results strongly suggest that disruption of AKH has very subtle effects on physiology, behavior and lipid status during aging.
Weinrich, T. W., Hogg, C. and Jeffery, G. (2018). The temporal sequence of improved mitochondrial function on the dynamics of respiration, mobility, and cognition in aged Drosophila. Neurobiol Aging 70: 140-147. PubMed ID: 30007163
Aging is associated with mitochondrial decline and reduced adenosine triphosphate (ATP) production leading to cellular dysfunction, but this is improved by long-wavelength light absorbed by cytochrome c oxidase, increasing cytochrome c oxidase activity, ATP production and improving metabolism, sensory motor function, and cognition. Yet, the sequence of these events is unknown. This study gave old flies a single 90-minute 670-nm pulse and measure temporal sequences of changes in respiration, ATP, motor, and cognitive ability. Respiration increased significantly 20 minutes after light initiation and remained elevated for 4 days. Measurable ATP increased at 1 hour, peaking at 3 hours, and then declined rapidly. Respiration improved before ATP increased, which indicates an early ATP sink. Flies explore environments stereotypically, which is lost with aging but is reestablished for 7 hours after light exposure. However, again, there are improvements before there are peaks in ATP production. Improved mobility and cognitive function persist after ATP levels return to normal. Hence, elevated ATP in age may initiate independent signaling mechanisms that result in improvements in aged metabolism and function.
Galikova, M., Dircksen, H. and Nassel, D. R. (2018). The thirsty fly: Ion transport peptide (ITP) is a novel endocrine regulator of water homeostasis in Drosophila. PLoS Genet 14(8): e1007618. PubMed ID: 30138334
Animals need to continuously adjust their water metabolism to the internal and external conditions. Homeostasis of body fluids thus requires tight regulation of water intake and excretion, and a balance between ingestion of water and solid food. This study investigated how these processes are coordinated in Drosophila melanogaster. The first thirst-promoting and anti-diuretic hormone of Drosophila was identified, encoded by the gene Ion transport peptide (ITP). This endocrine regulator belongs to the CHH (crustacean hyperglycemic hormone) family of peptide hormones. Using genetic gain- and loss-of-function experiments, this study showed that ITP signaling acts analogous to the human vasopressin and renin-angiotensin systems; expression of ITP is elevated by dehydration of the fly, and the peptide increases thirst while repressing excretion, promoting thus conservation of water resources. ITP responds to both osmotic and desiccation stress, and dysregulation of ITP signaling compromises the fly's ability to cope with these stressors. In addition to the regulation of thirst and excretion, ITP also suppresses food intake. Altogether, this work identifies ITP as an important endocrine regulator of thirst and excretion, which integrates water homeostasis with feeding of Drosophila.

Tuesday, September 26th - Behavior

Giraldo, Y. M., Leitch, K. J., Ros, I. G., Warren, T. L., Weir, P. T. and Dickinson, M. H. (2018). Sun navigation requires compass neurons in Drosophila. Curr Biol. PubMed ID: 30174187
Despite their small brains, insects can navigate over long distances by orienting using visual landmarks, skylight polarization, and sun position. Although Drosophila are not generally renowned for their navigational abilities, mark-and-recapture experiments in Death Valley revealed that they can fly nearly 15 km in a single evening. To accomplish such feats on available energy reserves, flies would have to maintain relatively straight headings, relying on celestial cues. Cues such as sun position and polarized light are likely integrated throughout the sensory-motor pathway, including the highly conserved central complex. Recently, a group of Drosophila central complex cells (E-PG neurons) have been shown to function as an internal compass, similar to mammalian head-direction cells. Using an array of genetic tools, this study set out to test whether flies can navigate using the sun and to identify the role of E-PG cells in this behavior. Using a flight simulator, it was found that Drosophila adopt arbitrary headings with respect to a simulated sun, thus performing menotaxis, and individuals remember their heading preference between successive flights-even over several hours. Imaging experiments performed on flying animals revealed that the E-PG cells track sun stimulus motion. When these neurons are silenced, flies no longer adopt and maintain arbitrary headings relative to the sun stimulus but instead exhibit frontal phototaxis. Thus, without the compass system, flies lose the ability to execute menotaxis and revert to a simpler, reflexive behavior.
Clemens, J., Coen, P., Roemschied, F. A., Pereira, T. D., Mazumder, D., Aldarondo, D. E., Pacheco, D. A. and Murthy, M. (2018). Discovery of a new song mode in Drosophila reveals hidden structure in the sensory and neural drivers of behavior. Curr Biol 28(15): 2400-2412.e2406. PubMed ID: 30057309
Deciphering how brains generate behavior depends critically on an accurate description of behavior. If distinct behaviors are lumped together, separate modes of brain activity can be wrongly attributed to the same behavior. Alternatively, if a single behavior is split into two, the same neural activity can appear to produce different behaviors. This study addresses this issue in the context of acoustic communicationa in Drosophila. During courtship, males vibrate their wings to generate time-varying songs, and females evaluate songs to inform mating decisions. For 50 years, Drosophila melanogaster song was thought to consist of only two modes, sine and pulse, but using unsupervised classification methods on large datasets of song recordings, this study now establishes the existence of at least three song modes: two distinct pulse types, along with a single sine mode. This seemingly subtle distinction affects the interpretation of the mechanisms underlying song production and perception. Specifically, this study showed that visual feedback influences the probability of producing each song mode and that male song mode choice affects female responses and contributes to modulating his song amplitude with distance. At the neural level, it was demonstrated how the activity of four separate neuron types within the fly's song pathway differentially affects the probability of producing each song mode. These results highlight the importance of carefully segmenting behavior to map the underlying sensory, neural, and genetic mechanisms.
Dawson, E. H., Bailly, T. P. M., Dos Santos, J., Moreno, C., Devilliers, M., Maroni, B., Sueur, C., Casali, A., Ujvari, B., Thomas, F., Montagne, J. and Mery, F. (2018). Social environment mediates cancer progression in Drosophila. Nat Commun 9(1): 3574. PubMed ID: 30177703
The influence of oncogenic phenomena on the ecology and evolution of animal species is becoming an important research topic. Similar to host-pathogen interactions, cancer negatively affects host fitness, which should lead to the selection of host control mechanisms, including behavioral traits that best minimize the proliferation of malignant cells. Social behavior is suggested to influence tumor progression. While the ecological benefits of sociality in gregarious species are widely acknowledged, only limited data are available on the role of the social environment on cancer progression. This study exposed adult Drosophila, with colorectal-like tumors, to different social environments. Subtle variations in social structure have dramatic effects on the progression of tumor growth. Finally, it is revealed that flies can discriminate between individuals at different stages of tumor development and selectively choose their social environment accordingly. This study demonstrates the reciprocal links between cancer and social interactions and how sociality may impact health and fitness in animals and its potential implications for disease ecology.
Cascallares, G., Riva, S., Franco, D. L., Risau-Gusman, S. and Gleiser, P. M. (2018).. Role of the circadian clock in the statistics of locomotor activity in Drosophila. PLoS One 13(8): e0202505. PubMed ID: 30138403
In many animals the circadian rhythm of locomotor activity is controlled by an endogenous circadian clock. Using custom made housing and video tracking software in order to obtain high spatial and temporal resolution, wthe statistical properties of the locomotor activity of wild type and two clock mutants of Drosophila melanogaster were studied. This study showed that the distributions of activity and quiescence bouts for the clock mutants in light-dark conditions (LD) are very different from the distributions obtained when there are no external cues from the environment (DD). In the wild type these distributions are very similar, showing that the clock controls this aspect of behavior in both regimes (LD and DD). Furthermore, the distributions are very similar to those reported for Wistar rats. For the timing of events important differences were observed, quantified by how the event rate distributions scale for increasing time windows. For the wild type these distributions can be rescaled by the same function in DD as in LD. Interestingly, the same function has been shown to rescale the rate distributions in Wistar rats. On the other hand, for the clock mutants it is not possible to rescale the rate distributions, which might indicate that the extent of circadian control depends on the statistical properties of activity and quiescence (Cascallares, 2018).
Elya, C., Lok, T. C., Spencer, Q. E., McCausland, H., Martinez, C. C. and Eisen, M. (2018). Robust manipulation of the behavior of Drosophila melanogaster by a fungal pathogen in the laboratory. Elife 7. PubMed ID: 30047862
Many microbes induce striking behavioral changes in their animal hosts, but how they achieve this is poorly understood, especially at the molecular level. Mechanistic understanding has been largely constrained by the lack of an experimental system amenable to molecular manipulation. A strain of the behavior-manipulating fungal pathogen Entomophthora muscae infects wild Drosophila, and methods were established to infect D. melanogaster in the lab. Lab-infected flies manifest the moribund behaviors characteristic of E. muscae infection: hours before death, they climb upward, extend their proboscides, affixing in place, then raise their wings, clearing a path for infectious spores to launch from their abdomens. E. muscae was found to invade the nervous system, suggesting a direct means by which the fungus could induce behavioral changes. Given the vast molecular toolkit available for D. melanogaster, this new system will enable rapid progress in understanding how E. muscae manipulates host behavior.
Hu, W., Peng, Y., Sun, J., Zhang, F., Zhang, X., Wang, L., Li, Q. and Zhong, Y. (2018). Fan-shaped body neurons in the Drosophila brain regulate both innate and conditioned nociceptive avoidance. Cell Rep 24(6): 1573-1584. PubMed ID: 30089267
Multiple brain regions respond to harmful nociceptive stimuli. However, it remains unclear as to whether behavioral avoidance of such stimuli can be modulated within the same or distinct brain networks. This study found subgroups of neurons localized within a well-defined brain region capable of mediating both innate and conditioned nociceptive avoidance in Drosophila. Neurons in the ventral, but not the dorsal, of the multiple-layer organized fan-shaped body (FB) are responsive to electric shock (ES). Silencing ES-responsive neurons, but not non-responsive neurons, leads to reduced avoidance of harmful stimuli, including ES and heat shock. Activating these neurons consistently triggers avoidance and can serve as an unconditional stimulus in an aversive classical conditioning task. Among the three groups of responsive neurons identified, two also have reduced activity in ES-conditioned odor avoidance. These results demonstrate that both innate and conditioned nociceptive avoidance might be represented within neurons confined to a single brain region.

Monday, September 24th - Adult Neural Development and Function

Chin, S. G., Maguire, S. E., Huoviala, P., Jefferis, G. and Potter, C. J. (2018). Olfactory neurons and brain centers directing oviposition decisions in Drosophila. Cell Rep 24(6): 1667-1678. PubMed ID: 30089274
The sense of smell influences many behaviors, yet how odors are represented in the brain remains unclear. A major challenge to studying olfaction is the lack of methods allowing activation of specific types of olfactory neurons in an ethologically relevant setting. To address this, a genetic method was developed in Drosophila called olfactogenetics in which a narrowly tuned odorant receptor, Or56a, is ectopically expressed in different olfactory neuron types. Stimulation with geosmin (the only known Or56a ligand) in an Or56a mutant background leads to specific activation of only target olfactory neuron types. This approach was used to identify olfactory sensory neurons (OSNs) that directly guide oviposition decisions. Five OSN-types (Or71a, Or47b, Or49a, Or67b, and Or7a) were identfied that, when activated alone, suppress oviposition. Projection neurons partnering with these OSNs share a region of innervation in the lateral horn, suggesting that oviposition site selection might be encoded in this brain region.
Contreras, E. G., Palominos, T., Glavic, A., Brand, A. H., Sierralta, J. and Oliva, C. (2018). The transcription factor SoxD controls neuronal guidance in the Drosophila visual system. Sci Rep 8(1): 13332. PubMed ID: 30190506
Precise control of neurite guidance during development is essential to ensure proper formation of neuronal networks and correct function of the central nervous system (CNS). How neuronal projections find their targets to generate appropriate synapses is not entirely understood. Although transcription factors are key molecules during neurogenesis, their entire function during the formation of networks in the CNS is not known. This study used the Drosophila melanogaster optic lobe as a model for understanding neurite guidance during development. The function of Sox102F/SoxD, the unique Drosophila orthologue of the vertebrate SoxD family of transcription factors, was assessed. SoxD is expressed in immature and mature neurons in the larval and adult lobula plate ganglia (one of the optic lobe neuropils), but is absent from glial cells, neural stem cells and progenitors of the lobula plate. SoxD RNAi knockdown in all neurons results in a reduction of the lobula plate neuropil, without affecting neuronal fate. This morphological defect is associated with an impaired optomotor response of adult flies. Moreover, knocking down SoxD only in T4/T5 neuronal types, which control motion vision, affects proper neurite guidance into the medulla and lobula. These findings suggest that SoxD regulates neurite guidance, without affecting neuronal fate.
Chang, Y. C., Tsao, C. K. and Sun, Y. H. (2018). Temporal and spatial order of photoreceptor and glia projections into optic lobe in Drosophila. Sci Rep 8(1): 12669. PubMed ID: 30140062
Photoreceptor (PR) axons project from the retina to the optic lobe in brain and form a precise retinotopic map in the Drosophila visual system. Yet the role of retinal basal glia in the retinotopic map formation is not previously known. This study examined the formation of the retinotopic map by marking single PR pairs and following their axonal projections. In addition to confirming previous studies that the spatial information is preserved from the retina to the optic stalk and then to the optic lamina, this study found that the young PR R3/4 axons transiently overshoot and then retract to their final destination, the lamina plexus. The process of wrapping glia (WG) membrane extension was examined in the eye disc, and the WG membrane extensions also follow the retinotopic map. The WG is important for the proper spatial distribution of PR axons in the optic stalk and lamina, suggesting an active role of wrapping glia in the retinotopic map formation.
Crittenden, J. R., Skoulakis, E. M. C., Goldstein, E. S. and Davis, R. L. (2018). Drosophila mef2 is essential for normal mushroom body and wing development. Biol Open. PubMed ID: 30115617
MEF2 (myocyte enhancer factor 2) transcription factors are found in the brain and muscle of insects and vertebrates and are essential for the differentiation of multiple cell types. In Drosophila, MEF2 is essential for the formation of mushroom bodies in the embryonic brain and for the normal development of wings in the adult. In embryos mutant for mef2, there is a striking reduction in the number of mushroom body neurons and their axon bundles are not detectable. The onset of MEF2 expression in neurons of the mushroom bodies coincides with their formation in the embryo and, in larvae, expression is restricted to post-mitotic neurons. In flies with a mef2 point mutation that disrupts nuclear localization, MEF2 was found to be restricted to a subset of Kenyon cells that project to the alpha/beta, and gamma axonal lobes of the mushroom bodies, but not to those forming the alpha'/beta' lobes.

Friday, September 21st - Transcriptional Regulation

De Castro, S., Peronnet, F., Gilles, J. F., Mouchel-Vielh, E. and Gibert, J. M. (2018). bric a brac (bab), a central player in the gene regulatory network that mediates thermal plasticity of pigmentation in Drosophila melanogaster. PLoS Genet 14(8): e1007573. PubMed ID: 30067846
Drosophila body pigmentation has emerged as a major Evo-Devo model. Using two Drosophila melanogaster lines, Dark and Pale, selected from a natural population, this study analysed the interaction between genetic variation and environmental factors to produce this complex trait. Indeed, pigmentation varies with genotype in natural populations and is sensitive to temperature during development. The bric a brac (bab) genes, that are differentially expressed between the two lines and whose expression levels vary with temperature, participate in the pigmentation difference between the Dark and Pale lines. The two lines differ in a bab regulatory sequence, the dimorphic element (called here bDE). Both bDE alleles are temperature-sensitive, but the activity of the bDE allele from the Dark line is lower than that of the bDE allele from the Pale line. These results suggest that this difference could partly be due to differential regulation by AbdB. bab has been previously reported to be a repressor of abdominal pigmentation. This study shows that one of its targets in this process is the pigmentation gene tan (t), regulated via the tan abdominal enhancer (t_MSE). Furthermore, t expression is strongly modulated by temperature in the two lines. Thus, temperature sensitivity of t expression is at least partly a consequence of bab thermal transcriptional plasticity. It is therefore proposed that a gene regulatory network integrating both genetic variation and temperature sensitivity modulates female abdominal pigmentation. Interestingly, both bDE and t_MSE were previously shown to have been recurrently involved in abdominal pigmentation evolution in drosophilids. It is proposed that the environmental sensitivity of these enhancers has turned them into evolutionary hotspots.
Mysore, K., Li, P. and Duman-Scheel, M. (2018). Identification of Aedes aegypti cis-regulatory elements that promote gene expression in olfactory receptor neurons of distantly related dipteran insects. Parasit Vectors 11(1): 406. PubMed ID: 29996889
Aedes aegypti is a vector mosquito that displays sexually dimorphic behaviors that contribute to pathogen transmission. Formaldehyde-assisted isolation of regulatory elements followed by sequencing (FAIRE-seq) has facilitated genome-wide discovery of A. aegypti cis-regulatory elements (CREs). The goal of this investigation was to identify FAIRE DNA elements that promote gene expression in the olfactory system. Eight A. aegypti CREs that promote gene expression in antennal olfactory receptor neurons (ORNs) were identified in a Drosophila melanogaster transgenic reporter screen. Four CREs identified in the screen were cloned upstream of GAL4 in a transgenic construct that is compatible with transformation of a variety of insect species. These constructs, which contained FAIRE DNA elements associated with the A. aegypti odorant coreceptor (orco), odorant receptor 1 (Or1), odorant receptor 8 (Or8) and fruitless (fru) genes, were used for transformation of A. aegypti. The CREs drove transgene expression in A. aegypti that corresponded to endogenous gene expression patterns of the orco, Or1, Or8 and fru genes in the mosquito antenna. These results provide further evidence that FAIRE-seq, which can be paired with D. melanogaster reporter screening to test FAIRE DNA element activity in select tissues, is a useful method for identification of mosquito cis-regulatory elements. These findings expand the genetic toolkit available for the study of Aedes neurobiology. Moreover, given that the CREs drive comparable olfactory neural expression in both A. aegypti and D. melanogaster, it is likely that they may function similarly in multiple dipteran insects, including other disease vector mosquito species.
Grover, S., Williams, M. E., Kaiser, R., Hughes, J. T., Gresham, L., Rebeiz, M. and Williams, T. M. (2018).. Augmentation of a wound response element accompanies the origin of a Hox-regulated Drosophila abdominal pigmentation trait. Dev Biol. PubMed ID: 29981311
A challenge for evolutionary research is to uncover how new morphological traits evolve the coordinated spatial and temporal expression patterns of genes that govern their formation during development. Detailed studies are often limited to characterizing how one or a few genes contributed to a trait's emergence, and thus knowledge of how entire GRNs evolve their coordinated expression of each gene remains unresolved. The melanic color patterns decorating the male abdominal tergites of Drosophila melanogaster evolved in part by novel expression patterns for genes acting at the terminus of a pigment metabolic pathway, driven by cis-regulatory elements (CREs) with distinct mechanisms of Hox regulation. This study examined the expression and evolutionary histories of two important enzymes in this pathway, encoded by the pale and Ddc genes. While both genes exhibit dynamic patterns of expression, a robust pattern of Ddc expression specifically evolved in the lineage of fruit flies with pronounced melanic abdomens. Derived Ddc expression requires the activity of a CRE previously shown to activate expression in response to epidermal wounding. A binding site for the Grainy head transcription factor that promotes the ancestral wound healing function of this CRE was also shown to be required for abdominal activity. Together with previous findings in this system, this work shows how the GRN for a novel trait emerged by assembling unique yet similarly functioning CREs from heterogeneous starting points.
Courgeon, M., He, D., Liu, H. H., Legent, K. and Treisman, J. E. (2018). The Drosophila Epidermal Growth Factor Receptor does not act in the nucleus. J Cell Sci. PubMed ID: 30158176
Mammalian members of the ErbB family, including the Epidermal Growth Factor Receptor (EGFR), can regulate transcription, DNA replication and repair through nuclear entry of either the full-length proteins or their cleaved cytoplasmic domains. In cancer cells, these nuclear functions contribute to tumor progression and drug resistance. This study examined whether the single Drosophila EGFR can also localize to the nucleus. A chimeric EGFR protein fused at its cytoplasmic C-terminus to DNA-binding and transcriptional activation domains strongly activated transcriptional reporters when overexpressed in cultured cells or in vivo. However, this activity was independent of cleavage and endocytosis. Without an exogenous activation domain, EGFR fused to a DNA-binding domain did not activate or repress transcription. Addition of the same DNA-binding and transcriptional activation domains to the endogenous Egfr locus by genome editing produced no detectable reporter expression in wild type or oncogenic contexts. These results show that when expressed at physiological levels, the cytoplasmic domain of the Drosophila EGFR does not have access to the nucleus. Nuclear EGFR functions are likely to have evolved after vertebrates and invertebrates diverged.

Thursday, September 20th - Evolution

Benton, M. A. (2018). A revised understanding of Tribolium morphogenesis further reconciles short and long germ development. PLoS Biol 16(7): e2005093. PubMed ID: 29969459
In Drosophila melanogaster, the germband forms directly on the egg surface and solely consists of embryonic tissue. In contrast, most insect embryos undergo a complicated set of tissue rearrangements to generate a condensed, multilayered germband. The ventral side of the germband is embryonic, while the dorsal side is thought to be an extraembryonic tissue called the amnion. While this tissue organisation has been accepted for decades and has been widely reported in insects, its accuracy has not been directly tested in any species. Using live cell tracking and differential cell labelling in the short germ beetle Tribolium castaneum, this study shows that most of the cells previously thought to be amnion actually give rise to large parts of the embryo. This process occurs via the dorsal-to-ventral flow of cells and contributes to germband extension (GBE). In addition, true 'amnion' cells in Tribolium were shown to originate from a small region of the blastoderm. Together, these findings show that development in the short germ embryos of Tribolium and the long germ embryos of Drosophila is more similar than previously proposed. Dorsal-to-ventral cell flow also occurs in Drosophila during GBE, and this study argues that the flow is driven by a conserved set of underlying morphogenetic events in both species. Furthermore, the revised Tribolium fate map that this study presents is far more similar to that of Drosophila than the classic Tribolium fate map. Lastly, these findings show that there is no qualitative difference between the tissue structure of the cellularised blastoderm and the short/intermediate germ germband. As such, the same tissue patterning mechanisms could function continuously throughout the cellularised blastoderm and germband stages, and easily shift between them over evolutionary time.
Battlay, P., Leblanc, P. B., Green, L., Garud, N. R., Schmidt, J. M., Fournier-Level, A. and Robin, C. (2018). Structural variants and selective sweep foci contribute to insecticide resistance in the Drosophila genetic reference panel. G3 (Bethesda). PubMed ID: 30190421
Patterns of nucleotide polymorphism within populations of Drosophila melanogaster suggest that insecticides have been the selective agents driving the strongest recent bouts of positive selection. However, there is a need to explicitly link selective sweeps to the particular insecticide phenotypes that could plausibly account for the drastic selective responses that are observed in these non-target insects. This study screened the Drosophila Genetic Reference Panel with two common insecticides; malathion (an organophosphate) and permethrin (a pyrethroid). Genome-wide association studies map survival on malathion to the two of the largest sweeps in the D. melanogaster genome; Ace and Cyp6g1. Malathion survivorship also correlates with lines which have high levels of Cyp12d1, Jheh1 and Jheh2 transcript abundance. Permethrin phenotypes map to the largest cluster of P450 genes in the Drosophila genome, however in contrast to a selective sweep driven by insecticide use, the derived allele seems to be associated with susceptibility. These results underscore previous findings that highlight the importance of structural variation to insecticide phenotypes: Cyp6g1 exhibits copy number variation and transposable element insertions, Cyp12d1 is tandemly duplicated, the Jheh loci are associated with a Bari1 transposable element insertion, and a Cyp6a17 deletion is associated with susceptibility.
Groth, B. R., Huang, Y., Monette, M. J. and Pool, J. E. (2018). Directional selection reduces developmental canalization against genetic and environmental perturbations in Drosophila wings. Evolution. PubMed ID: 29985527
Natural selection may enhance or weaken the robustness of phenotypes against genetic or environmental perturbations. However, important aspects of the relationship between adaptive evolution and canalization remain unclear. Recent work showed that the evolution of larger wing size in a high altitude natural population of Drosophila melanogaster was accompanied by decanalized wing development--specifically a loss of robustness to genetic perturbation. But that study did not address environmental robustness, and it compared populations that may have numerous biological differences. This study performed artificial selection on this same trait in D. melanogaster (larger wing length) and directly test whether this directional selection resulted in decanalization. In general, size-selected replicates show greater frequencies of wing defects than control replicates both after mutagenesis (genetic perturbation) and when subjected to high temperature stress (environmental perturbation), although the increase in defect frequency varies importantly among replicates. These results support the hypothesis that directional selection may result in the loss of both genetic and environmental robustness-offering a rare window into the relationship between adaptation and canalization.
Cooper, B. S., Sedghifar, A., Nash, W. T., Comeault, A. A. and Matute, D. R. (2018). A Maladaptive Combination of Traits Contributes to the Maintenance of a Drosophila Hybrid Zone. Curr Biol. PubMed ID: 30174184
Drosophila teissieri and D. yakuba diverged approximately 3 mya and are thought to share a large, ancestral, African range. These species now co-occur in parts of continental Africa and in west Africa on the island of Bioko. While D. yakuba is a human commensal, D. teissieri seems to be associated with Parinari fruits, restricting its range to forests. Genome data indicate introgression, despite no evidence of contemporary hybridization. This study reports the discovery of D. yakuba-D. teissieri hybrids at the interface of secondary forests and disturbed, open habitats on Bioko. Hybrids are the F1 progeny of D. yakuba females and D. teissieri males. At high temperatures like those found on Bioko, D. teissieri females are generally less receptive to mating, and in combination with temperature effects on egg lay and egg-to-adult viability, this decreases the potential for gene flow between female D. teissieri and male D. yakuba relative to the reciprocal cross. Field and laboratory experiments demonstrate that F1 hybrids have a maladaptive combination of D. yakuba behavior and D. teissieri physiology, generating additional barriers to gene flow. Nevertheless, analysis of introgressed and non-introgressed regions of the genome indicate that, while rare, gene flow is relatively recent. These observations identify precise intrinsic and extrinsic factors that, along with hybrid male sterility, limit gene flow and maintain these species. These data contribute to a growing body of literature that suggests the Gulf of Guinea may be a hotspot for hybridization.

Wednesday, September 19th - Disease Models

Chiku, T., Hayashishita, M., Saito, T., Oka, M., Shinno, K., Ohtake, Y., Shimizu, S., Asada, A., Hisanaga, S. I., Iijima, K. M. and Ando, K. (2018). S6K/p70S6K1 protects against tau-mediated neurodegeneration by decreasing the level of tau phosphorylated at Ser262 in a Drosophila model of tauopathy. Neurobiol Aging 71: 255-264. PubMed ID: 30172839
Abnormal accumulation of the microtubule-associated protein tau is thought to cause neuronal cell death in a group of age-associated neurodegenerative disorders. Tau is phosphorylated at multiple sites in diseased brains, and phosphorylation of tau at Ser262 initiates tau accumulation and toxicity. This study sought to identify novel factors that affect the metabolism and toxicity of tau phosphorylated at Ser262 (pSer262-tau). A biased screen using a Drosophila model of tau toxicity revealed that knockdown of S6K, the Drosophila homolog of p70S6K1, increased the level of pSer262-tau and enhanced tau toxicity. S6K can be activated by the insulin signaling, however, unlike knockdown of S6K, knockdown of insulin receptor or insulin receptor substrate nonselectively decreased total tau levels via autophagy. Importantly, activation of S6K significantly suppressed tau-mediated axon degeneration, whereas manipulation of either the insulin signaling pathway or autophagy did not. These results suggest that activation of S6K may be an effective therapeutic strategy for selectively decreasing the levels of toxic tau species and suppressing neurodegeneration.
Drombosky, K. W., Rode, S., Kodali, R., Jacob, T. C., Palladino, M. J. and Wetzel, R. (2018). Mutational analysis implicates the amyloid fibril as the toxic entity in Huntington's disease. Neurobiol Dis. PubMed ID: 30171891
In Huntington disease (HD), an expanded polyglutamine (polyQ>37) sequence within huntingtin (htt) exon1 leads to enhanced disease risk. It has proved difficult, however, to determine whether the toxic form generated by polyQ expansion is a misfolded or avid-binding monomer, an alpha-helix-rich oligomer, or a beta-sheet-rich amyloid fibril. This study describes an engineered htt exon1 analog featuring a short polyQ sequence that nonetheless quickly forms amyloid fibrils and causes HD-like toxicity in rat neurons and Drosophila. Additional modifications within the polyQ segment produce htt exon1 analogs that populate only spherical oligomers and are non-toxic in cells and flies. Furthermore, in mixture with expanded-polyQ htt exon1, the latter analogs in vitro suppress amyloid formation and promote oligomer formation, and in vivo rescue neurons and flies expressing mhtt exon1 from dysfunction and death. Thus, in these experiments, while htt exon1 toxicity tracks with aggregation propensity, it does so in spite of the toxic construct's possessing polyQ tracts well below those normally considered to be disease-associated. That is, aggregation propensity proves to be a more accurate surrogate for toxicity than is polyQ repeat length itself, strongly supporting a major toxic role for htt exon1 aggregation in HD. In addition, the results suggest that the aggregates that are most toxic in these model systems are amyloid-related. Small molecules with similar amyloid inhibitory properties might be developed into effective therapeutic agents.
Androschuk, A., He, R. X., Weber, S., Rosenfelt, C. and Bolduc, F. V. (2018). Stress odorant sensory response dysfunction in Drosophila Fragile X Syndrome mutants. Front Mol Neurosci 11: 242. PubMed ID: 30135642
Sensory processing dysfunction (SPD) is present in most patients with intellectual disability (ID) and autism spectrum disorder (ASD). Silencing expression of the Fragile X mental retardation 1 (FMR1) gene leads to Fragile X syndrome (FXS), the most common single gene cause of ID and ASD. Drosophila have a highly conserved FMR1 ortholog, dfmr1. dfmr1 mutants display cognitive and social defects reminiscent of symptoms seen in individuals with FXS. This study utilized a robust behavioral assay for sensory processing of the Drosophila stress odorant (dSO) to gain a better understanding of the molecular basis of SPD in FXS. This study shows that dfmr1 mutant flies present significant defects in dSO response. dfmr1 expression in mushroom bodies was found to be required for dSO processing. It was also shown that cyclic adenosine monophosphate (cAMP) signaling via PKA is activated after exposure to dSO and that several drugs regulating both cAMP and cyclic guanosine monophosphate (cGMP) levels significantly improved defects in dSO processing in dfmr1 mutant flies.
Cunningham, P. C., Waldeck, K., Ganetzky, B. and Babcock, D. T. (2018). Neurodegeneration and locomotor dysfunction in Drosophila scarlet mutants. J Cell Sci. PubMed ID: 30154211
Parkinson's Disease (PD) is characterized by the loss of dopaminergic neurons, resulting in progressive locomotor dysfunction. Identification of genes required for the maintenance of these neurons should help to identify potential therapeutic targets. However, little is known regarding the factors that render dopaminergic neurons selectively vulnerable to PD. This study shows that Drosophila melanogaster scarlet mutants exhibit an age-dependent progressive loss of dopaminergic neurons, along with subsequent locomotor defects and a shortened lifespan. Knockdown of Scarlet specifically within dopaminergic neurons is sufficient to produce this neurodegeneration, demonstrating a unique role for Scarlet beyond its well-characterized role in eye pigmentation. Both genetic and pharmacological manipulation of the kynurenine pathway rescued loss of dopaminergic neurons by promoting synthesis of the free radical scavenger Kynurenic Acid (KYNA) and limiting the production of the free radical generator 3-hydroxykynurenine (3-HK). Finally, this study showed that expression of wild-type Scarlet is neuroprotective in a model of PD, suggesting that manipulating kynurenine metabolism may be a potential therapeutic option in treating PD.

Tuesday, September 18th - Chromatin

Warecki, B. and Sullivan, W. (2018). Micronuclei formation is prevented by Aurora B-mediated exclusion of HP1a from late-segregating chromatin in Drosophila. Micronuclei formation is prevented by Aurora B-mediated exclusion of HP1a from late-segregating chromatin in Drosophila
While it is known that micronuclei pose a serious risk to genomic integrity by undergoing chromothripsis, mechanisms preventing micronucleus formation remain poorly understood. This study investigate how late-segregating acentric chromosomes that would otherwise form micronuclei instead reintegrate into daughter nuclei by passing through Aurora B kinase-dependent channels in the nuclear envelope of Drosophila melanogaster neuroblasts. :ocalized concentrations of Aurora B preferentially phosphorylate H3(S10) on acentrics and their associated DNA tethers. This phosphorylation event prevents HP1a from associating with heterochromatin and results in localized inhibition of nuclear envelope reassembly on endonuclease and X-irradiation-induced acentrics, promoting channel formation. Finally, this study found that HP1a also specifies initiation sites of nuclear envelope reassembly on undamaged chromatin. Taken together, these results demonstrate that Aurora B-mediated regulation of HP1a-chromatin interaction plays a key role maintaining genome integrity by locally preventing nuclear envelope assembly and facilitating incorporation of late-segregating acentrics into daughter nuclei.
Sureka, R., Wadhwa, R., Thakur, S. S., Pathak, R. U. and Mishra, R. K. (2018). Comparison of Nuclear Matrix and Mitotic Chromosome Scaffold proteins in Drosophila S2 cells - Transmission of hallmarks of nuclear organization through mitosis. Mol Cell Proteomics. PubMed ID: 29991507
Chromatin condenses several folds to form mitotic chromosomes during cell division and decondenses post-mitotically to re-occupy their nuclear territory and re-gain their specific transcriptional profile in a precisely lineage specific manner. This necessitates that the features of nuclear architecture and DNA topology persist through mitosis. This study compared the proteome of nuclease and high salt resistant fraction of interphase nucleus known as nuclear matrix (NuMat) and an equivalent biochemical fraction in the mitotic chromosome known as mitotic chromosome scaffold (MiCS). This study elucidates that as much as 67% of the NuMat proteins are retained in the MiCS indicating that the features of nuclear architecture in interphase nucleus are retained on the mitotic chromosomes. Proteins of the NuMat/MiCS have large dynamic range of MS signal and were detected in sub-femtomolar amounts. Chromatin/RNA binding proteins with hydrolase and helicase activity are highly enriched in NuMat as well as MiCS. While several transcription factors involved in functioning of interphase nucleus are present exclusively in NuMat, protein components responsible for assembly of membrane-less nuclear bodies are uniquely retained in MiCS. This study clearly indicates that the features of nuclear architecture, in the structural context of NuMat, are retained in MiCS and possibly play an important role in maintenance of cell lineage specific transcriptional status during cell division and thereby, serve as components of cellular memory.
Clemot, M., Molla-Herman, A., Mathieu, J., Huynh, J. R. and Dostatni, N. (2018). The replicative histone chaperone CAF1 is essential for the maintenance of identity and genome integrity in adult stem cells. Development 145(17). PubMed ID: 30093554
Chromatin packaging and modifications are important to define the identity of stem cells. How chromatin properties are retained over multiple cycles of stem cell replication, while generating differentiating progeny at the same time, remains a challenging question. The chromatin assembly factor CAF1 is a conserved histone chaperone, which assembles histones H3 and H4 onto newly synthesized DNA during replication and repair. This study has investigated the role of CAF1 in the maintenance of germline stem cells (GSCs) in Drosophila ovaries. P180, the large subunit of CAF1, was depleted in germ cells and found that it was required in GSCs to maintain their identity. In the absence of P180, GSCs still harbor stem cell properties but concomitantly express markers of differentiation. In addition, P180-depleted germ cells exhibit elevated levels of DNA damage and de-repression of the transposable I element. These DNA damages activate p53- and Chk2-dependent checkpoints pathways, leading to cell death and female sterility. Altogether, this work demonstrates that chromatin dynamics mediated by CAF1 play an important role in both the regulation of stem cell identity and genome integrity.
Baldi, S., Krebs, S., Blum, H. and Becker, P. B. (2018). Genome-wide measurement of local nucleosome array regularity and spacing by nanopore sequencing. Nat Struct Mol Biol 25(9): 894-901. PubMed ID: 30127356
The nature of chromatin as regular succession of nucleosomes has gained iconic status. However, since most nucleosomes in metazoans are poorly positioned it is unknown to which extent bulk genomic nucleosome repeat length reflects the regularity and spacing of nucleosome arrays at individual loci. This study describes a new approach to map nucleosome array regularity and spacing through sequencing oligonucleosome-derived DNA by Illumina sequencing and emergent nanopore technology. In Drosophila cells, this revealed modulation of array regularity and nucleosome repeat length depending on functional chromatin states independently of nucleosome positioning and even in unmappable regions. It was also found that nucleosome arrays downstream of silent promoters are considerably more regular than those downstream of highly expressed ones, despite more extensive nucleosome phasing of the latter. This approach is generally applicable and provides an important parameter of chromatin organization that so far had been missing.

Monday, September 17th - Synapse and Vesicles

Brusich, D. J., Spring, A. M., James, T. D., Yeates, C. J., Helms, T. H. and Frank, C. A. (2018). Drosophila CaV2 channels harboring human migraine mutations cause synapse hyperexcitability that can be suppressed by inhibition of a Ca2+ store release pathway. PLoS Genet 14(8): e1007577. PubMed ID: 30080864
Gain-of-function mutations in the human CaV2.1 gene CACNA1A cause familial hemiplegic migraine type 1 (FHM1). To characterize cellular problems potentially triggered by CaV2.1 gains of function, mutations encoding FHM1 amino-acid substitutions S218L (SL) and R192Q (RQ) were engineered into transgenes of Drosophila CaV2/cacophony. The transgenes were expressed pan-neuronally. Single mutant SL- and complex allele RQ,SL-expressing animals showed overt phenotypes, including sharply decreased viability. SL- and RQ,SL-expressing neuromuscular junctions (NMJs) exhibited enhanced evoked discharges, supernumerary discharges, and an increase in the amplitudes and frequencies of spontaneous events. Some spontaneous events were gigantic (10-40 mV), multi-quantal events. These were eliminated by application of TTX-or by lowered or chelated Ca2+-suggesting that gigantic events were elicited by spontaneous nerve firing. Some neuronal hyperexcitability phenotypes were reversed after knockdown of Drosophila phospholipase Cbeta (PLCbeta), IP3 receptor, or ryanodine receptor (RyR)-all factors known to mediate Ca2+ release from intracellular stores. Pharmacological inhibitors of intracellular Ca2+ store release produced similar effects. Interestingly, however, the decreased viability phenotype was not reversed by genetic impairment of intracellular Ca2+ release factors. On a cellular level, the data suggest inhibition of signaling that triggers intracellular Ca2+ release could counteract hyperexcitability induced by gains of CaV2.1 function.
Chandran, R. R., Scholl, A., Yang, Y. and Jiang, L. (2018). rebuff regulates apical luminal matrix to control tube size in Drosophila trachea. Biol Open 7(9). PubMed ID: 30185423
The Drosophila embryonic tracheal network is an excellent model to study tube size. The chitin-based apical luminal matrix and cell polarity are well known to regulate tube size in Drosophila trachea. Defects in luminal matrix and cell polarity lead to tube overexpansion. This study addressed the novel function of the rebuff (reb) gene, which encodes an evolutionarily conserved Smad-like protein. In reb mutants, tracheal tubes are moderately over-elongated. Despite the establishment of normal cell polarity, significantly reduced apical luminal matrix was observed in reb mutants. Among various luminal components, luminal Obstructor-A (ObstA) is drastically reduced. Interestingly, ObstA is localized in vesicle-like structures that are apically concentrated in reb mutants. To investigate the possibility that reb is involved in the endocytosis of ObstA, the co-localization of ObstA and endocytic markers was examined in reb mutants. It was observed that ObstA is localized in late endosomes and recycling endosomes. This suggests that in reb mutant trachea, endocytosed ObstA is degraded or recycled back to the apical region. However, ObstA vesicles are retained in the apical region and are failed to be secreted to the lumen. Taken together, these results suggest one function of reb is regulating the endocytosis of luminal matrix components.
Hahm, E. T., Nagaraja, R. Y., Waro, G. and Tsunoda, S. (2018). Cholinergic homeostatic synaptic plasticity drives the progression of Abeta-induced changes in neural activity. Cell Rep 24(2): 342-354. PubMed ID: 29996096
Homeostatic synaptic plasticity (HSP) is the ability of neurons to exert compensatory changes in response to altered neural activity. How pathologically induced activity changes are intertwined with HSP mechanisms is unclear. This study shows that, in cholinergic neurons from Drosophila, beta-amyloid (Abeta) peptides Abeta40 and Abeta42 (see Drosophila β amyloid protein precursor-like) both induce an increase in spontaneous activity. In a transgenic line expressing Abeta42, it was observed that this early increase in spontaneous activity is followed by a dramatic reduction in spontaneous events, a progression that has been suggested to occur in cholinergic brain regions of mammalian models of Alzheimer's disease. Evidence is presented that the early enhancement in synaptic activity is mediated by the Drosophila alpha7 nicotinic acetylcholine receptor (nAChR) and that, later, Abeta42-induced inhibition of synaptic events is a consequence of Dalpha7-dependent HSP mechanisms induced by earlier hyperactivity. Thus, while HSP may initially be an adaptive response, it may also drive maladaptive changes and downstream pathologies.
Mattingly, M., Weineck, K., Costa, J. and Cooper, R. L. (2018). Hyperpolarization by activation of halorhodopsin results in enhanced synaptic transmission: Neuromuscular junction and CNS circuit. PLoS One 13(7): e0200107. PubMed ID: 29969493
Optogenetics offers a unique method to regulate the activity of select neural circuits. However, the electrophysiological consequences of targeted optogenetic manipulation upon the entire circuit remain poorly understood. Analysis of the sensory-CNS-motor circuit in Drosophila larvae expressing eHpHR and ChR2-XXL revealed unexpected patterns of excitability. Optical stimulation of motor neurons targeted to express eNpHR resulted in inhibition followed by excitation of body wall contraction with repetitive stimulation in intact larvae. In situ preparations with direct electrophysiological measures showed an increased responsiveness to excitatory synaptic activity induced by sensory stimulation within a functional neural circuit. To ensure proper function of eNpHR and ChR2-XXL they were expressed in body wall muscle and direct electrophysiological measurements were obtained. Under eNpHR induced hyperpolarization the muscle remained excitable with increased amplitude of excitatory postsynaptic synaptic potentials. Theoretical models to explain the observations are presented. This study aids in increasing the understanding of the varied possible influences with light activated proteins within intact neural circuits.

Friday, September 14th - Embryonic and Larval Development

Mineo, A., Fuentes, E., Furriols, M. and Casanova, J. (2018). Holes in the Plasma Membrane Mimic Torso-Like Perforin in Torso Tyrosine Kinase Receptor Activation in the Drosophila Embryo. Genetics. PubMed ID: 30049783
Receptor tyrosine kinase (RTK) pathways play central roles in development, and when abnormally activated they can lead to pathological conditions, including oncogenesis. Thus, RTK activation, mediated by ligand binding, is under tight control, a critical step being the conversion of an inactive precursor into the active form of the ligand. A variety of mechanisms have been shown to be involved in this conversion; however, little attention has been paid to how mechanical phenomena may impinge on this process. This issue was addressed by studying Torso, an RTK activated at both poles of the Drosophila embryo at blastoderm stage. Torso activation is induced by a cleaved form of Trunk, a growth factor-like protein, but it also requires the accumulation of the Torso-like (Tsl) protein at both ends of the blastoderm. Tsl is the only known protein in Drosophila bearing a Membrane Attack Complex/Perforin (MACPF) domain, a motif present in proteins involved in pore formation at cell membranes. However, while different hypotheses have been put forward to account for the function of Tsl in Torso receptor activation, little is known about its molecular role and whether it indeed contributes to membrane pore formation. This study shows that mechanically induced holes in the Drosophila embryo can substitute for Tsl function. These results suggest that Tsl is required for an exchange between the interior of the Drosophila embryo and its surrounding milieu and that mechanically induced cell injuries may contribute to abnormal RTK activation.
Park, S. H., Lee, C. W., Lee, J. H., Park, J. Y., Roshandell, M., Brennan, C. A. and Choe, K. M. (2018). Requirement for and polarized localization of integrin proteins during Drosophila wound closure. Mol Biol Cell: mbcE17110635. PubMed ID: 29995573
Wound re-epithelialization is an evolutionarily conserved process in which skin cells migrate as sheets to heal the breach, and is critical to prevent infection, but impaired in chronic wounds. Integrin heterodimers mediate attachment between epithelia and underlying extracellular matrix, and also act in large signaling complexes. The complexity of the mammalian wound environment and evident redundancy among integrins has impeded determination of their specific contributions to re-epithelialization. Taking advantage of the genetic tools and smaller number of integrins in Drosophila, a systematic in vivo analysis of integrin requirements in the re-epithelialization of skin wounds was underrtaken in the larva. αPS2-βPS and αPS3-βPS were identified as the crucial integrin dimers, and talinnwas identified as the only integrin adhesion component required for re-epithelialization. The integrins rapidly accumulate in a JNK-dependent manner in a few rows of cells surrounding a wound. Intriguingly, the integrins localize to the distal margin in these cells, instead of the frontal or lamellipodial distribution expected for proteins providing traction, and also recruit nonmuscle myosin II to the same location. These findings indicate that signaling roles of integrins may be important for epithelial polarization around wounds, and lay the groundwork for using Drosophila to better understand integrin contributions to re-epithelialization.
Beaven, R. and Denholm, B. (2018). Release and spread of Wingless is required to pattern the proximo-distal axis of Drosophila renal tubules. Elife 7. PubMed ID: 30095068
Wingless/Wnts are signalling molecules, traditionally considered to pattern tissues as long-range morphogens. However, more recently the spread of Wingless was shown to be dispensable in diverse developmental contexts in Drosophila and vertebrates. This study demonstrates that release and spread of Wingless is required to pattern the proximo-distal (P-D) axis of Drosophila Malpighian tubules. Wingless signalling, emanating from the midgut, directly activates odd skipped expression several cells distant in the proximal tubule. Replacing Wingless with a membrane-tethered version that is unable to diffuse from the Wingless producing cells results in aberrant patterning of the Malpighian tubule P-D axis and development of short, deformed ureters. This work directly demonstrates a patterning role for a released Wingless signal. As well as extending the understanding about the functional modes by which Wnts shape animal development, it is anticipated that this mechanism is relevant to patterning epithelial tubes in other organs, such as the vertebrate kidney.
Mendoza-Ortiz, M. A., Murillo-Maldonado, J. M. and Riesgo-Escovar, J. R. (2018). aaquetzalli is required for epithelial cell polarity and neural tissue formation in Drosophila. PeerJ 6: e5042. PubMed ID: 29942698
Morphogenetic movements during embryogenesis require dynamic changes in epithelial cell polarity and cytoskeletal reorganization. Such changes involve, among others, rearrangements of cell-cell contacts and protein traffic. In Drosophila, neuroblast delamination is regulated by the Notch signaling pathway. Maintenance of epithelial cell polarity ensues proper Notch pathway activation during neurogenesis. This study characterized aaquetzalli (aqz), a gene whose mutations affect cell polarity and nervous system specification. The aqz locus encodes a protein that harbors a domain with significant homology to a proline-rich conserved domain of nuclear receptor co-activators. aqz expression occurs at all stages of the fly life cycle, and is dynamic. aqz mutants are lethal, showing a disruption of cell polarity during embryonic ventral neuroepithelium differentiation resulting in loss of epithelial integrity and mislocalization of membrane proteins (shown by mislocalization of Crumbs, DE-Cadherin, and Delta). As a consequence, aqz mutant embryos with compromised apical-basal cell polarity develop spotty changes of neuronal and epithelial numbers of cells.

Thursday September 13th - Behavior

Watanabe, K., Suzuki, Y., Inami, S., Ohashi, H. and Sakai, T. (2018). Light is required for proper female mate choice between winged and wingless males in Drosophila. Genes Genet Syst. PubMed ID: 29998908
In many animal species, females choose potential mating partners according to their own preferences. Thus, female preference-based mate choice affects intraspecific mating success and prevents interspecific mating. To clarify the neuronal basis of female mate choice, it is essential to identify the important relevant sensory cues. In the fruitfly Drosophila melanogaster, the courtship song of males promotes female sexual receptivity. When wild-type virgin females can freely choose one of two types of courting males (winged or wingless males), they prefer to mate with winged males. This study reports a crucial sensory cue relevant to this female mate choice. In a female choice test, female receptivity toward winged and wingless males was markedly reduced when females had auditory impairments, although females with visual or olfactory impairments showed normal receptivity similar to wild-type females. However, females with visual impairments did not show clear mate preference toward winged males. Thus, these findings suggest that females utilize visual cues in mate choice between winged and wingless males in Drosophila.
Zakharenko, L. P., Petrovskii, D. V. and Putilov, A. A. (2018). Larks, owls, swifts, and woodcocks among fruit flies: differential responses of four heritable chronotypes to long and hot summer days. Nat Sci Sleep 10: 181-191. PubMed ID: 29950910
This study examined the possibility to distinguish four extreme chronotypes among fruit flies and the possibility of the differential response of such chronotypes to light and heat stressors. Circadian rhythms of locomotor activity and sleep-wake pattern were tested in constant darkness, and four strains of fruit flies originating from three wild populations of Africa, Europe, and the USA were selected to represent four distinct chronotypes: "larks" (early morning and evening activity peaks), "owls" (late morning and evening peaks), "swifts" (early morning and late evening peaks), and "woodcocks" (late morning and early evening peaks). The circadian rhythms and sleep efficiency of the selected chronotypes were further tested under such extreme conditions as either long day (LD20:4 at 20 ° C) or a combination of LD20:4 with hot temperature (29 ° C). Despite the identity of such experimental conditions for four chronotypes, their circadian rhythms and sleep timing showed significantly distinct patterns of response to exposure to heat and/or long days. All two-way repeated measures analysis of variances yielded a significant interaction between chronotype and time of the day. It is concluded that an experimental study of heritable chronotypes in the fruit fly can facilitate a search for genetic underpinnings of individual variation in vulnerability to circadian misalignment, maladaptive sleep-wake behavior, and sleep disorders.
Ribeiro, I. M. A., Drews, M., Bahl, A., Machacek, C., Borst, A. and Dickson, B. J. (2018). Visual projection neurons mediating directed courtship in Drosophila. Cell 174(3): 607-621.e618. PubMed ID: 30033367
Many animals rely on vision to detect, locate, and track moving objects. In Drosophila courtship, males primarily use visual cues to orient toward and follow females and to select the ipsilateral wing for courtship song. This study shows that the LC10 visual projection neurons of the Lobula Column convey essential visual information during courtship. Males with LC10 neurons silenced are unable to orient toward or maintain proximity to the female and do not predominantly use the ipsilateral wing when singing. LC10 neurons preferentially respond to small moving objects using an antagonistic motion-based center-surround mechanism. Unilateral activation of LC10 neurons recapitulates the orienting and ipsilateral wing extension normally elicited by females, and the potency with which LC10 induces wing extension is enhanced in a state of courtship arousal controlled by male-specific P1 neurons. These data suggest that LC10 is a major pathway relaying visual input to the courtship circuits in the male brain.
Baik, L. S., Recinos, Y., Chevez, J. A. and Holmes, T. C. (2018). Circadian modulation of light-evoked avoidance/attraction behavior in Drosophila. PLoS One 13(8): e0201927. PubMed ID: 30106957
Many insects show strong behavioral responses to short wavelength light. Drosophila melanogaster exhibit Cryptochrome- and Hyperkinetic-dependent blue and ultraviolet (UV) light avoidance responses that vary by time-of-day, suggesting that these key sensory behaviors are circadian regulated. This study shows mutant flies lacking core clock genes exhibit defects in both time-of-day responses and valence of UV light avoidance/attraction behavior. Non-genetic environmental disruption of the circadian clock by constant UV light exposure leads to complete loss of rhythmic UV light avoidance/attraction behavior. Flies with ablated or electrically silenced circadian lateral ventral neurons have attenuated avoidance response to UV light. It is concluded that circadian clock proteins and the circadian lateral ventral neurons of the adult brain regulate both the timing and the valence of UV light avoidance/attraction. These results provide mechanistic support for Pittendrigh's "escape from light" hypothesis regarding the co-evolution of phototransduction and circadian systems.

Wednesday, September 12th - Disease Models

Sun, W, Samimi, H., Gamez, M., Zare, H. and Frost, B. (2018). Pathogenic tau-induced piRNA depletion promotes neuronal death through transposable element dysregulation in neurodegenerative tauopathies. Nat Neurosci 21(8): 1038-1048. PubMed ID: 30038280
Transposable elements, known colloquially as 'jumping genes', constitute approximately 45% of the human genome. Cells utilize epigenetic defenses to limit transposable element jumping, including formation of silencing heterochromatin and generation of piwi-interacting RNAs (piRNAs), small RNAs that facilitate clearance of transposable element transcripts. This study utilize Drosophila melanogaster and postmortem human brain samples to identify transposable element dysregulation as a key mediator of neuronal death in tauopathies, a group of neurodegenerative disorders that are pathologically characterized by deposits of tau protein in the brain. Mechanistically, it was found that heterochromatin decondensation and reduction of piwi and piRNAs drive transposable element dysregulation in tauopathy. A significant increase is reported in transcripts of the endogenous retrovirus class of transposable elements in human Alzheimer's disease and progressive supranuclear palsy, suggesting that transposable element dysregulation is conserved in human tauopathy. Taken together, these data identify heterochromatin decondensation, piwi and piRNA depletion and consequent transposable element dysregulation as a pharmacologically targetable, mechanistic driver of neurodegeneration in tauopathy.
Azuma, Y., Tokuda, T., Kushimura, Y., Yamamoto, I., Mizuta, I., Mizuno, T., Nakagawa, M., Ueyama, M., Nagai, Y., Iwasaki, Y., Yoshida, M., Pan, D., Yoshida, H. and Yamaguchi, M. (2018). Hippo, Drosophila MST, is a novel modifier of motor neuron degeneration induced by knockdown of Caz, Drosophila FUS. Exp Cell Res. PubMed ID: 30092221
Mutations in the Fused in Sarcoma (FUS) gene have been identified in familial ALS in human. Drosophila contains a single ortholog of human FUS called Cabeza (Caz). Drosophila models of ALS have been established targeted to Caz, which developed the locomotive dysfunction and caused anatomical defects in presynaptic terminals of motoneurons. Accumulating evidence suggests that ALS and cancer share defects in many cellular processes. The Hippo pathway was originally discovered in Drosophila and plays a role as a tumor suppressor in mammals. Whether Hippo pathway genes modify the ALS phenotype was determined using Caz knockdown flies. A genetic link was found between Caz and Hippo (hpo), the Drosophila ortholog of human Mammalian sterile 20-like kinase (MST) 1 and 2. Loss-of-function mutations of hpo rescued Caz knockdown-induced eye- and neuron-specific defects. The decreased Caz levels in nuclei induced by Caz knockdown were also rescued by loss of function mutations of hpo. Moreover, hpo mRNA level was dramatically increased in Caz knockdown larvae, indicating that Caz negatively regulated hpo. The results demonstrate that hpo, Drosophila MST, is a novel modifier of Drosophila FUS. Therapeutic targets that inhibit the function of MST could modify the pathogenic processes of ALS.
Xu, W. and Xu, J. (2018). C9orf72 dipeptide repeats cause selective neurodegeneration and cell-autonomous excitotoxicity in Drosophila glutamatergic neurons. J Neurosci. PubMed ID: 30037833
The arginine-rich dipeptide repeats (DPRs) are highly toxic products from the C9orf72 repeat expansion mutations, which are the most common causes of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). However, the effects of DPRs in the synaptic regulation and excitotoxicity remain elusive, and how they contribute to the development of FTD is largely unknown. By expressing DPRs with different toxicity strength in various neuronal populations in a Drosophila model, it was unexpectedly found that GR/PR with 36 repeats could lead to neurodegenerative phenotypes only when they were expressed in glutamatergic neurons, including motor neurons. Increased extracellular glutamate and intracellular calcium levels were detected in GR/PR-expressing larval ventral nerve cord and/or adult brain, accompanied by significant increase of synaptic boutons and active zones in larval neuromuscular junctions. Inhibiting the vesicular glutamate transporter (vGlut) expression or blocking the NMDA receptor in presynaptic glutamatergic motor neurons could effectively rescue the motor deficits and shortened life span caused by poly GR/PR, thus indicating a cell-autonomous excitotoxicity mechanism. Therefore, these results have revealed a novel mode of synaptic regulation by arginine-rich C9 DPRs expressed at more physiologically relevant toxicity levels and provided a mechanism that could contribute to the development of C9-related ALS and FTD.
Yamamoto, I., Azuma, Y., Kushimura, Y., Yoshida, H., Mizuta, I., Mizuno, T., Ueyama, M., Nagai, Y., Tokuda, T. and Yamaguchi, M. (2018). NPM-hMLF1 fusion protein suppresses defects of a Drosophila FTLD model expressing the human FUS gene. Sci Rep 8(1): 11291. PubMed ID: 30050143
Fused in sarcoma (FUS) was identified as a component of typical inclusions in frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). In FTLD, both nuclear and cytoplasmic inclusions with wild-type FUS exist, while cytoplasmic inclusions with a mutant-form of FUS occur in many ALS cases. These observations imply that FUS plays a role across these two diseases. This study examined the effect of several proteins including molecular chaperons on the aberrant eye morphology phenotype induced by overexpression of wild-type human FUS (hFUS) in Drosophila eye imaginal discs. By screening, it was found that the co-expression of nucleophosmin-human myeloid leukemia factor 1 (NPM-hMLF1) fusion protein could suppress the aberrant eye morphology phenotype induced by hFUS. The driving of hFUS expression at 28 degrees C down-regulated levels of hFUS and endogenous cabeza, a Drosophila homolog of hFUS. The down-regulation was mediated by proteasome dependent degradation. Co-expression of NPM-hMLF1 suppressed this down-regulation. In addition, co-expression of NPM-hMLF1 partially rescued pharate adult lethal phenotype induced by hFUS in motor neurons. These findings with a Drosophila model that mimics FTLD provide clues for the development of novel FTLD therapies.

Tuesday, September 11th - Adult Neural Function

Rudisill, S. S., Martin, B. R., Mankowski, K. M. and Tessier, C. R. (2018). Iron deficiency reduces synapse formation in the Drosophila clock circuit. Biol Trace Elem Res. PubMed ID: 30022428
Iron serves as a critical cofactor for proteins involved in a host of biological processes. In most animals, dietary iron is absorbed in enterocytes and then disseminated for use in other tissues in the body. The brain is particularly dependent on iron. Altered iron status correlates with disorders ranging from cognitive dysfunction to disruptions in circadian activity. The exact role iron plays in producing these neurological defects, however, remains unclear. Invertebrates provide an attractive model to study the effects of iron on neuronal development since many of the genes involved in iron metabolism are conserved, and the organisms are amenable to genetic and cytological techniques. This study has examined synapse growth specifically under conditions of iron deficiency in the Drosophila circadian clock circuit. This study shows that projections of the small ventrolateral clock neurons to the protocerebrum of the adult Drosophila brain are significantly reduced upon chelation of iron from the diet. This growth defect persists even when iron is restored to the diet. Genetic neuronal knockdown of ferritin 1 or ferritin 2, critical components of iron storage and transport, does not affect synapse growth in these cells. Together, these data indicate that dietary iron is necessary for central brain synapse formation in the fly and further validate the use of this model to study the function of iron homeostasis on brain development.
Al-Anzi, B. and Zinn, K. (2018). Identification and characterization of mushroom body neurons that regulate fat storage in Drosophila. Neural Dev 13(1): 18. PubMed ID: 30103787
Two neuronal populations, c673a and Fru-GAL4, regulate fat storage in fruit flies. Both populations partially overlap with a structure in the insect brain known as the mushroom body (MB), which plays a critical role in memory formation. This overlap prompted an examination of whether the MB is also involved in fat storage homeostasis. Using a variety of transgenic agents, the neural activity of different portions of the MB and associated neurons were selectively manipulated to decipher their roles in fat storage regulation. The data show that silencing of MB neurons that project into the &alpha:'β' lobes decreases de novo fatty acid synthesis and causes leanness, while sustained hyperactivation of the same neurons causes overfeeding and produces obesity. The &alpha:'β' neurons oppose and dominate the fat regulating functions of the c673a and Fru-GAL4 neurons. It was also shown that MB neurons that project into the γ lobe also regulate fat storage, probably because they are a subset of the Fru neurons. It was posible to identify input and output neurons whose activity affects fat storage, feeding, and metabolism. The activity of cholinergic output neurons that innervating the β'2 compartment (MBON-β'2mp and MBON-γ5β'2a) regulates food consumption, while glutamatergic output neurons innervating α' compartments (MBON-γ2α'1 and MBON-α'2) control fat metabolism. This study has identified a new fat storage regulating center, the α'β' lobes of the MB. The study also delineated the neuronal circuits involved in the actions of the α'β' lobes, and showed that food intake and fat metabolism are controlled by separate sets of postsynaptic neurons that are segregated into different output pathways.
Seeholzer, L. F., Seppo, M., Stern, D. L. and Ruta, V. (2018). Evolution of a central neural circuit underlies Drosophila mate preferences. Nature 559(7715): 564-569. PubMed ID: 29995860
Courtship rituals serve to reinforce reproductive barriers between closely related species. Drosophila melanogaster and Drosophila simulans exhibit reproductive isolation, owing in part to the fact that D. melanogaster females produce 7,11-heptacosadiene, a pheromone that promotes courtship in D. melanogaster males but suppresses courtship in D. simulans males. This study compares pheromone-processing pathways in D. melanogaster and D. simulans males to define how these sister species endow 7,11-heptacosadiene with the opposite behavioural valence to underlie species discrimination. Males of both species detect 7,11-heptacosadiene using homologous peripheral sensory neurons, but this signal is differentially propagated to P1 neurons, which control courtship behaviour. A change in the balance of excitation and inhibition onto courtship-promoting neurons transforms an excitatory pheromonal cue in D. melanogaster into an inhibitory cue in D. simulans. These results reveal how species-specific pheromone responses can emerge from conservation of peripheral detection mechanisms and diversification of central circuitry, and demonstrate how flexible nodes in neural circuits can contribute to behavioural evolution.
Barish, S., Nuss, S., Strunilin, I., Bao, S., Mukherjee, S., Jones, C. D. and Volkan, P. C. (2018). Combinations of DIPs and Dprs control organization of olfactory receptor neuron terminals in Drosophila. PLoS Genet 14(8): e1007560. PubMed ID: 30102700
In Drosophila, 50 classes of olfactory receptor neurons (ORNs) connect to 50 class-specific and uniquely positioned glomeruli in the antennal lobe. Despite the identification of cell surface receptors regulating axon guidance, how ORN axons sort to form 50 stereotypical glomeruli remains unclear. This study show that the heterophilic cell adhesion proteins, DIPs and Dprs, are expressed in ORNs during glomerular formation. Many ORN classes express a unique combination of DIPs/dprs, with neurons of the same class expressing interacting partners, suggesting a role in class-specific self-adhesion between ORN axons. Analysis of DIP/Dpr expression revealed that ORNs that target neighboring glomeruli have different combinations, and ORNs with very similar DIP/Dpr combinations can project to distant glomeruli in the antennal lobe. DIP/Dpr profiles are dynamic during development and correlate with sensilla type lineage for some ORN classes. Perturbations of DIP/dpr gene function result in local projection defects of ORN axons and glomerular positioning, without altering correct matching of ORNs with their target neurons. The results suggest that context-dependent differential adhesion through DIP/Dpr combinations regulate self-adhesion and sort ORN axons into uniquely positioned glomeruli.
Sun, Y., Jia, Y., Guo, Y., Chen, F. and Yan, Z. (2018). Taurine Transporter dEAAT2 is Required for Auditory Transduction in Drosophila. Neurosci Bull. PubMed ID: 30043098
Drosophila dEAAT2, a member of the excitatory amino-acid transporter (EAAT) family, has been described as mediating the high-affinity transport of taurine, which is a free amino-acid abundant in both insects and mammals. However, the role of taurine and its transporter in hearing is not clear. This study reports that dEAAT2 is required for the larval startle response to sound stimuli. dEAAT2 was found to be enriched in the distal region of chordotonal neurons where sound transduction occurs. The Ca(2+) imaging and electrophysiological results showed that disrupted dEAAT2 expression significantly reduced the response of chordotonal neurons to sound. More importantly, expressing dEAAT2 in the chordotonal neurons rescued these mutant phenotypes. Taken together, these findings indicate a critical role for Drosophila dEAAT2 in sound transduction by chordotonal neurons.
Alvarez-Salvado, E., Licata, A. M., Connor, E. G., McHugh, M. K., King, B. M., Stavropoulos, N., Victor, J. D., Crimaldi, J. P. and Nagel, K. I. (2018). Elementary sensory-motor transformations underlying olfactory navigation in walking fruit-flies. Elife 7. PubMed ID: 30129438
Odor attraction in walking Drosophila melanogaster is commonly used to relate neural function to behavior, but the algorithms underlying attraction are unclear. In this study, a high-throughput assay was developed to measure olfactory behavior in response to well-controlled sensory stimuli. Odor is shown to evokes two behaviors: an upwind run during odor (ON response), and a local search at odor offset (OFF response). Wind orientation requires antennal mechanoreceptors, but search is driven solely by odor. Using dynamic odor stimuli, the dependence of these two behaviors on odor intensity and history was measured. Based on these data, a navigation model was developed that recapitulates the behavior of flies in the apparatus, and generates realistic trajectories when run in a turbulent boundary layer plume. The ability to parse olfactory navigation into quantifiable elementary sensori-motor transformations provides a foundation for dissecting neural circuits that govern olfactory behavior.

Monday, September 10th - Autophagy and Apoptosis

Vishal, K., Bawa, S., Brooks, D., Bauman, K. and Geisbrecht, E. R. (2018). Thin is required for cell death in the Drosophila abdominal muscles by targeting DIAP1. Cell Death Dis 9(7): 740. PubMed ID: 29970915
In holometabolous insects, developmentally controlled programmed cell death (PCD) is a conserved process that destroys a subset of larval tissues for the eventual creation of new adult structures. This process of histolysis is relatively well studied in salivary gland and midgut tissues, while knowledge concerning larval muscle destruction is limited. This study examined the histolysis of a group of Drosophila larval abdominal muscles called the dorsal external oblique muscles (DEOMs). Previous studies have defined apoptosis as the primary mediator of DEOM breakdown, whose timing is controlled by ecdysone signaling. However, very little is known about other factors that contribute to DEOM destruction. In this paper, the role of thin (tn), which encodes for the Drosophila homolog of mammalian TRIM32, was examined in the regulation of DEOM histolysis. Loss of Tn blocks DEOM degradation independent of ecdysone signaling. Instead, tn genetically functions in a pathway with the death-associated inhibitor of apoptosis (DIAP1), Dronc, and death-associated APAF1-related killer (Dark) to regulate apoptosis. Importantly, blocking Tn results in the absence of active Caspase-3 immunostaining, upregulation of DIAP1 protein levels, and inhibition of Dronc activation. DIAP1 and Dronc mRNA levels are not altered in tn mutants, showing that Tn acts post-transcriptionally on DIAP1 to regulate apoptosis. This study also found that the RING domain of Tn is required for DEOM histolysis as loss of this domain results in higher DIAP1 levels. Together, these results suggest that the direct control of DIAP1 levels, likely through the E3 ubiquitin ligase activity of Tn, provides a mechanism to regulate caspase activity and to facilitate muscle cell death.
Issa, A. R., Sun, J., Petitgas, C., Mesquita, A., Dulac, A., Robin, M., Mollereau, B., Jenny, A., Cherif-Zahar, B. and Birman, S. (2018). The lysosomal membrane protein LAMP2A promotes autophagic flux and prevents SNCA-induced Parkinson disease-like symptoms in the Drosophila brain. Autophagy. PubMed ID: 29989488
The autophagy-lysosome pathway plays a fundamental role in the clearance of aggregated proteins and protection against cellular stress and neurodegenerative conditions. Alterations in autophagy processes, including macroautophagy and chaperone-mediated autophagy (CMA), have been described in Parkinson disease (PD). CMA is a selective autophagic process that depends on LAMP2A (Lysosomal associated membrane protein 2A), a mammal and bird-specific membrane glycoprotein that translocates cytosolic proteins containing a KFERQ-like peptide motif across the lysosomal membrane. Drosophila reportedly lack CMA and use endosomal microautophagy (eMI) as an alternative selective autophagic process. This study reports that neuronal expression of human LAMP2A protected Drosophila against starvation and oxidative stress, and delayed locomotor decline in aging flies without extending their lifespan. LAMP2A also prevented the progressive locomotor and oxidative defects induced by neuronal expression of PD-associated human SNCA (synuclein alpha) with alanine-to-proline mutation at position 30 (SNCA(A30P)). LAMP2A expression stimulated selective autophagy in the adult brain and not in the larval fat body. Noteworthy, neurally expressed LAMP2A markedly upregulated levels of Drosophila Atg5, a key macroautophagy initiation protein, and of the Atg5-containing complex, and that it increased the density of Atg8a/LC3-positive puncta, which reflects the formation of autophagosomes. Furthermore, LAMP2A efficiently prevented accumulation of the autophagy defect marker Ref(2)P/p62 in the adult brain under acute oxidative stress. These results indicate that LAMP2A can promote autophagosome formation and potentiate autophagic flux in the Drosophila brain, leading to enhanced stress resistance and neuroprotection.
Gorelick-Ashkenazi, A., Weiss, R., Sapozhnikov, L., Florentin, A., Tarayrah-Ibraheim, L., Dweik, D., Yacobi-Sharon, K. and Arama, E. (2018). Caspases maintain tissue integrity by an apoptosis-independent inhibition of cell migration and invasion. Nat Commun 9(1): 2806. PubMed ID: 30022065
Maintenance of tissue integrity during development and homeostasis requires the precise coordination of several cell-based processes, including cell death. In animals, the majority of such cell death occurs by apoptosis, a process mediated by caspase proteases. To elucidate the role of caspases in tissue integrity, this study investigated the behavior of Drosophila epithelial cells that are severely compromised for caspase activity. These cells acquire migratory and invasive capacities, either within 1-2 days following irradiation or spontaneously during development. Importantly, low levels of effector caspase activity, which are far below the threshold required to induce apoptosis, can potently inhibit this process, as well as a distinct, developmental paradigm of primordial germ cell migration. These findings may have implications for radiation therapy in cancer treatment. Furthermore, given the presence of caspases throughout metazoa, the results could imply that preventing unwanted cell migration constitutes an ancient non-apoptotic function of these proteases.
Chi, C., Wang, L., Lan, W., Zhao, L. and Su, Y. (2018). PpV, acting via the JNK pathway, represses apoptosis during normal development of Drosophila wing. Apoptosis. PubMed ID: 30159848
Apoptosis is one of the main fundamental biological processes required for development of multicellular organisms. Inappropriate regulation of apoptosis can lead to severe developmental abnormalities and diseases. Therefore, the control of apoptosis, not only for its activation but also for its inhibition, is critically important during development. In contrast to the extensive studies of apoptosis induction, its inhibitory mechanisms that are even more vital in certain populations of cells actually are very far from being well understood. This study reports an inhibitory role of protein phosphatase V (PpV), a serine/threonine protein phosphatase, in controlling the apoptosis during Drosophila wing development. Inhibition of ppv by RNAi in wing imaginal discs induced ectopic cell death and caspase activation, thus, resulted in a defective adult wing. Moreover, knocking-down ppv triggered the activation of c-Jun N-terminal kinase (JNK) signal, an evolutionarily conserved intracellular signaling that has been implicated to modulate the apoptotic machinery in many biological and experimental systems. Disrupting the JNK signal transduction was adequate to suppress the ppv effects for wing development. Together, this study provides the evidence to demonstrate that ppv is required for normal wing development in maintaining the silence of apoptotic signal possibly through JNK pathway.

Friday, September 7th - Signaling

Xun, Q., Bi, C., Cui, X., Wu, H., Wang, M., Liao, Y., Wang, R., Xie, H., Shen, Z. and Fang, M. (2018). MagT1 is essential for Drosophila development through the shaping of Wnt and Dpp signaling pathways. Biochem Biophys Res Commun. PubMed ID: 29959918
Magnesium transporter subtype 1 (MagT1) is a magnesium membrane transporter with channel like properties. MagT1 (CG7830) has been identified in Drosophila genome, and its protein product has been characterized by electrophysiological means. This study reports the generation of fly MagT1 mutants and shows that MagT1 is essential for early embryonic development. In wings and primordial wings, by clonal analysis and RNAi knock down of MagT1, it was found that loss of MagT1 results enhanced/ectopic Wingless (Wg, a fly Wnt) signaling and disrupted Decapentaplegic (Dpp) signaling, indicating the crucial role of MagT1 for fly development at later stages. Finally, it was demonstrated directly that magnesium transportations are proportional with the MagT1 expressional levels in Drosophila Kc167cells. Taken together, these findings may suggest that MagT1 is a major magnesium transporter/channel profoundly involved in fly development by affecting developmental signaling pathways, such as Wg and Dpp signaling.
Zhang, Z., Krauchunas, A. R., Huang, S. and Wolfner, M. F. (2018). Maternal proteins that are phosphoregulated upon egg activation include crucial factors for oogenesis, egg activation and embryogenesis in Drosophila melanogaster. G3 (Bethesda). PubMed ID: 30012668
Egg activation is essential for the successful transition from a mature oocyte to a developmentally competent egg. It consists of a series of events including the resumption and completion of meiosis, initiation of translation of some maternal mRNAs and destruction of others, and changes to the vitelline envelope. This study used germline-specific RNAi to examine the function of 189 maternal proteins that are phosphoregulated during egg activation in Drosophila melanogaster. 53 genes were identified whose knockdown reduced or abolished egg production and caused a range of defects in ovarian morphology, as well as 51 genes whose knockdown led to significant impairment or abolishment of the egg hatchability. Different stages of developmental arrest were observed in the embryos and various defects in spindle morphology and aberrant centrosome activities in the early arrested embryos. The results revealed 15 genes with newly discovered roles in egg activation and early embryogenesis in Drosophila. It is suggested that the phosphoregulated proteins may provide a rich pool of candidates for the identification of important players in the egg-to-embryo transition.
You, S., Li, H., Hu, Z. and Zhang, W. (2018). eIF2alpha kinases PERK and GCN2 act on FOXO to potentiate FOXO activity. Genes Cells. PubMed ID: 30043468
PERK and GCN2 are eIF2alpha kinases known to mediate the effects of ER stress and respond to an array of diverse stress stimuli. Previously, it has been reported that ER stress potentiates insulin resistance through PERK-mediated FOXO phosphorylation. Inhibition of PERK improves cellular insulin responsiveness at the level of FOXO activity. This study provides further evidence that FOXO is required for the functional output of PERK by showing that lowering FOXO activity ameliorates a PERK gain-of-function phenotype in Drosophila. More importantly, results are presented demonstrating that GCN2 acts similarly to PERK to promote FOXO activity. Regulation of FOXO by GCN2 is evolutionarily conserved and can be compensated for by PERK. The combination of these mechanisms may contribute to the complex regulatory network between PERK, GCN2, and FOXO, which has been implicated in the development and progression of a variety of diseases.
Zulbahar, S., Sieglitz, F., Kottmeier, R., Altenhein, B., Rumpf, S. and Klambt, C. (2018). Differential expression of the Drosophila Ntan/Obek controls ploidy in the blood-brain barrier. Development. PubMed ID: 30002129
During development tissue growth is mediated by either cell proliferation or cell growth, coupled with polyploidy. Both strategies are employed by the cell types comprising the Drosophila blood-brain barrier. During larval growth, the perineurial glia proliferate, whereas the subperineurial glia expand enormously and become polyploid. This study shows that the level of ploidy in subperineurial glia is controlled by the N-terminal asparagine amidohydrolase homolog Obek, where high Obek levels are required to limit replication. In contrast, perineurial glia express moderate levels of Obek, and increased expression blocks their proliferation. Interestingly, other dividing cells are not affected by alteration of Obek expression. In glia, Obek counteracts FGF- and Hippo-signaling to differentially affect cell growth and number. A mechanism is proposed where growth signals are integrated differentially in a glia-specific manner through different levels of Obek protein to adjust cell proliferation versus endoreplication in the blood-brain barrier.

Thursday, September 6th - RNA

Rahman, R., Xu, W., Jin, H. and Rosbash, M. (2018). Identification of RNA-binding protein targets with HyperTRIBE. Nat Protoc. PubMed ID: 30013039
RNA-binding proteins (RBPs) accompany RNA from birth to death, affecting RNA biogenesis and functions. Identifying RBP-RNA interactions is essential to understanding their complex roles in different cellular processes. However, detecting in vivo RNA targets of RBPs, especially in a small number of discrete cells, has been a technically challenging task. A novel technique called TRIBE (targets of RNA-binding proteins identified by editing) has been developed to overcome this problem. TRIBE expresses a fusion protein consisting of a queried RBP and the catalytic domain of the RNA-editing enzyme ADAR (adenosine deaminase acting on RNA) (ADARcd), which marks target RNA transcripts by converting adenosine to inosine near the RBP binding sites. These marks can be subsequently identified via high-throughput sequencing. In spite of its usefulness, TRIBE is constrained by a low editing efficiency and editing-sequence bias from the ADARcd. Therefore, HyperTRIBE was developed by incorporating a previously characterized hyperactive mutation, E488Q, into the ADARcd. This strategy increases the editing efficiency and reduces sequence bias, which markedly increases the sensitivity of this technique without sacrificing specificity. HyperTRIBE provides a more powerful strategy for identifying RNA targets of RBPs with an easy experimental and computational protocol at low cost, that can be performed not only in flies, but also in mammals. The HyperTRIBE experimental protocol can be carried out in cultured Drosophila S2 cells in 1 week, using tools available in a common molecular biology laboratory; the computational analysis requires 3 more days (Rahman, 2018).
Stegeman, R., Hall, H., Escobedo, S. E., Chang, H. C. and Weake, V. M. (2018). Proper splicing contributes to visual function in the aging Drosophila eye. Aging Cell: e12817. PubMed ID: 30003673
Changes in splicing patterns are a characteristic of the aging transcriptome; however, it is unclear whether these age-related changes in splicing facilitate the progressive functional decline that defines aging. In Drosophila, visual behavior declines with age and correlates with altered gene expression in photoreceptors, including downregulation of genes encoding splicing factors. This study characterized the significance of these age-regulated splicing-associated genes in both splicing and visual function. To do this, differential splicing events were identified in either the entire eye or photoreceptors of young and old flies. Intriguingly, aging photoreceptors show differential splicing of a large number of visual function genes. In addition, as shown previously for aging photoreceptors, aging eyes showed increased accumulation of circular RNAs, which result from noncanonical splicing events. To test whether proper splicing was necessary for visual behavior, age-regulated splicing factors were knocked down in photoreceptors in young flies and phototaxis was examined. Notably, many of the age-regulated splicing factors tested were necessary for proper visual behavior. In addition, knockdown of individual splicing factors resulted in changes in both alternative splicing at age-spliced genes and increased accumulation of circular RNAs. Together, these data suggest that cumulative decreases in splicing factor expression could contribute to the differential splicing, circular RNA accumulation, and defective visual behavior observed in aging photoreceptors.
Zhou, L., Lim, M. Y., Kaur, P., Saj, A., Bortolamiol-Becet, D., Gopal, V., Tolwinski, N., Tucker-Kellogg, G. and Okamura, K. (2018). Importance of miRNA stability and alternative primary miRNA isoforms in gene regulation during Drosophila development. Elife 7. PubMed ID: 30024380
Mature microRNAs (miRNAs) are processed from primary transcripts (pri-miRNAs), and their expression is controlled at transcriptional and post-transcriptional levels. However, how regulation at multiple levels achieves precise control remains elusive. Using published and new datasets, this study profiled a time course of mature and pri-miRNAs in Drosophila embryos and revealed the dynamics of miRNA production and degradation as well as dynamic changes in pri-miRNA isoform selection. 5' nucleotides influence stability of mature miRNAs. Furthermore, distinct half-lives of miRNAs from the mir-309 cluster shape their temporal expression patterns, and the importance of rapid degradation of the miRNAs in gene regulation is detected as distinct evolutionary signatures at the target sites in the transcriptome. Finally, rapid degradation of miR-3/-309 may be important for regulation of the planar cell polarity pathway component Vang. Altogether, the results suggest that complex mechanisms regulate miRNA expression to support normal development.
Osman, I. and Pek, J. W. (2018). A sisRNA/miRNA axis prevents loss of germline stem cells during starvation in Drosophila. Stem Cell Reports 11(1): 4-12. PubMed ID: 30008327
Animal reproduction responds to nutritional status. During starvation, Drosophila and Caenorhabditis elegans enter a period of reproductive diapause with increase apoptosis, while maintaining a stable pool of germline stem cells (GSCs). How GSCs are protected is not understood. This study shows that a sisRNA/miRNA axis maintains ovarian GSCs during starvation in Drosophila. Starvation induces the expression of an ovary-enriched sisRNA sisR-2, which negatively regulates GSC maintenance via a fatty acid metabolism gene dFAR1. sisR-2 promotes the expression of bantam, which in turn inhibits the activity of ssisR-2, forming a negative feedback loop. Therefore, bantam acts as a buffer to counteract sisR-2 activity to prevent GSC loss during starvation. It is proposed that the sisR-2/bantam axis confers robustness to GSCs in Drosophila (Osman, 2018).

Wednesday, September 5th - Cytoskeleton

Roper, J. C., Mitrossilis, D., Stirnemann, G., Waharte, F., Brito, I., Fernandez-Sanchez, M. E., Baaden, M., Salamero, J. and Farge, E. (2018). The major beta-catenin/E-cadherin junctional binding site is a primary molecular mechano-transductor of differentiation in vivo. Elife 7. PubMed ID: 30024850
In vivo, the primary molecular mechanotransductive events mechanically initiating cell differentiation remain unknown. This study finds the molecular stretching of the highly conserved Y654-beta-catenin-D665-E-cadherin binding site as mechanically induced by tissue strain. It triggers the increase of accessibility of the Y654 site, target of the Src42A kinase phosphorylation leading to irreversible unbinding. Molecular dynamics simulations of the beta-catenin/E-cadherin complex under a force mimicking a 6 pN physiological mechanical strain predict a local 45% stretching between the two alpha-helices linked by the site and a 15% increase in accessibility of the phosphorylation site. Both are quantitatively observed using FRET lifetime imaging and non-phospho Y654 specific antibody labelling, in response to the mechanical strains developed by endogenous and magnetically mimicked early mesoderm invagination of gastrulating Drosophila embryos. This is followed by the predicted release of 16% of beta-catenin from junctions, observed in FRAP, which initiates the mechanical activation of the beta-catenin pathway process.
Tovey, C. A., Tubman, C. E., Hamrud, E., Zhu, Z., Dyas, A. E., Butterfield, A. N., Fyfe, A., Johnson, E. and Conduit, P. T. (2018). gamma-TuRC heterogeneity revealed by analysis of Mozart1. Curr Biol 28(14): 2314-2323.e2316. PubMed ID: 29983314
Microtubules are essential for various cell processes and are nucleated by multi-protein gamma-tubulin ring complexes (gamma-TuRCs) at various microtubule organizing centers (MTOCs), including centrosomes. MOZART1 (Mzt1) was recently identified as a gamma-TuRC component and is conserved in nearly all eukaryotes. Mzt1 has so far been studied in cultured human cells, yeast, and plants; its absence leads to failures in gamma-TuRC recruitment and cell division, resulting in cell death. Mzt1 is small (approximately 8.5 kDa), binds directly to core gamma-TuRC components, and appears to mediate the interaction between gamma-TuRCs and proteins that tether gamma-TuRCs to MTOCs. This study used Drosophila to investigate the function of Mzt1 in a multicellular animal for the first time. Surprisingly, Drosophila Mzt1 was found to be expressed only in the testes and is present in gamma-TuRCs recruited to basal bodies, but not to mitochondria, in developing sperm cells. mzt1 mutants are viable but have defects in basal body positioning and gamma-TuRC recruitment to centriole adjuncts; sperm formation is affected and mutants display a rapid age-dependent decline in sperm motility and male fertility. These results reveal that tissue-specific and MTOC-specific gamma-TuRC heterogeneity exist in Drosophila and highlight the complexity of gamma-TuRC recruitment in a multicellular animal.
Sanchez-Corrales, Y. E., Blanchard, G. B. and Roper, K. (2018). Radially-patterned cell behaviours during tube budding from an epithelium. Elife 7. PubMed ID: 30015616
The budding of tubular organs from flat epithelial sheets is a vital morphogenetic process. Cell behaviours that drive such processes are only starting to be unraveled. Using live-imaging and novel morphometric methods this study shows that in addition to apical constriction, radially-oriented directional intercalation of cells plays a major contribution to early stages of invagination of the salivary gland tube in the Drosophila embryo. Extending analyses in 3D, it was found that near the pit of invagination, isotropic apical constriction leads to strong cell-wedging. Further from the pit cells interleave circumferentially, suggesting apically-driven behaviours. Supporting this, junctional myosin is enriched in, and neighbour exchanges are biased towards the circumferential orientation. In a mutant failing pit specification, neither are biased due to an inactive pit. Thus, tube budding involves radially-patterned pools of apical myosin, medial as well as junctional, and radially-patterned 3D-cell behaviours, with a close mechanical interplay between invagination and intercalation.
Vanderleest, T. E., Smits, C. M., Xie, Y., Jewett, C. E., Blankenship, J. T. and Loerke, D. (2018). Vertex sliding drives intercalation by radial coupling of adhesion and actomyosin networks during Drosophila germband extension. Elife 7. PubMed ID: 29985789
Oriented cell intercalation is an essential developmental process that shapes tissue morphologies through the directional insertion of cells between their neighbors. Previous research has focused on properties of cell-cell interfaces, while the function of tricellular vertices has remained unaddressed. This study identifies a highly novel mechanism in which vertices demonstrate independent sliding behaviors along cell peripheries to produce the topological deformations responsible for intercalation. Through systematic analysis, it was found that the motion of vertices connected by contracting interfaces is not physically coupled, but instead possess strong radial coupling. E-cadherin and Myosin II exist in previously unstudied populations at cell vertices and undergo oscillatory cycles of accumulation and dispersion that are coordinated with changes in cell area. Additionally, peak enrichment of vertex E-cadherin/Myosin II coincides with interface length stabilization. These results suggest a model in which asymmetric radial force balance directs the progressive, ratcheted motion of individual vertices to drive intercalation.

Tuesday, September 4th - Signaling

Praktiknjo, S. D., Saad, F., Maier, D., Ip, P. and Hipfner, D. R. (2018). Activation of Smoothened in the Hedgehog pathway unexpectedly increases Galphas-dependent cAMP levels in Drosophila. J Biol Chem. PubMed ID: 30018136
Hedgehog (Hh) signaling plays a key role in the development and maintenance of animal tissues. This signaling is mediated by the atypical G protein-coupled receptor (GPCR) Smoothened (Smo). Smo activation leads to signaling through several well-characterized effectors to activate Hh target gene expression. Recent studies have implicated activation of the heterotrimeric G protein subunit Galphai and the subsequent decrease in cellular 3',5'-cyclic adenosine monophosphate (cAMP) levels in promoting the Hh response in flies and mammals. Although Hh stimulation decreases cAMP levels in some insect cell lines, here using a bioluminescence resonance energy transfer (BRET)-based assay it was found that this stimulation had no detectable effect in Drosophila S2-R+ cells. However, an unexpected and significant Galphas-dependent increase in cAMP levels was observed in response to strong Smo activation in Smo-transfected cells. This effect was mediated by Smo's broadly conserved core, and was specifically activated in response to phosphorylation of the Smo C-terminus by GPCR kinase 2 (Gprk2). Genetic analysis of heterotrimeric G protein function in the developing Drosophila wing revealed a positive role for cAMP in the endogenous Hh response. Specifically, it was found that mutation or depletion of Galphas diminished low-threshold Hh responses in Drosophila, whereas depletion of Galphai potentiated them (in contrast to previous findings). This analysis suggested that regulated cAMP production is important for controlling the sensitivity of cellular responses to Hh in Drosophila.
Ramos-Lewis, W., LaFever, K. S. and Page-McCaw, A. (2018). A scar-like lesion is apparent in basement membrane after wound repair in vivo. Matrix Biol. PubMed ID: 29981372
Basement membrane is a highly conserved sheet-like extracellular matrix in animals, underlying simple and complex epithelia, and wrapping around tissues like muscles and nerves. Like the tissues they support, basement membranes become damaged by environmental insults. Although it is clear that basement membranes are repaired after damage, virtually nothing is known about this process. For example, it is not known how repaired basement membranes compare to undamaged ones, whether basement membrane components are necessary for epithelial wound closure, or whether there is a hierarchy of assembly that repairing basement membranes follow, similar to the hierarchy of assembly of embryonic basement membranes. This report addresses these questions using the basement membrane of the Drosophila larval epidermis as a model system. By analyzing the four main basement membrane proteins - laminin, collagen IV, perlecan, and nidogen - it was found that although basement membranes are repaired within a day after mechanical damage in vivo, thickened and disorganized matrix scars are evident with all four protein components. The new matrix proteins that repair damaged basement membranes are provided by distant adipose and muscle tissues rather than by the local epithelium, the same distant tissues that provide matrix proteins for growth of unwounded epithelial basement membranes. To identify a hierarchy of repair, the dependency of each of the basement membrane proteins on the others was tested for incorporation after damage. For proper incorporation after damage, nidogen requires laminin, and perlecan requires collagen IV, but surprisingly collagen IV does not to depend on laminin. Thus, the rules of basement membrane repair are subtly different than those of de novo assembly.
Richier, B., Inoue, Y., Dobramysl, U., Friedlander, J., Brown, N. H. and Gallop, J. L. (2018). Integrin signaling downregulates filopodia in muscle-tendon attachment. J Cell Sci. PubMed ID: 30054384
Cells need to sense their environment to ensure accurate targeting to specific destinations. This occurs in developing muscles, which need to attach to tendon cells before muscle contractions can begin. Elongating myotube tips form filopodia, which are presumed to have sensory roles, and are later suppressed upon building the attachment site. This study used live imaging and quantitative image analysis of lateral transverse (LT) myotubes in Drosophila to show that filopodia suppression occurs as a result of integrin signaling. Loss of the integrin subunits alphaPS2 and betaPS increased filopodia number and length at stages when they are normally suppressed. Conversely, inducing integrin signaling, achieved by expression of constitutively dimerised betaPS cytoplasmic domain (dibeta), prematurely suppressed filopodia. The integrin signal is transmitted through the protein Git (G-protein receptor coupled interacting protein) and its downstream kinase Pak (p21-activated kinase). Absence of these proteins causes profuse filopodia and prevents filopodial inhibition by dibeta. Thus, integrin signaling terminates the exploratory behaviour of myotubes seeking tendons, enabling the actin machinery to focus on forming a strong attachment and assembling the contractile apparatus.
Rust, K., Tiwari, M. D., Mishra, V. K., Grawe, F. and Wodarz, A. (2018). Myc and the Tip60 chromatin remodeling complex control neuroblast maintenance and polarity in Drosophila. Embo J. PubMed ID: 29997178
Stem cells establish cortical polarity and divide asymmetrically to simultaneously maintain themselves and generate differentiating offspring cells. Several chromatin modifiers have been identified as stemness factors in mammalian pluripotent stem cells, but whether these factors control stem cell polarity and asymmetric division has not been investigated so far. This question was addressed in Drosophila neural stem cells called neuroblasts. The Tip60 chromatin remodeling complex and its interaction partner Myc were identified as regulators of genes required for neuroblast maintenance. Knockdown of Tip60 complex members results in loss of cortical polarity, symmetric neuroblast division, and premature differentiation through nuclear entry of the transcription factor Prospero. aPKC is the key target gene of Myc and the Tip60 complex subunit Domino plays a roll regulating neuroblast polarity. Transcriptome analysis further showed that Domino regulates the expression of mitotic spindle genes previously identified as direct Myc targets. These findings reveal an evolutionarily conserved functional link between Myc, the Tip60 complex, and the molecular network controlling cell polarity and asymmetric cell division. Home page: The Interactive Fly© 1996-2015 Thomas B. Brody, Ph.D.

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