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Thursday, March 31st, 2022 - Disease models

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Kotian, N., Troike, K. M., Curran, K. N., Lathia, J. D. and McDonald, J. A. (2021). A Drosophila RNAi screen reveals conserved glioblastoma-related adhesion genes that regulate collective cell migration. G3 (Bethesda) 12(1). PubMed ID: 34849760
Summary:
Migrating cell collectives are key to embryonic development but also contribute to invasion and metastasis of a variety of cancers. Cell collectives can invade deep into tissues, leading to tumor progression and resistance to therapies. Collective cell invasion is also observed in the lethal brain tumor glioblastoma, which infiltrates the surrounding brain parenchyma leading to tumor growth and poor patient outcomes. Drosophila border cells, which migrate as a small cell cluster in the developing ovary, are a well-studied and genetically accessible model used to identify general mechanisms that control collective cell migration within native tissue environments. Most cell collectives remain cohesive through a variety of cell-cell adhesion proteins during their migration through tissues and organs. This study first identified cell adhesion, cell matrix, cell junction, and associated regulatory genes that are expressed in human brain tumors. RNAi knockdown of the Drosophila orthologs was performed in border cells to evaluate if migration and/or cohesion of the cluster was impaired. From this screen, eight adhesion-related genes were identified that disrupted border cell collective migration upon RNAi knockdown. Bioinformatics analyses further demonstrated that subsets of the orthologous genes were elevated in the margin and invasive edge of human glioblastoma patient tumors. These data together show that conserved cell adhesion and adhesion regulatory proteins with potential roles in tumor invasion also modulate collective cell migration. This dual screening approach for adhesion genes linked to glioblastoma and border cell migration thus may reveal conserved mechanisms that drive collective tumor cell invasion.
Parker-Character, J., Hager, D. R., Call, T. B., Pickup, Z. S., Turnbull, S. A., Marshman, E. M., Korch, S. B., Chaston, J. M. and Call, G. B. (2021). An altered microbiome in a Parkinson's disease model Drosophila melanogaster has a negative effect on development. Sci Rep 11(1): 23635. PubMed ID: 34880269
Summary:
Parkinson's disease (PD) is the second most common neurodegenerative disease, besides Alzheimer's Disease, characterized by multiple symptoms, including the well-known motor dysfunctions. It is well-established that there are differences in the fecal microbiota composition between Parkinson's disease (PD) patients and control populations, but the mechanisms underlying these differences are not yet fully understood. To begin to close the gap between description and mechanism, the relationship between the microbiota and PD was studied in a model organism, Drosophila melanogaster. First, fecal transfers were performed with a D. melanogaster model of PD that had a mutation in the parkin (park25) gene. Results indicate that the PD model feces had a negative effect on both pupation and eclosion in both control and park25 flies, with a greater effect in PD model flies. Analysis of the microbiota composition revealed differences between the control and park25 flies, consistent with many human studies. Conversely, gnotobiotic treatment of axenic embryos with feces-derived bacterial cultures did not affect eclosure. It is speculated that this result might be due to similarities in bacterial prevalence between mutant and control feces. Further, a bacteria-potentiated impact on mutant and control fly phenotypes was confirmed by measuring eclosure rate in park25 flies that were mono-associated with members of the fly microbiota. Both the fecal transfer and the mono-association results indicate a host genotype-microbiota interaction. Overall, this study concludes functional effects of the fly microbiota on PD model flies, providing support to the developing body of knowledge regarding the influence of the microbiota on PD.
Rouka, E., Gourgoulianni, N., Lüpold, S., Hatzoglou, C., Gourgoulianis, K. I. and Zarogiannis, S. G. (2022). Prediction and enrichment analyses of the Homo sapiens-Drosophila melanogaster COPD-related orthologs: potential for modeling of human COPD genomic responses with the fruit fly. Am J Physiol Regul Integr Comp Physiol 322(1): R77-r82. PubMed ID: 34877887
Summary:
The significant similarities in airway epithelial cells between mammals and the fruit fly Drosophila melanogaster have rendered the latter an important model organism for studies of chronic inflammatory lung diseases. Focusing on the chronic obstructive pulmonary disease (COPD), this study mapped human gene orthologs associated with this disease in D. melanogaster to identify functionally equivalent genes for immediate, further screening with the fruit fly model. The DIOPT-DIST tool was accessed for the prediction of the COPD-associated orthologs between humans and Drosophila. Enrichment analyses with respect to pathways of the retrieved functional homologs were performed using the ToppFun and FlyMine tools, identifying 73 unique human genes as well as 438 fruit fly genes. The ToppFun analysis verified that the human gene list is associated with COPD phenotypes. Furthermore, the FlyMine investigation highlighted that the Drosophila genes are functionally connected mainly with the "ABC-family proteins mediated transport" and the "β-catenin-independent WNT signaling pathway." These results suggest an evolutionarily conserved role toward responses to inhaled toxicants and CO(2) in both species. It is reasoned that the predicted orthologous genes should be further studied in the Drosophila models of cigarette smoke-induced COPD.
Coombs, G. S., Rios-Monterrosa, J. L., Lai, S., Dai, Q., Goll, A. C., Ketterer, M. R., Valdes, M. F., Uche, N., Benjamin, I. J. and Wallrath, L. L. (2021). Modulation of muscle redox and protein aggregation rescues lethality caused by mutant lamins. Redox Biol 48: 102196. PubMed ID: 34872044
Summary:
Mutations in the human LMNA gene cause a collection of diseases called laminopathies, which includes muscular dystrophy and dilated cardiomyopathy. The LMNA gene encodes lamins, filamentous proteins that form a meshwork on the inner side of the nuclear envelope. How mutant lamins cause muscle disease is not well understood, and treatment options are currently limited. To understand the pathological functions of mutant lamins so that therapies can be developed, new Drosophila models and human iPS cell-derived cardiomyocytes were generated. In the Drosophila models, muscle-specific expression of the mutant lamins caused nuclear envelope defects, cytoplasmic protein aggregation, activation of the Nrf2/Keap1 redox pathway, and reductive stress. These defects reduced larval motility and caused death at the pupal stage. Patient-derived cardiomyocytes expressing mutant lamins showed nuclear envelope deformations. The Drosophila models allowed for genetic and pharmacological manipulations at the organismal level. Genetic interventions to increase autophagy, decrease Nrf2/Keap1 signaling, or lower reducing equivalents partially suppressed the lethality caused by mutant lamins. Moreover, treatment of flies with pamoic acid, a compound that inhibits the NADPH-producing malic enzyme, partially suppressed lethality. Taken together, these studies have identified multiple new factors as potential therapeutic targets for LMNA-associated muscular dystrophy.
Maslov, D. L., Zemskaya, N. V., Trifonova, O. P., Lichtenberg, S., Balashova, E. E., Lisitsa, A. V., Moskalev, A. A. and Lokhov, P. G. (2021). Comparative Metabolomic Study of Drosophila Species with Different Lifespans. Int J Mol Sci 22(23). PubMed ID: 34884677
Summary:
The increase in life expectancy, leading to a rise in the proportion of older people, is accompanied by a prevalence of age-related disorders among the world population, the fight against which today is one of the leading biomedical challenges. Exploring the biological insights concerning the lifespan is one of the ways to provide a background for designing an effective treatment for the increase in healthy years of life. Untargeted direct injection mass spectrometry-based metabolite profiling of 12 species of Drosophila with significant variations in natural lifespans was conducted in this research. A cross-comparison study of metabolomic profiles revealed lifespan signatures of flies. These signatures indicate that lifespan extension is associated with the upregulation of amino acids, phospholipids, and carbohydrate metabolism. Such information provides a metabolome-level view on longevity and may provide a molecular measure of organism age in age-related studies.
Moulton, M. J., Barish, S., Ralhan, I., Chang, J., Goodman, L. D., Harland, J. G., Marcogliese, P. C., Johansson, J. O., Ioannou, M. S. and Bellen, H. J. (2021). Neuronal ROS-induced glial lipid droplet formation is altered by loss of Alzheimer's disease-associated genes. Proc Natl Acad Sci U S A 118(52). PubMed ID: 34949639
Summary:
A growing list of Alzheimer's disease (AD) genetic risk factors is being identified, but the contribution of each variant to disease mechanism remains largely unknown. Previous work has shown that elevated levels of reactive oxygen species (ROS) induces lipid synthesis in neurons leading to the sequestration of peroxidated lipids in glial lipid droplets (LD), delaying neurotoxicity. This neuron-to-glia lipid transport is APOD/E-dependent. To identify proteins that modulate these neuroprotective effects, the role was tested of AD risk genes in ROS-induced LD formation, and several genes were demonstrated to impact neuroprotective LD formation, including homologs of human ABCA1 (Drosophila ATP binding cassette subfamily A member 3), ABCA7, VLDLR, VPS26, VPS35, AP2A, PICALM, and CD2AP. These data also show that ROS enhances A&betal42 phenotypes in flies and mice. Finally, a peptide agonist of ABCA1 restores glial LD formation in a humanized APOE4 fly model, highlighting a potentially therapeutic avenue to prevent ROS-induced neurotoxicity. This study places many AD genetic risk factors in a ROS-induced neuron-to-glia lipid transfer pathway with a critical role in protecting against neurotoxicity.

Wednesday, March 30th - Larval and adult neural development and function

Prisco, L., Deimel, S. H., Yeliseyeva, H., Fiala, A. and Tavosanis, G. (2021). The anterior paired lateral neuron normalizes odour-evoked activity in the Drosophila mushroom body calyx. Elife 10. PubMed ID: 34964714
Summary:
To identify and memorize discrete but similar environmental inputs, the brain needs to distinguish between subtle differences of activity patterns in defined neuronal populations. The Kenyon cells (KCs) of the Drosophila adult mushroom body (MB) respond sparsely to complex olfactory input, a property that is thought to support stimuli discrimination in the MB. To understand how this property emerges, the role was explored of the inhibitory anterior paired lateral (APL) neuron in the input circuit of the MB, the calyx. Within the calyx, presynaptic boutons of projection neurons (PNs) form large synaptic microglomeruli (MGs) with dendrites of postsynaptic KCs. Combining electron microscopy (EM) data analysis and in vivo calcium imaging, it was shown that APL, via inhibitory and reciprocal synapses targeting both PN boutons and KC dendrites, normalizes odour-evoked representations in MGs of the calyx. APL response scales with the PN input strength and is regionalized around PN input distribution. These data indicate that the formation of a sparse code by the KCs requires APL-driven normalization of their MG postsynaptic responses. This work provides experimental insights on how inhibition shapes sensory information representation in a higher brain centre, thereby supporting stimuli discrimination and allowing for efficient associative memory formation.
Pineiro, M., Mena, W., Ewer, J. and Orio, P. (2021). Extracting temporal relationships between weakly coupled peptidergic and motoneuronal signaling: Application to Drosophila ecdysis behavior. PLoS Comput Biol 17(12): e1008933. PubMed ID: 34910730
Summary:
Neuromodulators, such as neuropeptides, can regulate and reconfigure neural circuits to alter their output, affecting in this way animal physiology and behavior. This study presents a quantitative framework to study the relationships between the temporal pattern of activity of peptidergic neurons and of motoneurons during Drosophila ecdysis behavior, a highly stereotyped motor sequence that is critical for insect growth. This study analyzed, in the time and frequency domains, simultaneous intracellular calcium recordings of peptidergic CCAP (crustacean cardioactive peptide) neurons and motoneurons obtained from isolated central nervous systems throughout fictive ecdysis behavior induced ex vivo by Ecdysis triggering hormone. The activity of both neuronal populations was found to be tightly coupled in a cross-frequency manner, suggesting that CCAP neurons modulate the frequency of motoneuron firing. To explore this idea further, a probabilistic logistic model was used to show that calcium dynamics in CCAP neurons can predict the oscillation of motoneurons, both in a simple model and in a conductance-based model capable of simulating many features of the observed neural dynamics. Finally, an algorithm was developed to quantify the motor behavior observed in videos of pupal ecdysis, and their features were compared to the patterns of neuronal calcium activity recorded ex vivo. The motor activity of the intact animal was found to be more regular than the motoneuronal activity recorded from ex vivo preparations during fictive ecdysis behavior; the analysis of the patterns of movement also allowed to identification of a new post-ecdysis phase.
Raisch, T., Brockmann, A., Ebbinghaus-Kintscher, U., Freigang, J., Gutbrod, O., Kubicek, J., Maertens, B., Hofnagel, O. and Raunser, S. (2021). Small molecule modulation of the Drosophila Slo channel elucidated by cryo-EM. Nat Commun 12(1): 7164. PubMed ID: 34887422
Summary:
Slowpoke (Slo) potassium channels display extraordinarily high conductance, are synergistically activated by a positive transmembrane potential and high intracellular Ca(2+) concentrations and are important targets for insecticides and antiparasitic drugs. However, it is unknown how these compounds modulate ion translocation and whether there are insect-specific binding pockets. This study reports structures of Drosophila Slo in the Ca(2+)-bound and Ca(2+)-free form and in complex with the fungal neurotoxin verruculogen and the anthelmintic drug emodepside. Whereas the architecture and gating mechanism of Slo channels are conserved, potential insect-specific binding pockets exist. Verruculogen inhibits K(+) transport by blocking the Ca(2+)-induced activation signal and precludes K(+) from entering the selectivity filter. Emodepside decreases the conductance by suboptimal K(+) coordination and uncouples ion gating from Ca(2+) and voltage sensing. These results expand the mechanistic understanding of Slo regulation and lay the foundation for the rational design of regulators of Slo and other voltage-gated ion channels.
Pannunzi, M. and Nowotny, T. (2021). Non-synaptic interactions between olfactory receptor neurons, a possible key feature of odor processing in flies. PLoS Comput Biol 17(12): e1009583. PubMed ID: 34898600
Summary:
When flies explore their environment, they encounter odors in complex, highly intermittent plumes. To navigate a plume and, for example, find food, they must solve several challenges, including reliably identifying mixtures of odorants and their intensities, and discriminating odorant mixtures emanating from a single source from odorants emitted from separate sources and just mixing in the air. Lateral inhibition in the antennal lobe is commonly understood to help solving these challenges. With a computational model of the Drosophila olfactory system, this study analyzed the utility of an alternative mechanism for solving them: Non-synaptic ('ephaptic') interactions (NSIs) between olfactory receptor neurons that are stereotypically co-housed in the same sensilla. NSIs were found to improve mixture ratio detection and plume structure sensing and do so more efficiently than the traditionally considered mechanism of lateral inhibition in the antennal lobe. The best performance is achieved when both mechanisms work in synergy. However, it was also found that NSIs decrease the dynamic range of co-housed ORNs, especially when they have similar sensitivity to an odorant. These results shed light, from a functional perspective, on the role of NSIs, which are normally avoided between neurons, for instance by myelination.
Li, Q., Jang, H., Lim, K. Y., Lessing, A. and Stavropoulos, N. (2021). insomniac links the development and function of a sleep-regulatory circuit. Elife 10. PubMed ID: 34908527
Summary:
Although many genes are known to influence sleep, when and how they impact sleep-regulatory circuits remain ill-defined. This study shows that insomniac (inc), a conserved adaptor for the autism-associated Cul3 ubiquitin ligase, acts in a restricted period of neuronal development to impact sleep in adult Drosophila. The loss of inc causes structural and functional alterations within the mushroom body (MB), a center for sensory integration, associative learning, and sleep regulation. In inc mutants, MB neurons are produced in excess, develop anatomical defects that impede circuit assembly, and are unable to promote sleep when activated in adulthood. These findings link neurogenesis and postmitotic development of sleep-regulatory neurons to their adult function and suggest that developmental perturbations of circuits that couple sensory inputs and sleep may underlie sleep dysfunction in neurodevelopmental disorders.
Musso, P. Y., Junca, P. and Gordon, M. D. (2021). A neural circuit linking two sugar sensors regulates satiety-dependent fructose drive in Drosophila. Sci Adv 7(49): eabj0186. PubMed ID: 34851668
Summary:
In flies, neuronal sensors detect prandial changes in circulating fructose levels and either sustain or terminate feeding, depending on internal state. This study describes a three-part neural circuit that imparts satiety-dependent modulation of fructose sensing. Dorsal fan-shaped body neurons display oscillatory calcium activity when hemolymph glucose is high, and these oscillations require glutamatergic input from SLP-AB or 'Janus' neurons projecting from the protocerebrum to the asymmetric body. Suppression of activity in this circuit, either by starvation or by genetic silencing, promotes specific drive for fructose ingestion. This is achieved through neuropeptidergic signaling by tachykinin, which is released from the fan-shaped body when glycemia is high. Tachykinin, in turn, signals to Gr43a-positive fructose sensors to modulate their response to fructose. Together, these results demonstrate how a three-layer neural circuit links the detection of two sugars to produce precise satiety-dependent control of feeding behavior.

Monday, March 28th, Larval and Adult Physiology and Metabolism

Oberhofer, G., Ivy, T. and Hay, B. A. (2021). Gene drive that results in addiction to a temperature-sensitive version of an essential gene triggers population collapse in Drosophila. Proc Natl Acad Sci U S A 118(49). PubMed ID: 34845012
Summary:
One strategy for population suppression seeks to use gene drive to spread genes that confer conditional lethality or sterility, providing a way of combining population modification with suppression. Stimuli of potential interest could be introduced by humans, such as an otherwise benign virus or chemical, or occur naturally on a seasonal basis, such as a change in temperature. Cleave and Rescue (ClvR) selfish genetic elements use Cas9 and guide RNAs (gRNAs) to disrupt endogenous versions of an essential gene while also including a Rescue version of the essential gene resistant to disruption. ClvR spreads by creating loss-of-function alleles of the essential gene that select against those lacking it, resulting in populations in which the Rescue provides the only source of essential gene function. As a consequence, if function of the Rescue, a kind of Trojan horse now omnipresent in a population, is condition dependent, so too will be the survival of that population. To test this idea, a ClvR was created in Drosophila in which Rescue activity of an essential gene, dribble, requires splicing of a temperature-sensitive intein (TS-ClvRdbe). This element spreads to transgene fixation at 23 °C, but when populations now dependent on Ts-ClvRdbe are shifted to 29 °C, death and sterility result in a rapid population crash. These results show that conditional population elimination can be achieved. A similar logic, in which Rescue activity is conditional, could also be used in homing-based drive and to bring about suppression and/or killing of specific individuals in response to other stimuli.
Majlish, A. N. K., Lye, S. H., Cytron, E., Bolus, H., Marotto, K. and Chtarbanova, S. (2021). The Drosophila K(+) -dependent Na(+) /Ca(2+) exchanger Nckx30C is implicated in temperature sensitive paralysis and age-dependent neurodegeneration. Alzheimers Dement 17 Suppl 12: e058564. PubMed ID: 34971107
Summary:
Increasing evidence links epileptic seizures to neurodegenerative disorders such as Alzheimer's disease (AD). Perturbed cellular Ca(2+) homeostasis is known to be implicated in AD pathogenesis. Previous studies in the model organism Drosophila have established a connection between mutations linked to temperature-sensitive (TS) paralysis, which is reminiscent of vertebrate epileptiform behavior, and neurodegeneration. An unbiased genetic screen was performed to identify mutants that exhibit paralytic behavior at 38°C. Gene mapping and DNA sequencing were used to locate the mutation in the Nckx30C gene. Mutant line 426 exhibited TS paralysis and progressive neurodegeneration in comparison to controls. The mutation was mapped to the Nckx30C locus, in the region encoding the predicted ion-binding domain. In comparison to wild type, 426 flies showed reduced lifespan, impaired climbing and changes in NMJ morphology, indicative of synaptic dysfunction. Lifespan reduction and early-onset neurodegeneration were found in flies carrying two other Nckx30C alleles. Neuron- but not glia-specific knockdown of Nckx30C recapitulates the TS-paralytic phenotype of 426 flies. Knockdown of Nckx30C in glia and neurons led to early onset climbing defects. It is concluded that the Drosophila Nckx30C gene encodes for a K(+) -dependent Na(+) /Ca(2+) exchanger with enriched expression in brain tissue. Nckx30C is homologous to mammalian Solute Carrier Family 24 (SLC24) proteins of which pathophysiological involvement and function in the brain is poorly understood. These studies of Nckx30C in flies could help to decipher the function of SLC24 proteins in both seizures and neurodegenerative conditions such as AD.
Pan, H. Y., Ye, Z. W., Zheng, Q. W., Yun, F., Tu, M. Z., Hong, W. G., Chen, B. X., Guo, L. Q. and Lin, J. F. (2022). Ergothioneine exhibits longevity-extension effect in Drosophila melanogaster via regulation of cholinergic neurotransmission, tyrosine metabolism, and fatty acid oxidation. Food Funct 13(1): 227-241. PubMed ID: 34877949
Summary:
Many studies have demonstrated the protective effect of ergothioneine (EGT), the unique sulfur-containing antioxidant found in mushrooms, on several aging-related diseases. Nevertheless, to date, no single study has explored the potential role of EGT in the lifespan of animal models. This study shows that EGT consistently extends fly lifespan in diverse genetic backgrounds and both sexes, as well as in a dose and gender-dependent manner. Additionally, EGT is shown to increases the climbing activity of flies, enhance acetylcholinesterase (AchE) activity, and maintain the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) of aged flies. The increase in lifespan by EGT is gut microorganism dependent. This study proposed potential mechanisms of lifespan extension in Drosophila by EGT through RNA-seq analysis: preservation of the normal status of the central nervous system via the coordination of cholinergic neurotransmission, tyrosine metabolism, and peroxisomal proteins, regulation of autophagic activity by altering the lysosomal protein CTSD, and the preservation of normal mitochondrial function through controlled substrate feeding into the tricarboxylic acid (TCA) cycle, the major energy-yielding metabolic process in cells.
Murakami, K., Palermo, J., Stanhope, B. A., Gibbs, A. G. and Keene, A. C. (2021). A screen for sleep and starvation resistance identifies a wake-promoting role for the auxiliary channel unc79. G3 (Bethesda) 11(8). PubMed ID: 34849820
Summary:
The regulation of sleep and metabolism are highly interconnected, and dysregulation of sleep is linked to metabolic diseases that include obesity, diabetes, and heart disease. Furthermore, both acute and long-term changes in diet potently impact sleep duration and quality. To identify novel factors that modulate interactions between sleep and metabolic state, a genetic screen was performed for their roles in regulating sleep duration, starvation resistance, and starvation-dependent modulation of sleep. This screen identified a number of genes with potential roles in regulating sleep, metabolism, or both processes. One such gene encodes the auxiliary ion channel UNC79, which was implicated in both the regulation of sleep and starvation resistance. Genetic knockdown or mutation of unc79 results in flies with increased sleep duration, as well as increased starvation resistance. Previous findings have shown that unc79 is required in pacemaker for 24-hours circadian rhythms. This study found that unc79 functions in the mushroom body, but not pacemaker neurons, to regulate sleep duration and starvation resistance. Together, these findings reveal spatially localized separable functions of unc79 in the regulation of circadian behavior, sleep, and metabolic function.
Potter, R., Meade, A., Potter, S. and Cooper, R. L. (2021). Rapid and Direct Action of Lipopolysaccharide (LPS) on Skeletal Muscle of Larval Drosophila. Biology (Basel) 10(12). PubMed ID: 34943150
Summary:
The endotoxin lipopolysaccharide (LPS) from Gram-negative bacteria exerts a direct and rapid effect on tissues. While most attention is given to the downstream actions of the immune system in response to LPS, this study focuses on the direct actions of LPS on skeletal muscle in Drosophila melanogaster. It was noted in earlier studies that the membrane potential rapidly hyperpolarizes in a dose-dependent manner with exposure to LPS from Pseudomonas aeruginosa and Serratia marcescens. The response is transitory while exposed to LPS, and the effect does not appear to be due to calcium-activated potassium channels, activated nitric oxide synthase (NOS), or the opening of Cl(-) channels. The purpose of this study was to further investigate the mechanism of the hyperpolarization of the larval Drosophila muscle due to exposure of LPS using several different experimental paradigms. It appears this response is unlikely related to activation of the Na-K pump or Ca(2+) influx. The unknown activation of a K(+) efflux could be responsible. This will be an important factor to consider in treatments of bacterial septicemia and cellular energy demands.
Li, Y., Wang, W. and Lim, H. Y. (2021). Drosophila Solute Carrier 5A5 Regulates Systemic Glucose Homeostasis by Mediating Glucose Absorption in the Midgut. Int J Mol Sci 22(22). PubMed ID: 34830305
Summary:
The small intestine is the initial site of glucose absorption and thus represents the first of a continuum of events that modulate normal systemic glucose homeostasis. A better understanding of the regulation of intestinal glucose transporters is therefore pertinent to efforts in curbing metabolic disorders. Using molecular genetic approaches, this study investigated the role of Drosophila Solute Carrier 5A5 (dSLC5A5) in regulating glucose homeostasis by mediating glucose uptake in the fly midgut. By genetically knocking down dSLC5A5 in flies, it was found that systemic and circulating glucose and trehalose levels are significantly decreased, which correlates with an attenuation in glucose uptake in the enterocytes. Reciprocally, overexpression of dSLC5A5 significantly increases systemic and circulating glucose and trehalose levels and promotes glucose uptake in the enterocytes. dSLC5A5 was shown to undergo apical endocytosis in a dynamin-dependent manner, which is essential for glucose uptake in the enterocytes. Furthermore, this study showed that the dSLC5A5 level in the midgut is upregulated by glucose and that dSLC5A5 critically directs systemic glucose homeostasis on a high-sugar diet. Together, these studies have uncovered the first Drosophila glucose transporter in the midgut and revealed new mechanisms that regulate glucose transporter levels and activity in the enterocyte apical membrane.

Friday, March 25th - Enhancers and transcriptional regulation

Li, L., Waymack, R., Gad, M. and Wunderlich, Z. (2021). Two promoters integrate multiple enhancer inputs to drive wild-type knirps expression in the Drosophila melanogaster embryo. Genetics 219(4). PubMed ID: 34849867
Summary:
Proper development depends on precise spatiotemporal gene expression patterns. Most developmental genes are regulated by multiple enhancers and often by multiple core promoters that generate similar transcripts. It was hypothesized that multiple promoters may be required either because enhancers prefer a specific promoter or because multiple promoters serve as a redundancy mechanism. To test these hypotheses, the expression of the knirps locus in the early Drosophila melanogaster embryo was studied; this expression is mediated by multiple enhancers and core promoters. One of these promoters resembles a typical "sharp" developmental promoter, while the other resembles a "broad" promoter usually associated with housekeeping genes. Using synthetic reporter constructs, this study found that some, but not all, enhancers in the locus show a preference for one promoter, indicating that promoters provide both redundancy and specificity. By analyzing the reporter dynamics, specific burst properties during the transcription process were identified, namely burst size and frequency, that are most strongly tuned by the combination of promoter and enhancer. Using locus-sized reporters, it was discovered that enhancers with no promoter preference in a synthetic setting have a preference in the locus context. These results suggest that the presence of multiple promoters in a locus is due both to enhancer preference and a need for redundancy and that "broad" promoters with dispersed transcription start sites are common among developmental genes. They also imply that it can be difficult to extrapolate expression measurements from synthetic reporters to the locus context, where other variables shape a gene's overall expression pattern.
Hildebrandt, K., Kolb, D., Kloppel, C., Kaspar, P., Wittling, F., Hartwig, O., Federspiel, J., Findji, I. and Walldorf, U. (2022). Regulatory modules mediating the complex neural expression patterns of the homeobrain gene during Drosophila brain development. Hereditas 159(1): 2. PubMed ID: 34983686
Summary:
The homeobox gene homeobrain (hbn) is located in the 57B region together with two other homeobox genes, Drosophila Retinal homeobox (DRx) and orthopedia (otp). All three genes encode transcription factors with important functions in brain development. Hbn mutants are embryonic lethal and characterized by a reduction in the anterior protocerebrum, including the mushroom bodies, and a loss of the supraoesophageal brain commissure. This study conducted detailed expression analysis of Hbn in later developmental stages. In the larval brain, Hbn is expressed in all type II lineages and the optic lobes, including the medulla and lobula plug. The gene is expressed in the cortex of the medulla and the lobula rim in the adult brain. A new hbnKOGal4 enhancer trap strain was generated by reintegrating Gal4 in the hbn locus through gene targeting, which reflects the complete hbn expression during development. Eight different enhancer-Gal4 strains covering 12 kb upstream of hbn, the two large introns and 5 kb downstream of the gene, were established and hbn expression was investigated. Several enhancers were characterized that drive expression in specific areas of the brain throughout development, from embryo to the adulthood. Finally, deletions of four of these enhancer regions were created through gene targeting, and their effects on the expression and function of hbn were analyzed. The complex expression of Hbn in the developing brain is regulated by several specific enhancers within the hbn locus. Each enhancer fragment drives hbn expression in several specific cell lineages, and with largely overlapping patterns, suggesting the presence of shadow enhancers and enhancer redundancy. Specific enhancer deletion strains generated by gene targeting display developmental defects in the brain. This analysis opens an avenue for a deeper analysis of hbn regulatory elements in the future.
Yokoshi, M., Kawasaki, K., Cambon, M. and Fukaya, T. (2022). Dynamic modulation of enhancer responsiveness by core promoter elements in living Drosophila embryos. Nucleic Acids Res 50(1): 92-107. PubMed ID: 34897508
Summary:
Regulatory interactions between enhancers and core promoters are fundamental for the temporal and spatial specificity of gene expression in development. The central role of core promoters is to initiate productive transcription in response to enhancer's activation cues. However, it has not been systematically assessed how individual core promoter elements affect the induction of transcriptional bursting by enhancers. This study provides evidence that each core promoter element differentially modulates functional parameters of transcriptional bursting in developing Drosophila embryos. Quantitative live imaging analysis revealed that the timing and the continuity of burst induction are common regulatory steps on which core promoter elements impact. It was further shown that the upstream TATA also affects the burst amplitude. On the other hand, Inr, MTE and DPE mainly contribute to the regulation of the burst frequency. Genome editing analysis of the pair-rule gene fushi tarazu revealed that the endogenous TATA and DPE are both essential for its correct expression and function during the establishment of body segments in early embryos. It is suggested that core promoter elements serve as a key regulatory module in converting enhancer activity into transcription dynamics during animal development.
Folkendt, L., Lohmann, I. and Domsch, K. (2021). An Evolutionary Perspective on Hox Binding Site Preferences in Two Different Tissues. J Dev Biol 9(4). PubMed ID: 34940504
Summary:
Transcription factor (TF) networks define the precise development of multicellular organisms. While many studies focused on TFs expressed in specific cell types to elucidate their contribution to cell specification and differentiation, it is less understood how broadly expressed TFs perform their precise functions in the different cellular contexts. To uncover differences that could explain tissue-specific functions of such TFs, this study analyzed genomic chromatin interactions of the broadly expressed Drosophila Hox TF Ultrabithorax (Ubx) in the mesodermal and neuronal tissues using bioinformatics. This investigations showed that Ubx preferentially interacts with multiple yet tissue-specific chromatin sites in putative regulatory regions of genes in both tissues. Importantly, the classical Hox/Ubx DNA binding motif was found to be enriched only among the neuronal Ubx chromatin interactions, whereas a novel Ubx-like motif with rather low predicted Hox affinities was identified among the regions bound by Ubx in the mesoderm. Finally, this analysis revealed that tissues-specific Ubx chromatin sites are also different with regards to the distribution of active and repressive histone marks. Based on these data, it is proposed that the tissue-related differences in Ubx binding behavior could be a result of the emergence of the mesoderm as a new germ layer in triploblastic animals, which might have required the Hox TFs to relax their binding specificity.
Waters, C. T., Gisselbrecht, S. S., Sytnikova, Y. A., Cafarelli, T. M., Hill, D. E. and Bulyk, M. L. (2021). Quantitative-enhancer-FACS-seq (QeFS) reveals epistatic interactions among motifs within transcriptional enhancers in developing Drosophila tissue. Genome Biol 22(1): 348. PubMed ID: 34930411
Summary:
Understanding the contributions of transcription factor DNA binding sites to transcriptional enhancers is a significant challenge. Quantitative enhancer-FACS-Seq was developed for highly parallel quantification of enhancer activities from a genomically integrated reporter in Drosophila melanogaster embryos. The contributions were investigated of the DNA binding motifs of four poorly characterized TFs to the activities of twelve embryonic mesodermal enhancers. Quantitative changes were measured in enhancer activity, and a range was discovered of epistatic interactions among the motifs, both synergistic and alleviating. Understanding the regulatory consequences of TF binding motifs requires that they be investigated in combination across enhancer contexts.
Naville, M. and Merabet, S. (2021). In-Depth Annotation of the Drosophila Bithorax-Complex Reveals the Presence of Several Alternative ORFs That Could Encode for Motif-Rich Peptides. Cells 10(11). PubMed ID: 34831206
Summary:
It is recognized that a large proportion of eukaryotic RNAs and proteins is not produced from conventional genes but from short and alternative (alt) open reading frames (ORFs). This study presents an in silico prediction of altORFs by applying several selecting filters based on evolutionary conservation and annotations of previously characterized altORF peptides. This work was performed in the Bithorax-complex (BX-C). Several altORFs could be predicted from coding and non-coding sequences of BX-C. In addition, the selected altORFs encode for proteins that contain several interesting molecular features, such as the presence of transmembrane helices or a general propensity to be rich in short interaction motifs. Of particular interest, one altORF encodes for a protein that contains a peptide sequence found in specific isoforms of two Drosophila Hox proteins. This work thus suggests that several altORF proteins could be produced from a particular genomic region known for its critical role during Drosophila embryonic development. The molecular signatures of these altORF proteins further suggests that several of them could make numerous protein-protein interactions and be of functional importance in vivo.

Thursday, March 24th - Evolution

Kinsey, S. D., Vinluan, J. P., Shipman, G. A. and Verheyen, E. M. (2021). Expression of human HIPKs in Drosophila demonstrates their shared and unique functions in a developmental model. G3 (Bethesda) 11(12). PubMed ID: 34849772
Summary:
Homeodomain-interacting protein kinases (HIPKs) are a family of four conserved proteins essential for vertebrate development, as demonstrated by defects in the eye, brain, and skeleton that culminate in embryonic lethality when multiple HIPKs are lost in mice. While HIPKs are essential for development, functional redundancy between the four vertebrate HIPK paralogues has made it difficult to compare their respective functions. Because understanding the unique and shared functions of these essential proteins could directly benefit the fields of biology and medicine, this study addressed the gap in knowledge of the four vertebrate HIPK paralogues by studying them in the fruit fly Drosophila melanogaster, where reduced genetic redundancy simplifies functional assessment. The single hipk present in the fly allowed the performance of rescue experiments with human HIPK genes that provide new insight into their individual functions not easily assessed in vertebrate models. Furthermore, the abundance of genetic tools and established methods for monitoring specific developmental pathways and gross morphological changes in the fly allowed for functional comparisons in endogenous contexts. Rescue experiments were performed to demonstrate the extent to which each of the human HIPKs can functionally replace Drosophila Hipk for survival and morphological development. The ability of each human HIPK to modulate Armadillo/β-catenin levels, JAK/STAT activity, proliferation, growth, and death, each of which have previously been described for Hipks, but never all together in comparable tissue contexts. Finally, novel developmental phenotypes induced by human HIPKs were characterized to gain insight to their unique functions. Together, these experiments provide the first direct comparison of all four vertebrate HIPKs to determine their roles in a developmental context.
Kotwica-Rolinska, J., Chodakova, L., Smykal, V., Damulewicz, M., Provazník, J., Wu, B. C., Hejnikova, M., Chvalova, D. and Dolezel, D. (2022). Loss of Timeless Underlies an Evolutionary Transition within the Circadian Clock. Mol Biol Evol 39(1). PubMed ID: 34893879
Summary:
Most organisms possess time-keeping devices called circadian clocks. At the molecular level, circadian clocks consist of transcription-translation feedback loops (TTFLs). Although some components of the negative TTFL are conserved across the animals, important differences exist between typical models, such as mouse and the fruit fly. In Drosophila, the key components are PERIOD (PER) and TIMELESS (TIM-d) proteins, whereas the mammalian clock relies on PER and CRYPTOCHROME (CRY-m). Importantly, how the clock has maintained functionality during evolutionary transitions between different states remains elusive. Therefore, this studdy systematically described the circadian clock gene setup in major bilaterian lineages and identified marked lineage-specific differences in their clock constitution. Then a thorough functional analysis was performed of the linden bug Pyrrhocoris apterus, an insect species comprising features characteristic of both the Drosophila and the mammalian clocks. Unexpectedly, the knockout of timeless-d, a gene essential for the clock ticking in Drosophila, did not compromise rhythmicity in P. apterus, it only accelerated its pace. Furthermore, silencing timeless-m, the ancestral timeless type ubiquitously present across animals, resulted in a mild gradual loss of rhythmicity, supporting its possible participation in the linden bug clock, which is consistent with timeless-m role suggested by research on mammalian models. The dispensability of timeless-d in P. apterus allows drawing a scenario in which the clock has remained functional at each step of transition from an ancestral state to the TIM-d-independent PER + CRY-m system operating in extant vertebrates, including humans.
Hotzy, C., Fowler, E., Kiehl, B., Francis, R., Mason, J., Moxon, S., Rostant, W., Chapman, T. and Immler, S. (2021). Evolutionary history of sexual selection affects microRNA profiles in Drosophila sperm. Evolution. PubMed ID: 34874067
Summary:
The presence of small RNAs in sperm is a relatively recent discovery and little is currently known about their importance and functions. Environmental changes including social conditions and dietary manipulations are known to affect the composition and expression of some small RNAs in sperm and may elicit a physiological stress response resulting in an associated change in gamete miRNA profiles. This study tested how microRNA profiles in sperm are affected by variation in both sexual selection and dietary regimes in Drosophila melanogaster selection lines. The selection lines were exposed to standard versus low yeast diet treatments and three different population sex ratios (male-biased, female-biased, or equal sex) in a full-factorial design. After 38 generations of selection, all males were maintained on their selected diet and in a common garden male-only environment prior to sperm sampling. Transcriptome analyses were performed on miRNAs in purified sperm samples. 11 differentially expressed miRNAs were found with the majority showing differences between male- and female-biased lines. Dietary treatment only had a significant effect on miRNA expression levels in interaction with sex ratio. These findings suggest that long-term adaptation may affect miRNA profiles in sperm and that these may show varied interactions with short-term environmental changes.
Lange, J. D., Bastide, H., Lack, J. B. and Pool, J. E. (2021). A Population Genomic Assessment of Three Decades of Evolution in a Natural Drosophila Population. Mol Biol Evol. PubMed ID: 34971382
Summary:
Population genetics seeks to illuminate the forces shaping genetic variation, often based on a single snapshot of genomic variation. However, utilizing multiple sampling times to study changes in allele frequencies can help clarify the relative roles of neutral and non-neutral forces on short time scales. This study compares whole-genome sequence variation of recently collected natural population samples of Drosophila melanogaster against a collection made approximately 35 years prior from the same locality-encompassing roughly 500 generations of evolution. The allele frequency changes between these time points would suggest a relatively small local effective population size on the order of 10,000, significantly smaller than the global effective population size of the species. Some loci display stronger allele frequency changes than would be expected anywhere in the genome under neutrality-most notably the tandem paralogs Cyp6a17 and Cyp6a23, which are impacted by structural variation associated with resistance to pyrethroid insecticides. A genome-wide excess of outliers was found for high genetic differentiation between old and new samples, but a larger number of adaptation targets may have affected SNP-level differentiation versus window differentiation. Evidence was found for strengthening latitudinal allele frequency clines: northern-associated alleles have increased in frequency by an average of nearly 2.5% at SNPs previously identified as clinal outliers, but no such pattern is observed at random SNPs. This project underscores the scientific potential of using multiple sampling time points to investigate how evolution operates in natural populations, by quantifying how genetic variation has changed over ecologically relevant timescales.
Matsunaga, T., Reisenman, C. E., Goldman-Huertas, B., Brand, P., Miao, K., Suzuki, H. C., Verster, K. I., Ramírez, S. R. and Whiteman, N. K. (2021). Evolution of olfactory receptors tuned to mustard oils in herbivorous Drosophilidae. Mol Biol Evol. PubMed ID: 34963012
Summary:
The diversity of herbivorous insects is attributed to their propensity to specialize on toxic plants. In an evolutionary twist, toxins betray the identity of their bearers when herbivores co-opt them as cues for host-plant finding, but the evolutionary mechanisms underlying this phenomenon are poorly understood. This study focused on Scaptomyza flava, an herbivorous drosophilid specialized on isothiocyanate (ITC)-producing (Brassicacales) plants, and identified Or67b paralogs that were triplicated as mustard-specific herbivory evolved. Using in vivo heterologous systems for the expression of olfactory receptors, it was found that S. flava Or67bs, but not the homologs from microbe-feeding relatives, responded selectively to ITCs, each paralog detected different ITC subsets. Consistent with this, S. flava was attracted to ITCs, as was Drosophila melanogaster expressing S. flava Or67b3 in the homologous Or67b olfactory circuit. ITCs were likely co-opted as olfactory attractants through gene duplication and functional specialization (neofunctionalization and subfunctionalization) in S. flava, a recently-derived herbivore.
Mirsalehi, A., Markova, D. N., Eslamieh, M. and Betran, E. (2021). Nuclear transport genes recurrently duplicate by means of RNA intermediates in Drosophila but not in other insects. BMC Genomics 22(1): 876. PubMed ID: 34863092
Summary:
The nuclear transport machinery is involved in a well-known male meiotic drive system in Drosophila. Fast gene evolution and gene duplications have been major underlying mechanisms in the evolution of meiotic drive systems, and this might include some nuclear transport genes in Drosophila. So, using a comprehensive, detailed phylogenomic study, this study examined 51 insect genomes for the duplication of the same nuclear transport genes. Most of the nuclear transport duplications in Drosophila are of a few classes of nuclear transport genes, RNA mediated and fast evolving. Many pseudogenes for the Ran gene were obtained. Some of the duplicates are relatively young and likely contributing to the turnover expected for genes under strong but changing selective pressures. These duplications are potentially revealing what features of nuclear transport are under selection. Unlike in flies, only a few duplications were found when the Drosophila duplicated nuclear transport genes were studied in dipteran species outside of Drosophila, and none in other insects. These findings strengthen the hypothesis that nuclear transport gene duplicates in Drosophila evolve either as drivers or suppressors of meiotic drive systems or as other male-specific adaptations circumscribed to flies and involving a handful of nuclear transport functions.

Wednesday, March 23rd - Gonadogenesis

Ibaraki, K., Nakatsuka, M., Ohsako, T., Watanabe, M., Miyazaki, Y., Shirakami, M., Karr, T. L., Sanuki, R., Tomaru, M. and Takano-Shimizu-Kouno, T. (2021). A cross-species approach for the identification of Drosophila male sterility genes. G3 (Bethesda) 11(8). PubMed ID: 34849808
Summary:
Male reproduction encompasses many essential cellular processes and interactions. As a focal point for these events, sperm offer opportunities for advancing understanding of sexual reproduction at multiple levels during development. Using male sterility genes identified in human, mouse, and fruit fly databases as a starting point, 103 Drosophila melanogaster genes were screened for their association with male sterility by tissue-specific RNAi knockdown and CRISPR/Cas9-mediated mutagenesis. This list included 56 genes associated with male infertility in the human databases, but not found in the Drosophila database, resulting in the discovery of 63 new genes associated with male fertility in Drosophila. The phenotypes identified were categorized into six distinct classes affecting sperm development. Interestingly, the second largest class (Class VI) caused sterility despite apparently normal testis and sperm morphology suggesting that these proteins may have functions in the mature sperm following spermatogenesis. This study focused on one such gene, Rack 1, and found that it plays an important role in two developmental periods, in early germline cells or germline stem cells and in spermatogenic cells or sperm. Taken together, many genes are yet to be identified and their role in male reproduction, especially after ejaculation, remains to be elucidated in Drosophila, where a wealth of data from human and other model organisms would be useful.
Bauerly, E., Akiyama, T., Staber, C., Yi, K. and Gibson, M. C. (2022). Impact of cilia-related genes on mitochondrial dynamics during Drosophila spermatogenesis. Dev Biol 482: 17-27. PubMed ID: 34822845
Summary:
Spermatogenesis is a dynamic process of cellular differentiation that generates the mature spermatozoa required for reproduction. Errors that arise during this process can lead to sterility due to low sperm counts and malformed or immotile sperm. While it is estimated that 1 out of 7 human couples encounter infertility, the underlying cause of male infertility can only be identified in 50% of cases. This study described and examined the genetic requirements for missing minor mitochondria (mmm), sterile affecting ciliogenesis (sac), and testes of unusual size (tous), three previously uncharacterized genes in Drosophila that are predicted to be components of the flagellar axoneme. Using Drosophila, it was demonstrated that these genes are essential for male fertility and that loss of mmm, sac, or tous results in complete immotility of the sperm flagellum. Cytological examination uncovered additional roles for sac and tous during cytokinesis and transmission electron microscopy of developing spermatids in mmm, sac, and tous mutant animals revealed defects associated with mitochondria and the accessory microtubules, required for the proper elongation of the mitochondria and flagella during ciliogenesis. This study highlights the complex interactions of cilia-related proteins within the cell body and advances understanding of male infertility by uncovering novel mitochondrial defects during spermatogenesis.
Kim, J., Hyun, M., Hibi, M. and You, Y. J. (2021). Maintenance of quiescent oocytes by noradrenergic signals. Nat Commun 12(1): 6925. PubMed ID: 34836956
Summary:
All females adopt an evolutionary conserved reproduction strategy; under unfavorable conditions such as scarcity of food or mates, oocytes remain quiescent. However, the signals to maintain oocyte quiescence are largely unknown. This paper reports that in four different species - Caenorhabditis elegans, Caenorhabditis remanei, Drosophila melanogaster, and Danio rerio - octopamine and norepinephrine play an essential role in maintaining oocyte quiescence. In the absence of mates, the oocytes of Caenorhabditis mutants lacking octopamine signaling fail to remain quiescent, but continue to divide and become polyploid. Upon starvation, the egg chambers of D. melanogaster mutants lacking octopamine signaling fail to remain at the previtellogenic stage, but grow to full-grown egg chambers. Upon starvation, D. rerio lacking norepinephrine fails to maintain a quiescent primordial follicle and activates an excessive number of primordial follicles. This study reveals an evolutionarily conserved function of the noradrenergic signal in maintaining quiescent oocytes.
Bordet, G., Kotova, E. and Tulin, A. V. (2022). Poly(ADP-ribosyl)ating pathway regulates development from stem cell niche to longevity control. Life Sci Alliance 5(3). PubMed ID: 34949666
Summary:
The regulation of poly(ADP-ribose) polymerase, the enzyme responsible for the synthesis of homopolymer ADP-ribose chains on nuclear proteins, has been extensively studied over the last decades for its involvement in tumorigenesis processes. However, the regulation of poly(ADP-ribose) glycohydrolase (PARG), the enzyme responsible for removing this posttranslational modification, has attracted little attention. This study identified that PARG activity is partly regulated by two phosphorylation sites, ph1 and ph2, in Drosophila. The disruption of these sites affects the germline stem-cells maintenance/differentiation balance as well as embryonic and larval development, but also the synchronization of egg production with the availability of a calorically sufficient food source. Moreover, these PARG phosphorylation sites play an essential role in the control of fly survivability from larvae to adults. PARG was also shown to be phosphorylated by casein kinase 2, and this phosphorylation seems to protect PARG protein against degradation in vivo. Taken together, these results suggest that the regulation of PARG protein activity plays a crucial role in the control of several developmental processes.
Colonnetta, M. M., Goyal, Y., Johnson, H. E., Syal, S., Schedl, P. and Deshpande, G. (2022). Preformation and epigenesis converge to specify primordial germ cell fate in the early Drosophila embryo. PLoS Genet 18(1): e1010002. PubMed ID: 34986144
Summary:
A critical step in animal development is the specification of primordial germ cells (PGCs), the precursors of the germline. Two seemingly mutually exclusive mechanisms are implemented across the animal kingdom: epigenesis and preformation. In epigenesis, PGC specification is non-autonomous and depends on extrinsic signaling pathways. The BMP pathway provides the key PGC specification signals in mammals. Preformation is autonomous and mediated by determinants localized within PGCs. In Drosophila, a classic example of preformation, constituents of the germ plasm localized at the embryonic posterior are thought to be both necessary and sufficient for proper determination of PGCs. Contrary to this longstanding model, this study shows that these localized determinants are insufficient by themselves to direct PGC specification in blastoderm stage embryos. Instead, it was found that the BMP signaling pathway is required at multiple steps during the specification process and functions in conjunction with components of the germ plasm to orchestrate PGC fate.
McCarthy, A., Sarkar, K., Martin, E. T., Upadhyay, M., Jang, S., Williams, N. D., Forni, P. E., Buszczak, M. and Rangan, P. (2022). Msl3 promotes germline stem cell differentiation in female Drosophila. Development 149(1). PubMed ID: 34878097
Summary:
Gamete formation from germline stem cells (GSCs) is essential for sexual reproduction. However, the regulation of GSC differentiation is incompletely understood. Set2, which deposits H3K36me3 modifications, is required for GSC differentiation during Drosophila oogenesis. The H3K36me3 reader Male-specific lethal 3 (Msl3) and histone acetyltransferase complex Ada2a-containing (ATAC) cooperate with Set2 to regulate GSC differentiation in female Drosophila. Msl3, acting independently of the rest of the male-specific lethal complex, promotes transcription of genes, including a germline-enriched ribosomal protein S19 paralog RpS19b. RpS19b upregulation is required for translation of RNA-binding Fox protein 1 (Rbfox1), a known meiotic cell cycle entry factor. Thus, Msl3 regulates GSC differentiation by modulating translation of a key factor that promotes transition to an oocyte fate.

Tuesday, March 22nd - Synapse and Vesicles

Maldonado-Díaz, C., Vazquez, M. and Marie, B. (2021). A comparison of three different methods of eliciting rapid activity-dependent synaptic plasticity at the Drosophila NMJ. PLoS One 16(11): e0260553. PubMed ID: 34847197
Summary:
The Drosophila NMJ is a system of choice for investigating the mechanisms underlying the structural and functional modifications evoked during activity-dependent synaptic plasticity. Because fly genetics allows considerable versatility, many strategies can be employed to elicit this activity. This study compared three different stimulation methods for eliciting activity-dependent changes in structure and function at the Drosophila NMJ. It was found that the method using patterned stimulations driven by a K+-rich solution creates robust structural modifications but reduces muscle viability, as assessed by resting potential and membrane resistance. It is argued that, using this method, electrophysiological studies that consider the frequency of events, rather than their amplitude, are the only reliable studies. These results were contrasted with the expression of CsChrimson channels and red-light stimulation at the NMJ, as well as with the expression of TRPA channels and temperature stimulation. With both these methods reliable modifications were observed of synaptic structures, along with consistent changes in electrophysiological properties. Indeed, a rapid appearance was observed of immature boutons that lack postsynaptic differentiation, and a potentiation of spontaneous neurotransmission frequency. Surprisingly, a patterned application of temperature changes alone is sufficient to provoke both structural and functional plasticity. In this context, temperature-dependent TRPA channel activation induces additional structural plasticity but no further increase in the frequency of spontaneous neurotransmission, suggesting an uncoupling of these mechanisms.
Hurbain, I., Mace, A. S., Romao, M., Prince, E., Sengmanivong, L., Ruel, L., Basto, R., Therond, P. P., Raposo, G. and D'Angelo, G. (2022). Microvilli-derived extracellular vesicles carry Hedgehog morphogenic signals for Drosophila wing imaginal disc development. Curr Biol 32(2): 361-373.e366. PubMed ID: 34890558
Summary:
Morphogens are secreted molecules that regulate and coordinate major developmental processes, such as cell differentiation and tissue morphogenesis. Depending on the mechanisms of secretion and the nature of their carriers, morphogens act at short and long range. This study investigated the paradigmatic long-range activity of Hedgehog (Hh), a well-known morphogen, and its contribution to the growth and patterning of the Drosophila wing imaginal disc. Extracellular vesicles (EVs) contribute to Hh long-range activity; however, the nature, the site, and the mechanisms underlying the biogenesis of these vesicular carriers remain unknown. Through the analysis of mutants and a series of Drosophila RNAi-depleted wing imaginal discs using fluorescence and live-imaging electron microscopy, including tomography and 3D reconstruction, this study demonstrated that microvilli of the wing imaginal disc epithelium are the site of generation of small EVs that transport Hh across the tissue. Further, it was shown that the Prominin-like (PromL) protein is critical for microvilli integrity. Together with actin cytoskeleton and membrane phospholipids, PromL maintains microvilli architecture that is essential to promote its secretory function. Importantly, the distribution of Hh to microvilli and its release via these EVs contribute to the proper morphogenesis of the wing imaginal disc. These results demonstrate that microvilli-derived EVs are carriers for Hh long-range signaling in vivo. By establishing that members of the Prominin protein family are key determinants of microvilli formation and integrity, these findings support the view that microvilli-derived EVs conveying Hh may provide a means for exchanging signaling cues of high significance in tissue development and cancer.
Hernandez-Diaz, S., Ghimire, S., Sanchez-Mirasierra, I., Montecinos-Oliva, C., Swerts, J., Kuenen, S., Verstreken, P. and Soukup, S. F. (2022). Endophilin-B regulates autophagy during synapse development and neurodegeneration. Neurobiol Dis 163: 105595. PubMed ID: 34933093
Summary:
Synapses are critical for neuronal communication and brain function. To maintain neuronal homeostasis, synapses rely on autophagy. Autophagic alterations cause neurodegeneration and synaptic dysfunction is a feature in neurodegenerative diseases. In Parkinson's disease (PD), where the loss of synapses precedes dopaminergic neuron loss, various PD-causative proteins are involved in the regulation of autophagy. So far only a few factors regulating autophagy at the synapse have been identified and the molecular mechanisms underlying autophagy at the synapse is only partially understood. this study describes Endophilin-B (EndoB) as a novel player in the regulation of synaptic autophagy in health and disease. EndoB is required for autophagosome biogenesis at the synapse, whereas the loss of EndoB blocks the autophagy induction promoted by the PD mutation LRRK2(G2019S). EndoB is required to prevent neuronal loss. Moreover, loss of EndoB in the Drosophila visual system leads to an increase in synaptic contacts between photoreceptor terminals and their post-synaptic synapses. These data confirm the role of autophagy in synaptic contact formation and neuronal survival.
Hayne, M. and DiAntonio, A. (2022). Protein phosphatase 2A restrains DLK signaling to promote proper Drosophila synaptic development and mammalian cortical neuron survival. Neurobiol Dis 163: 105586. PubMed ID: 34923110
Summary:
Protein phosphatase 2A (PP2A) is a major cellular phosphatase with many protein substrates. In post-mitotic neurons, the microtubule associated protein Tau is a particularly well-studied PP2A substrate as hyperphosphorylation of Tau is a hallmark of Alzheimer's disease. This study finds that activation of a single pathway can explain important aspects of the PP2A loss-of-function phenotype in neurons. PP2A inhibits activation of the neuronal stress kinase DLK and its Drosophila ortholog Wallenda. In the fly, PP2A inhibition activates a DLK/Wallenda-regulated transcriptional program that induces synaptic terminal overgrowth at the neuromuscular junction. In cultured mammalian neurons, PP2A inhibition activates a DLK-dependent apoptotic program that induces cell death. Contrary to expectations, in the absence of Tau PP2A inhibition still activates DLK and induces neuronal cell death, demonstrating that hyperphosphorylated Tau is not required for cell death in this model. Moreover, hyperphosphorylation of Tau following PP2A inhibition does not require DLK. Hence, loss of PP2A function in cortical neurons triggers two independent neuropathologies: 1) Tau hyperphosphorylation and 2) DLK activation and subsequent neuronal cell death. These findings demonstrate that inhibition of the DLK pathway is an essential function of PP2A required for normal Drosophila synaptic terminal development and mammalian cortical neuron survival.
Liu, T. X., Davoudian, P. A., Lizbinski, K. M. and Jeanne, J. M. (2021). Connectomic features underlying diverse synaptic connection strengths and subcellular computation. Curr Biol. PubMed ID: 34914905
Summary:
Connectomes generated from electron microscopy images of neural tissue unveil the complex morphology of every neuron and the locations of every synapse interconnecting them. These wiring diagrams may also enable inference of synaptic and neuronal biophysics, such as the functional weights of synaptic connections, but this requires integration with physiological data to properly parameterize. Working with a stereotyped olfactory network in the Drosophila brain, direct comparisons were made of the anatomy and physiology of diverse neurons and synapses with subcellular and subthreshold resolution. Synapse density and location jointly predict the amplitude of the somatic postsynaptic potential evoked by a single presynaptic spike. Biophysical models fit to data predict that electrical compartmentalization allows axon and dendrite arbors to balance independent and interacting computations. These findings begin to fill the gap between connectivity maps and activity maps, which should enable new hypotheses about how network structure constrains network function.
Kiral, F. R., Dutta, S. B., Linneweber, G. A., Hilgert, S., Poppa, C., Duch, C., von Kleist, M., Hassan, B. A. and Hiesinger, P. R. (2021). Brain connectivity inversely scales with developmental temperature in Drosophila. Cell Rep 37(12): 110145. PubMed ID: 34936868
Summary:
Variability of synapse numbers and partners despite identical genes reveals the limits of genetic determinism. This study used developmental temperature as a non-genetic perturbation to study variability of brain wiring and behavior in Drosophila. Unexpectedly, slower development at lower temperatures increases axo-dendritic branching, synapse numbers, and non-canonical synaptic partnerships of various neurons, while maintaining robust ratios of canonical synapses. Using R7 photoreceptors as a model, this study showed that changing the relative availability of synaptic partners using a DIPγ mutant that ablates R7's preferred partner leads to temperature-dependent recruitment of non-canonical partners to reach normal synapse numbers. Hence, R7 synaptic specificity is not absolute but based on the relative availability of postsynaptic partners and presynaptic control of synapse numbers. Behaviorally, movement precision is temperature robust, while movement activity is optimized for the developmentally encountered temperature. These findings suggest genetically encoded relative and scalable synapse formation to develop functional, but not identical, brains and behaviors.

Monday, March 21st - Adult neural development and function

Lee, W. P., Chiang, M. H., Chang, L. Y., Shyu, W. H., Chiu, T. H., Fu, T. F., Wu, T. and Wu, C. L. (2021). Serotonin Signals Modulate Mushroom Body Output Neurons for Sustaining Water-Reward Long-Term Memory in Drosophila. Front Cell Dev Biol 9:755574. PubMed ID: 34858982
Summary:
Memory consolidation is a time-dependent process through which an unstable learned experience is transformed into a stable long-term memory; however, the circuit and molecular mechanisms underlying this process are poorly understood. The Drosophila mushroom body (MB) is a huge brain neuropil that plays a crucial role in olfactory memory. The MB neurons can be generally classified into three subsets: γ, αβ, and α'β'. This study reports that water-reward long-term memory (wLTM) consolidation requires activity from α'β'-related mushroom body output neurons (MBONs) in a specific time window. wLTM consolidation requires neurotransmission in MBON-γ3β'1 during the 0-2 h period after training, and neurotransmission in MBON-α'2 is required during the 2-4 h period after training. Moreover, neurotransmission in MBON-α'1α'3 is required during the 0-4 h period after training. Intriguingly, blocking neurotransmission during consolidation or inhibiting serotonin biosynthesis in serotoninergic dorsal paired medial (DPM) neurons also disrupted the wLTM, suggesting that wLTM consolidation requires serotonin signals from DPM neurons. The GFP Reconstitution Across Synaptic Partners (GRASP) data showed the connectivity between DPM neurons and MBON-γ3β'1, MBON-α'2, and MBON-α'1α'3, and RNAi-mediated silencing of serotonin receptors in MBON-γ3β'1, MBON-α'2, or MBON-α'1α'3 disrupted wLTM. Taken together, these results suggest that serotonin released from DPM neurons modulates neuronal activity in MBON-γ3β'1, MBON-α'2, and MBON-&alpha'1&alpha'3 at specific time windows, which is critical for the consolidation of wLTM in Drosophila.
Lu, J., Behbahani, A. H., Hamburg, L., Westeinde, E. A., Dawson, P. M., Lyu, C., Maimon, G., Dickinson, M. H., Druckmann, S. and Wilson, R. I. (2022). Transforming representations of movement from body- to world-centric space. Nature 601(7891): 98-104. PubMed ID: 34912123
Summary:
When an animal moves through the world, its brain receives a stream of information about the body's translational velocity from motor commands and sensory feedback signals. These incoming signals are referenced to the body, but ultimately, they must be transformed into world-centric coordinates for navigation. This study shows that this computation occurs in the fan-shaped body in the brain of Drosophila melanogaster. Two cell types, PFNd and PFNv were identified, that conjunctively encode translational velocity and heading as a fly walks. In these cells, velocity signals are acquired from locomotor brain regions and are multiplied with heading signals from the compass system. PFNd neurons prefer forward-ipsilateral movement, whereas PFNv neurons prefer backward-contralateral movement, and perturbing PFNd neurons disrupts idiothetic path integration in walking flies. Downstream, PFNd and PFNv neurons converge onto hδB neurons, with a connectivity pattern that pools together heading and translation direction combinations corresponding to the same movement in world-centric space. This network motif effectively performs a rotation of the brain's representation of body-centric translational velocity according to the current heading direction. Consistent with these predictions, it was observe that hΔB neurons form a representation of translational velocity in world-centric coordinates. By integrating this representation over time, it should be possible for the brain to form a working memory of the path travelled through the environment.
Kind, E., Longden, K. D., Nern, A., Zhao, A., Sancer, G., Flynn, M. A., Laughland, C. W., Gezahegn, B., Ludwig, H. D., Thomson, A. G., Obrusnik, T., Alarcón, P. G., Dionne, H., Bock, D. D., Rubin, G. M., Reiser, M. B. and Wernet, M. F. (2021). Synaptic targets of photoreceptors specialized to detect color and skylight polarization in Drosophila. Elife 10. PubMed ID: 34913436
Summary:
Color and polarization provide complementary information about the world and are detected by specialized photoreceptors. However, the downstream neural circuits that process these distinct modalities are incompletely understood in any animal. Using electron microscopy, this study had systematically reconstructed the synaptic targets of the photoreceptors specialized to detect color and skylight polarization in Drosophila, and light microscopy was used to confirm many of the findings. Known and novel downstream targets were identified that are selective for different wavelengths or polarized light, and their projections were followed to other areas in the optic lobes and the central brain. The results revealed many synapses along the photoreceptor axons between brain regions, new pathways in the optic lobes, and spatially segregated projections to central brain regions. Strikingly, photoreceptors in the polarization-sensitive dorsal rim area target fewer cell types, and lack strong connections to the lobula, a neuropil involved in color processing. This reconstruction identifies shared wiring and modality-specific specializations for color and polarization vision, and provides a comprehensive view of the first steps of the pathways processing color and polarized light inputs.
Lyu, C., Abbott, L. F. and Maimon, G. (2022). Building an allocentric travelling direction signal via vector computation. Nature 601(7891): 92-97. PubMed ID: 34912112
Summary:
Many behavioural tasks require the manipulation of mathematical vectors, but, outside of computational models, it is not known how brains perform vector operations. This study shows how the Drosophila central complex, a region implicated in goal-directed navigation, performs vector arithmetic. First, a neural signal in the fan-shaped body is described that explicitly tracks the allocentric travelling angle of a fly, that is, the travelling angle in reference to external cues. Past work has identified neurons in Drosophila and mammals that track the heading angle of an animal referenced to external cues (for example, head direction cells), but this new signal illuminates how the sense of space is properly updated when travelling and heading angles differ (for example, when walking sideways). A neuronal circuit was characterized that performs an egocentric-to-allocentric (that is, body-centred to world-centred) coordinate transformation and vector addition to compute the allocentric travelling direction. This circuit operates by mapping two-dimensional vectors onto sinusoidal patterns of activity across distinct neuronal populations, with the amplitude of the sinusoid representing the length of the vector and its phase representing the angle of the vector. The principles of this circuit may generalize to other brains and to domains beyond navigation where vector operations or reference-frame transformations are required (Lyu 2022).
Tseng, C. Y., Burel, M., Cammer, M., Harsh, S., Flaherty, M. S., Baumgartner, S. and Bach, E. A. (2022). chinmo-mutant spermatogonial stem cells cause mitotic drive by evicting non-mutant neighbors from the niche. Dev Cell 57(1): 80-94.e87. PubMed ID: 34942115
Summary:
Niches maintain a finite pool of stem cells via restricted space and short-range signals. Stem cells compete for limited niche resources, but the mechanisms regulating competition are poorly understood. Using the Drosophila testis model, this study showed that germline stem cells (GSCs) lacking the transcription factor Chinmo gain a competitive advantage for niche access. Surprisingly, chinmo(-/-) GSCs rely on a new mechanism of competition in which they secrete the extracellular matrix protein Perlecan to selectively evict non-mutant GSCs and then upregulate Perlecan-binding proteins to remain in the altered niche. Over time, the GSC pool can be entirely replaced with chinmo(-/-) cells. As a consequence, the mutant chinmo allele acts as a gene drive element; the majority of offspring inherit the allele despite the heterozygous genotype of the parent. These results suggest that the influence of GSC competition may extend beyond individual stem cell niche dynamics to population-level allelic drift and evolution.
Li, B., Li, S., Zheng, H. and Yan, Z. (2021). Nanchung and Inactive define pore properties of the native auditory transduction channel in Drosophila. Proc Natl Acad Sci U S A 118(49). PubMed ID: 34848538
Summary:
Auditory transduction is mediated by chordotonal (Cho) neurons in Drosophila larvae, but the molecular identity of the mechanotransduction (MET) channel is elusive. This study established a whole-cell recording system of Cho neurons and showed that two transient receptor potential vanilloid (TRPV) channels, Nanchung (NAN) and Inactive (IAV), are essential for MET currents in Cho neurons. NAN and IAV form active ion channels when expressed simultaneously in S2 cells. Point mutations in the pore region of NAN-IAV change the reversal potential of the MET currents. Particularly, residues 857 through 990 in the IAV carboxyl terminus regulate the kinetics of MET currents in Cho neurons. In addition, TRPN channel NompC contributes to the adaptation of auditory transduction currents independent of its ion-conduction function. These results indicate that NAN-IAV, rather than NompC, functions as essential pore-forming subunits of the native auditory transduction channel in Drosophila and provide insights into the gating mechanism of MET currents in Cho neurons.

Thursday, March 17th - Gonads

Majane, A. C., Cridland, J. M. and Begun, D. J. (2021). Single-nucleus transcriptomes reveal evolutionary and functional properties of cell types in the Drosophila accessory gland. Genetics. PubMed ID: 34849871
Summary:
Many traits responsible for male reproduction evolve quickly, including gene expression phenotypes in germline and somatic male reproductive tissues. Rapid male evolution in polyandrous species is thought to be driven by competition among males for fertilizations and conflicts between male and female fitness interests that manifest in post-copulatory phenotypes. In Drosophila, seminal fluid proteins secreted by three major cell types of the male accessory gland and ejaculatory duct are required for female sperm storage and use, and influence female post-copulatory traits. Recent work has shown that these cell types have overlapping but distinct effects on female post-copulatory biology, yet relatively little is known about their evolutionary properties. This study used single-nucleus RNA-Seq of the accessory gland and ejaculatory duct from Drosophila melanogaster and two closely related species to comprehensively describe the cell diversity of these tissues and their transcriptome evolution for the first time. Seminal fluid transcripts were found to be strongly partitioned across the major cell types, and expression of many other genes additionally define each cell type. Previously undocumented diversity in main cells is reported. Transcriptome divergence was found to be heterogeneous across cell types and lineages, revealing a complex evolutionary process. Furthermore, protein adaptation varied across cell types, with potential consequences for understanding of selection on male post-copulatory traits.
Bauerly, E., Akiyama, T., Staber, C., Yi, K. and Gibson, M. C. (2022). Impact of cilia-related genes on mitochondrial dynamics during Drosophila spermatogenesis. Dev Biol 482: 17-27. PubMed ID: 34822845
Summary:
Spermatogenesis is a dynamic process of cellular differentiation that generates the mature spermatozoa required for reproduction. Errors that arise during this process can lead to sterility due to low sperm counts and malformed or immotile sperm. While it is estimated that 1 out of 7 human couples encounter infertility, the underlying cause of male infertility can only be identified in 50% of cases. This study described and examined the genetic requirements for missing minor mitochondria (mmm), sterile affecting ciliogenesis (sac), and testes of unusual size (tous), three previously uncharacterized genes in Drosophila that are predicted to be components of the flagellar axoneme. Using Drosophila, it was demonstrated that these genes are essential for male fertility and that loss of mmm, sac, or tous results in complete immotility of the sperm flagellum. Cytological examination uncovered additional roles for sac and tous during cytokinesis and transmission electron microscopy of developing spermatids in mmm, sac, and tous mutant animals revealed defects associated with mitochondria and the accessory microtubules required for the proper elongation of the mitochondria and flagella during ciliogenesis. This study highlights the complex interactions of cilia-related proteins within the cell body and advances understanding of male infertility by uncovering novel mitochondrial defects during spermatogenesis.
Bordet, G., Kotova, E. and Tulin, A. V. (2022). Poly(ADP-ribosyl)ating pathway regulates development from stem cell niche to longevity control. Life Sci Alliance 5(3). PubMed ID: 34949666
Summary:
The regulation of poly(ADP-ribose) polymerase, the enzyme responsible for the synthesis of homopolymer ADP-ribose chains on nuclear proteins, has been extensively studied over the last decades for its involvement in tumorigenesis processes. However, the regulation of poly(ADP-ribose) glycohydrolase (PARG), the enzyme responsible for removing this posttranslational modification, has attracted little attention. This study identified that PARG activity is partly regulated by two phosphorylation sites, ph1 and ph2, in Drosophila. Disruption of these sites affects the germline stem-cells maintenance/differentiation balance as well as embryonic and larval development, but also the synchronization of egg production with the availability of a calorically sufficient food source. Moreover, these PARG phosphorylation sites play an essential role in the control of fly survivability from larvae to adults. It wa also showed that PARG is phosphorylated by casein kinase 2 and that this phosphorylation seems to protect PARG protein against degradation in vivo. Taken together, these results suggest that the regulation of PARG protein activity plays a crucial role in the control of several developmental processes.
Ibaraki, K., Nakatsuka, M., Ohsako, T., Watanabe, M., Miyazaki, Y., Shirakami, M., Karr, T. L., Sanuki, R., Tomaru, M. and Takano-Shimizu-Kouno, T. (2021). A cross-species approach for the identification of Drosophila male sterility genes. G3 (Bethesda) 11(8). PubMed ID: 34849808
Summary:
Using male sterility genes identified in human, mouse, and fruit fly databases as a starting point, 103 Drosophila melanogaster genes were screened for their association with male sterility by tissue-specific RNAi knockdown and CRISPR/Cas9-mediated mutagenesis. This list included 56 genes associated with male infertility in the human databases, but not found in the Drosophila database, resulting in the discovery of 63 new genes associated with male fertility in Drosophila. The phenotypes identified were categorized into six distinct classes affecting sperm development. Interestingly, the second largest class (Class VI) caused sterility despite apparently normal testis and sperm morphology suggesting that these proteins may have functions in the mature sperm following spermatogenesis. Focus was placed on one such gene, Rack 1, and it was found to play an important role in two developmental periods, in early germline cells or germline stem cells and in spermatogenic cells or sperm. Taken together, many genes are yet to be identified and their role in male reproduction, especially after ejaculation, remains to be elucidated in Drosophila, where a wealth of data from human and other model organisms would be useful.
Colonnetta, M. M., Goyal, Y., Johnson, H. E., Syal, S., Schedl, P. and Deshpande, G. (2022). Preformation and epigenesis converge to specify primordial germ cell fate in the early Drosophila embryo. PLoS Genet 18(1): e1010002. PubMed ID: 34986144
Summary:
A critical step in animal development is the specification of primordial germ cells (PGCs), the precursors of the germline. Two seemingly mutually exclusive mechanisms are implemented across the animal kingdom: epigenesis and preformation. In epigenesis, PGC specification is non-autonomous and depends on extrinsic signaling pathways. The BMP pathway provides the key PGC specification signals in mammals. Preformation is autonomous and mediated by determinants localized within PGCs. In Drosophila, a classic example of preformation, constituents of the germ plasm localized at the embryonic posterior are thought to be both necessary and sufficient for proper determination of PGCs. Contrary to this longstanding model, this study shows that these localized determinants are insufficient by themselves to direct PGC specification in blastoderm stage embryos. Instead, it was found that the BMP signaling pathway is required at multiple steps during the specification process and functions in conjunction with components of the germ plasm to orchestrate PGC fate.
Kim, J., Hyun, M., Hibi, M. and You, Y. J. (2021). Maintenance of quiescent oocytes by noradrenergic signals. Nat Commun 12(1): 6925. PubMed ID: 34836956
Summary:
All females adopt an evolutionary conserved reproduction strategy; under unfavorable conditions such as scarcity of food or mates, oocytes remain quiescent. However, the signals to maintain oocyte quiescence are largely unknown. This study reports that in four different species - Caenorhabditis elegans, Caenorhabditis remanei, Drosophila melanogaster, and Danio rerio - octopamine and norepinephrine play an essential role in maintaining oocyte quiescence. In the absence of mates, the oocytes of Caenorhabditis mutants lacking octopamine signaling fail to remain quiescent, but continue to divide and become polyploid. Upon starvation, the egg chambers of D. melanogaster mutants lacking octopamine signaling fail to remain at the previtellogenic stage, but grow to full-grown egg chambers. Upon starvation, D. rerio lacking norepinephrine fails to maintain a quiescent primordial follicle and activates an excessive number of primordial follicles.This study reveals an evolutionarily conserved function of the noradrenergic signal in maintaining quiescent oocytes.

Wednesday, March 16th - Signaling

La Marca, J. E., Willoughby, L. F., Allan, K., Portela, M., Goh, P. K., Tiganis, T. and Richardson, H. E. (2021). PTP61F Mediates Cell Competition and Mitigates Tumorigenesis. Int J Mol Sci 22(23). PubMed ID: 34884538
Summary:
Tissue homeostasis via the elimination of aberrant cells is fundamental for organism survival. Cell competition is a key homeostatic mechanism, contributing to the recognition and elimination of aberrant cells, preventing their malignant progression and the development of tumors. Using Drosophila as a model organism, this study have defined a role for protein tyrosine phosphatase 61F (PTP61F) (orthologue of mammalian PTP1B and TCPTP) in the initiation and progression of epithelial cancers. A Ptp61F null mutation confers cells with a competitive advantage relative to neighbouring wild-type cells, while elevating PTP61F levels has the opposite effect. Furthermore, it was shown that knockdown of Ptp61F affects the survival of clones with impaired cell polarity, and that this occurs through regulation of the JAK-STAT signalling pathway. Importantly, PTP61F plays a robust non-cell-autonomous role in influencing the elimination of adjacent polarity-impaired mutant cells. Moreover, in a neoplastic RAS-driven polarity-impaired tumor model, it was shown that PTP61F levels determine the aggressiveness of tumors, with Ptp61F knockdown or overexpression, respectively, increasing or reducing tumor size. These effects correlate with the regulation of the RAS-MAPK and JAK-STAT signalling by PTP61F. Thus, PTP61F acts as a tumor suppressor that can function in an autonomous and non-cell-autonomous manner to ensure cellular fitness and attenuate tumorigenesis.
Kaltezioti, V., Vakaloglou, K. M., Charonis, A. S. and Zervas, C. G. (2021). Evidence of Swim secretion and association with extracellular matrix in the Drosophila embryo. Int J Dev Biol. PubMed ID: 34881800
Summary:
Secreted wingless-interacting protein (Swim) is the Drosophila ortholog gene of the mammalian Tubulointerstitial Nephritis Antigen Like 1 (TINAGL1). Swim and TINAGL1 proteins share a significant homology, including the somatomedin B and the predictive inactive C1 cysteine peptidase domains. In mammals, both TINAGL1 and its closely related homolog TINAG have been identified in basement membranes, where they may function as modulators of integrin-mediated adhesion. In Drosophila, Swim was initially identified in the eggshell matrix. Further biochemical analysis indicated that Swim binds to wingless (wg) in a lipid-dependent manner. This observation together with RNAi knockdown studies suggested that Swim is an essential cofactor of wg-signalling. However, recent elegant genetic studies ruled out the possibility that Swim is required alone to facilitate wg signalling in Drosophila, because flies without Swim are viable and fertile. This study used the UAS/Gal4 expression system together with confocal imaging to analyze the in vivo localization of a chimeric Swim-GFP in the developing Drosophila embryo. The data fully support the notion that Swim is an extracellular matrix component that upon ectopic expression is secreted and preferentially associates with the basement membranes of various organs and with the specialized tendon matrix at the muscle attachment sites (MAS). In conclusion, Swim is an extracellular matrix component, and it is possible that Swim exhibits overlapping functions in concert with other undefined components.
Langton, P. F., Baumgartner, M. E., Logeay, R. and Piddini, E. (2021). Xrp1 and Irbp18 trigger a feed-forward loop of proteotoxic stress to induce the loser status. PLoS Genet 17(12): e1009946. PubMed ID: 34914692
Summary:
Cell competition induces the elimination of less-fit "loser" cells by fitter "winner" cells. In Drosophila, cells heterozygous mutant in ribosome genes, Rp/+, known as Minutes, are outcompeted by wild-type cells. Rp/+ cells display proteotoxic stress and the oxidative stress response, which drive the loser status. Minute cell competition also requires the transcription factors Irbp18 and Xrp1, but how these contribute to the loser status is partially understood. This study provided evidence that initial proteotoxic stress in RpS3/+ cells is Xrp1-independent. However, Xrp1 is sufficient to induce proteotoxic stress in otherwise wild-type cells and is necessary for the high levels of proteotoxic stress found in RpS3/+ cells. Surprisingly, Xrp1 is also induced downstream of proteotoxic stress, and is required for the competitive elimination of cells suffering from proteotoxic stress or overexpressing Nrf2. These data suggests that a feed-forward loop between Xrp1, proteotoxic stress, and Nrf2 drives Minute cells to become losers.
Liu, J., Tao, X., Zhu, Y., Li, C., Ruan, K., Diaz-Perez, Z., Rai, P., Wang, H. and Zhai, R. G. (2021). NMNAT promotes glioma growth through regulating post-translational modifications of P53 to inhibit apoptosis. Elife 10. PubMed ID: 34919052
Summary:
Gliomas are highly malignant brain tumors with poor prognosis and short survival. NAD(+) has been shown to impact multiple processes that are dysregulated in cancer; however, anti-cancer therapies targeting NAD(+) synthesis have had limited success due to insufficient mechanistic understanding. This study adapted a Drosophila glial neoplasia model and discovered the genetic requirement for NAD(+) synthase nicotinamide mononucleotide adenylyltransferase (NMNAT) in glioma progression in vivo and in human glioma cells. Overexpressing enzymatically active NMNAT significantly promotes glial neoplasia growth and reduces animal viability. Mechanistic analysis suggests that NMNAT interferes with DNA damage-p53-caspase-3 apoptosis signaling pathway by enhancing NAD(+)-dependent posttranslational modifications (PTMs) poly(ADP-ribosyl)ation (PARylation) and deacetylation of p53. Since PARylation and deacetylation reduce p53 pro-apoptotic activity, modulating p53 PTMs could be a key mechanism by which NMNAT promotes glioma growth. These findings reveal a novel tumorigenic mechanism involving protein complex formation of p53 with NAD(+) synthetic enzyme NMNAT and NAD(+)-dependent PTM enzymes that regulates glioma growth.
Kerekes, K., Trexler, M., Bányai, L. and Patthy, L. (2021). Wnt Inhibitory Factor 1 Binds to and Inhibits the Activity of Sonic Hedgehog. Cells 10(12). PubMed ID: 34944004
Summary:
The hedgehog (Hh) and Wnt pathways, crucial for the embryonic development and stem cell proliferation of Metazoa, have long been known to have similarities that argue for their common evolutionary origin. A surprising additional similarity of the two pathways came with the discovery that WIF1 proteins are involved in the regulation of both the Wnt and Hh pathways. Originally, WIF1 (Wnt Inhibitory Factor 1) was identified as a Wnt antagonist of vertebrates, but subsequent studies have shown that in Drosophila, the WIF1 ortholog, Shifted) serves primarily to control the distribution of Hh. The present work characterized the interaction of the human WIF1 protein with human sonic hedgehog (Shh) using Surface Plasmon Resonance spectroscopy and reporter assays monitoring the signaling activity of human Shh. These studies have shown that human WIF1 protein binds human Shh with high affinity and inhibits its signaling activity efficiently. The observation that the human WIF1 protein is a potent antagonist of human Shh suggests that the known tumor suppressor activity of WIF1 may not be ascribed only to its role as a Wnt inhibitor.
Karkali, K. and Martin-Blanco, E. (2021).. Dissection of the Regulatory Elements of the Complex Expression Pattern of Puckered, a Dual-Specificity JNK Phosphatase. Int J Mol Sci 22(22). PubMed ID: 34830088
Summary:
For developmental processes, most of the gene networks controlling specific cell responses. It still has to be determined how these networks cooperate and how signals become integrated. The JNK pathway is one of the key elements modulating cellular responses during development. Yet, still little is known about how the core components of the pathway interact with additional regulators or how this network modulates cellular responses in the whole organism in homeostasis or during tissue morphogenesis. A promoter analysis was performed, searching for potential regulatory sequences of puckered (puc) and identified different specific enhancers directing gene expression in different tissues and at different developmental times. Remarkably, some of these domains respond to the JNK activity, but not all. Altogether, these analyses show that puc expression regulation is very complex and that JNK activities participate in non-previously known processes during the development of Drosophila.

Tuesday, March 15th - Adult Physiology

Hoshino, R. and Niwa, R. (2021). Regulation of Mating-Induced Increase in Female Germline Stem Cells in the Fruit Fly Drosophila melanogaster. Front Physiol 12: 785435. PubMed ID: 34950056
Summary:
In many insect species, mating stimuli can lead to changes in various behavioral and physiological responses, including feeding, mating refusal, egg-laying behavior, energy demand, and organ remodeling, which are collectively known as the post-mating response. Recently, an increase in germline stem cells (GSCs) has been identified as a new post-mating response in both males and females of the fruit fly, Drosophila melanogaster. Mating-induced increase in female GSCs of D. melanogaster were extensively studied at the molecular, cellular, and systemic levels. After mating, the male seminal fluid peptide [e.g. sex peptide (SP)] is transferred to the female uterus. This is followed by binding to the sex peptide receptor (SPR), which evokes post-mating responses, including increase in number of female GSCs. Downstream of SP-SPR signaling, the following three hormones and neurotransmitters have been found to act on female GSC niche cells to regulate mating-induced increase in female GSCs: (1) neuropeptide F, a peptide hormone produced in enteroendocrine cells; (2) octopamine, a monoaminergic neurotransmitter synthesized in ovary-projecting neurons; and (3) ecdysone, a steroid hormone produced in ovarian follicular cells. These humoral factors are secreted from each organ and are received by ovarian somatic cells and regulate the strength of niche signaling in female GSCs. This review provides an overview of the latest findings on the inter-organ relationship to regulate mating-induced female GSC increase in D. melanogaster as a model.
Justs, K. A., Lu, Z., Chouhan, A. K., Borycz, J. A., Lu, Z., Meinertzhagen, I. A. and Macleod, G. T. (2021). Presynaptic Mitochondrial Volume and Packing Density Scale with Presynaptic Power Demand. J Neurosci. PubMed ID: 34907026
Summary:
Stable neural function requires an energy supply that can meet the intense episodic power demands of neuronal activity. Neurons have presumably optimized the volume of their bioenergetic machinery to ensure these power demands are met, but the relationship between presynaptic power demands and the volume available to the bioenergetic machinery has never been quantified. This study estimated the power demands of six motor nerve terminals in female Drosophila larvae through direct measurements of neurotransmitter release and Ca(2+) entry, and via theoretical estimates of Na(+) entry and power demands at rest. Electron microscopy revealed that terminals with the highest power demands contained the greatest volume of mitochondria, indicating that mitochondria are allocated according to presynaptic power demands. In addition, terminals with the greatest power demand-to-volume ratio (∼66 nmol·min-1·μL-1) harbor the largest mitochondria packed at the greatest density. If sequential and complete oxidation of glucose by glycolysis and oxidative phosphorylation is assumed, then these mitochondria are required to produce ATP at a rate of 52 nmol·min-1·μL-1 at rest, rising to 963 during activity. Glycolysis would contribute ATP at 0.24 nmol·min-1·μL-1 of cytosol at rest, rising to 4.36 during activity. These data provide a quantitative framework for presynaptic bioenergetics in situ, and reveal that, beyond an immediate capacity to accelerate ATP output from glycolysis and oxidative phosphorylation, over longer time periods presynaptic terminals optimize mitochondrial volume and density to meet power demand.
Anqueira-González, A., Acevedo-Gonzalez, J. P., Montes-Mercado, A., Irizarry-Hernandez, C., Fuenzalida-Uribe, N. L. and Ghezzi, A. (2021). Transcriptional Correlates of Chronic Alcohol Neuroadaptation in Drosophila Larvae. Front Behav Neurosci 15: 768694. PubMed ID: 34803626
Summary:
When presented with the choice, Drosophila melanogaster females will often prefer to lay eggs on food containing a significant amount of alcohol. While, in some cases, this behavioral decision can provide a survival advantage to the developing larvae, it can also lead to developmental and cognitive problems. Alcohol consumption can affect executive functions, episodic memory, and other brain function capacities. However, in the fruit fly, the initial cognitive effects of alcohol consumption have been shown to reverse upon persistent exposure to alcohol. Using an olfactory conditioning assay where an odorant is implemented as a conditioned stimulus and paired with a heat shock as an unconditioned stimulus, a previous study has shown that when exposed to a short acute dose of alcohol, Drosophila larvae can no longer learn this association. Interestingly, upon prolonged chronic alcohol exposure, larvae seem to successfully avoid the conditioned stimulus just as well as control alcohol-naive larvae, suggestive of alcohol-induced neuroadaptations. However, the mechanisms by which Drosophila adapt to the presence of alcohol remains unknown. This study explores the transcriptional correlates of neuroadaptation in Drosophila larvae exposed to chronic alcohol to understand the genetic and cellular components responsible for this adaptation. For this, RNA sequencing technology was employed to evaluate differences in gene expression in the brain of larvae chronically exposed to alcohol. The results suggest that alcohol-induced neuroadaptations are modulated by a diverse array of synaptic genes within the larval brain through a series of epigenetic modulators.
Groen, C. M., Podratz, J. L., Pathoulas, J., Staff, N. and Windebank, A. J. (2021). Genetic Reduction of Mitochondria Complex I Subunits is Protective Against Cisplatin-Induced Neurotoxicity in Drosophila. J Neurosci. PubMed ID: 34893548
Summary:
Chemotherapy-induced peripheral neuropathy (CIPN) is a prevalent side effect of widely used platinum-based anti-cancer agents. There are few predictable risk factors to identify susceptible patients. Effective preventive measures or treatments are not available. This study used a model of CIPN in Drosophila melanogaster to identify genetic changes that confer resistance to cisplatin-induced neuronal damage but not in the rapidly dividing cells of the ovary. The Drosophila strain attP40, used as a genetic background for creation of RNAi lines, is resistant to cisplatin damage compared to the similar attP2 background strain. attP40 flies have reduced mRNA expression of ND-13A, a component of the mitochondria electron transport chain complex I. Reduction of ND-13A via neuron-specific RNAi leads to resistance to the dose-dependent climbing deficiencies and neuronal apoptosis observed in control flies. These flies are also resistant to acute oxidative stress, suggesting a mechanism for resistance to cisplatin. The mitochondria of attP40 flies function similarly to control attP2 mitochondria under normal conditions. Mitochondria are damaged by cisplatin, leading to reduced activity, but attP40 mitochondria are able to retain function and even increase basal respiration rates in response to this stress. This retained mitochondrial activity is likely mediated by Sirt1 and PGC1α, and is key to cisplatin resistance. These findings represent potential for both identification of susceptible patients and prevention of CIPN through the targeting of mitochondria.
Hughson, B. N. (2022). PKG acts in the adult corpora cardiaca to regulate nutrient stress-responsivity through adipokinetic hormone. J Insect Physiol 136: 104339. PubMed ID: 34856210
Summary:
In Drosophila melanogaster, the adipokinetic hormone (AKH) is a glucagon-like peptide that acts antagonistically with insulin-like peptides to maintain metabolic homeostasis. AKH is biosynthesized in and secreted from the corpora cardiaca (CC). This report describes a CC-specific role for dg2 - which encodes a cGMP-dependent protein kinase (PKG) - as a regulator of AKH during adulthood. Transcriptional silencing of dg2 during adulthood decreased starvation resistance, increased sucrose responsiveness, and decreased whole body lipid content. PKG protein was localized to CC cell membranes, and starvation caused a significant decrease in CC intracellular AKH content. Strikingly, reduced CC-dg2 expression caused a significant decrease in intracellular AKH content in adults fed ad libitum. This work demonstrated that dysregulation of CC-specific dg2 expression during adult life impaired metabolic homeostasis, and that dg2 acted in the CC to regulate systemic AKH activity.
Brown, J. B., Langley, S. A. ..., Mao, J. H. and Celniker, S. E. (2021). An integrated host-microbiome response to atrazine exposure mediates toxicity in Drosophila. Commun Biol 4(1): 1324. PubMed ID: 34819611
Summary:
The gut microbiome produces vitamins, nutrients, and neurotransmitters, and helps to modulate the host immune system-and also plays a major role in the metabolism of many exogenous compounds, including drugs and chemical toxicants. However, the extent to which specific microbial species or communities modulate hazard upon exposure to chemicals remains largely opaque. Focusing on the effects of collateral dietary exposure to the widely used herbicide atrazine, integrated omics and phenotypic screening were applied to assess the role of the gut microbiome in modulating host resilience in Drosophila melanogaster. Sequencing the genomes of all abundant microbes in the fly gut revealed an enzymatic pathway responsible for atrazine detoxification unique to Acetobacter tropicalis. Acetobacter tropicalis alone, in gnotobiotic animals, wad found to be sufficient to rescue increased atrazine toxicity to wild-type, conventionally reared levels. This work points toward the derivation of biotic strategies to improve host resilience to environmental chemical exposures.

Monday, March 14th - Disease Models

Huggett, S. B., Hatfield, J. S., Walters, J. D., McGeary, J. E., Welsh, J. W., Mackay, T. F. C., Anholt, R. R. H. and Palmer, R. H. C. (2021). Ibrutinib as a potential therapeutic for cocaine use disorder. Transl Psychiatry 11(1): 623. PubMed ID: 34880215
Summary:
Cocaine use presents a worldwide public health problem with high socioeconomic cost. No current pharmacologic treatments are available for cocaine use disorder (CUD) or cocaine toxicity. To explore pharmaceutical treatments for this disorder and its sequelae gene expression data was analyzed from post-mortem brain tissue of individuals with CUD who died from cocaine-related causes with matched cocaine-free controls (n = 71, M(age) = 39.9, 100% male, 49% with CUD, 3 samples/brain regions). To match molecular signatures from brain pathology with potential therapeutics, this study leveraged the L1000 database honing in on neuronal mRNA profiles of 825 repurposable compounds (e.g., FDA approved). 16 compounds were identified that were negatively associated with CUD gene expression patterns across all brain regions, all of which outperformed current targets undergoing clinical trials for CUD. An additional 43 compounds were positively associated with CUD expression. An in silico follow-up potential therapeutics was performed using independent transcriptome-wide in vitro and in vivo (mouse cocaine self-administration) datasets to prioritize candidates for experimental validation. Among these medications, ibrutinib was consistently linked with the molecular profiles of both neuronal cocaine exposure and mouse cocaine self-administration. The therapeutic efficacy of ibrutinib was assessed using the Drosophila melanogaster model. Ibrutinib reduced cocaine-induced startle response and cocaine-induced seizures, despite increasing cocaine consumption. These results suggest that ibrutinib could be used for the treatment of cocaine use disorder.
Iyengar, A., Ruan, H. and Wu, C. F. (2022). Distinct Aging-Vulnerable and -Resilient Trajectories of Specific Motor Circuit Functions in Oxidation- and Temperature-Stressed Drosophila. eNeuro 9(1). PubMed ID: 34876473
Summary:
In Drosophila, molecular pathways affecting longevity have been extensively studied. However, corresponding neurophysiological changes underlying aging-related functional and behavioral deteriorations remain to be fully explored. This study examined different motor circuits in Drosophila across the life span and uncovered distinctive age-resilient and age-vulnerable trajectories in their established functional properties. In the giant fiber (GF) and downstream circuit elements responsible for the jump-and-flight escape reflex, relatively mild deterioration was observed toward the end of the life span. In contrast, more substantial age-dependent modifications were seen in the plasticity of GF afferent processing, specifically in use dependence and habituation properties. In addition, there were profound changes in different afferent circuits that drive flight motoneuron activities, including flight pattern generation and seizure spike discharges evoked by electroconvulsive stimulation. Importantly, in high-temperature (HT)-reared flies (29°C), the general trends in these age-dependent trajectories were largely maintained, albeit over a compressed time scale, lending support for the common practice of HT rearing for expediting Drosophila aging studies. It was discovered that shortened life spans in Cu/Zn superoxide dismutase (Sod) mutant flies were accompanied by altered aging trajectories in motor circuit properties distinct from those in HT-reared flies, highlighting differential effects of oxidative versus temperature stressors. This work helps to identify several age-vulnerable neurophysiological parameters that may serve as quantitative indicators for assessing genetic and environmental influences on aging progression in Drosophila.
Epiney, D. G., Salameh, C., Cassidy, D., Zhou, L. T., Kruithof, J., Milutinović, R., Andreani, T. S., Schirmer, A. E. and Bolterstein, E. (2021). Characterization of Stress Responses in a Drosophila Model of Werner Syndrome. Biomolecules 11(12). PubMed ID: 34944512
Summary:
As organisms age, their resistance to stress decreases while their risk of disease increases. This can be shown in patients with Werner syndrome (WS), which is a genetic disease characterized by accelerated aging along with increased risk of cancer and metabolic disease. WS is caused by mutations in WRN, a gene involved in DNA replication and repair. Recent research has shown that WRN mutations contribute to multiple hallmarks of aging including genomic instability, telomere attrition, and mitochondrial dysfunction. However, questions remain regarding the onset and effect of stress on early aging. This study used a fly model of WS (WRNexo(Δ)) to investigate stress response during different life stages; stress sensitivity was found to vary according to age and stressor. While larvae and young WRNexo(Δ) adults are not sensitive to exogenous oxidative stress, high antioxidant activity suggests high levels of endogenous oxidative stress. WRNexo(Δ) adults are sensitive to stress caused by elevated temperature and starvation suggesting abnormalities in energy storage and a possible link to metabolic dysfunction in WS patients. Higher levels of sleep were observed in aged WRNexo(Δ) adults suggesting an additional adaptive mechanism to protect against age-related stress. It is suggested that stress response in WRNexo(Δ) is multifaceted and evokes a systemic physiological response to protect against cellular damage. These data further validate WRNexo(Δ) flies as a WS model with which to study mechanisms of early aging and provide a foundation for development of treatments for WS and similar diseases.
Dhankhar, J., Agrawal, N. and Shrivastava, A. (2022). Pan-neuronal expression of human mutant huntingtin protein in Drosophila impairs immune response of hemocytes. J Neuroimmunol 363: 577801. PubMed ID: 34973473
Summary:
Huntington's disease (HD) is a late-onset; progressive, dominantly inherited neurological disorder marked by an abnormal expansion of polyglutamine (poly Q) repeats in Huntingtin (HTT) protein. The pathological effects of mutant Huntingtin (mHTT) are not restricted to the nervous system but systemic abnormalities including immune dysregulation have been evidenced in clinical and experimental settings of HD. Indeed, mHTT is ubiquitously expressed and could induce cellular toxicity by directly acting on immune cells. However, it is still unclear if selective expression of mHTT exon1 in neurons could induce immune responses and hemocytes' function. This study intended to monitor perturbations in the hemocytes' population and their physiological functions in Drosophila, caused by pan-neuronal expression of mHTT protein. A measure of hemocyte count and their physiological activities caused by pan-neuronal expression of mHTT protein highlighted the extent of immune dysregulation occurring with disease progression. It was found that pan-neuronal expression of mHTT significantly alters crystal cells and plasmatocyte count in larvae and adults with disease progression. Interestingly, plasmatocytes isolated from diseased conditions exhibit a gradual decline in phagocytic activity ex vivo at progressive stages of the disease as compared to age-matched control groups. In addition, diseased flies displayed elevated reactive oxygen species (ROS) in circulating plasmatocytes at the larval stage and in sessile plasmatocytes of hematopoietic pockets at terminal stages of disease. These findings strongly implicate that neuronal expression of mHTT alone is sufficient to induce non-cell-autonomous immune dysregulation in vivo.
Joshi, R., Banerjee, S. J., Curtiss, J. and Ashley, A. K. (2022). DNA ligase IV mutations confer shorter lifespan and increased sensitivity to nutrient stress in Drosophila melanogaster. J Appl Genet 63(1): 141-144. PubMed ID: 34817771
Summary:
The nonhomologous end-joining pathway is a primary DNA double-strand break repair pathway in eukaryotes. DNA ligase IV (Lig4) catalyzes the final step of DNA end ligation in this pathway. Partial loss of Lig4 in mammals causes Lig4 syndrome, while complete loss is embryonically lethal. DNA ligase 4 (DNAlig4) null Drosophila melanogaster is viable, but sensitive to ionizing radiation during early development. It is proposed to explore if DNAlig4 loss induced other long-term sensitivities and defects in D. melanogaster. This study demonstrated that DNAlig4 mutant strains had decreased lifespan and lower resistance to nutrient deprivation, indicating Lig4 is required for maintaining health and longevity in D. melanogaster.
Kunkeaw, T., Suttisansanee, U., Trachootham, D., Karinchai, J., Chantong, B., Potikanond, S., Inthachat, W., Pitchakarn, P. and Temviriyanukul, P. (2021). Diplazium esculentum (Retz.) Sw. reduces BACE-1 activities and amyloid peptides accumulation in Drosophila models of Alzheimer's disease. Sci Rep 11(1): 23796. PubMed ID: 34893659
Summary:
Alzheimer's disease (AD), one type of dementia, is a complex disease affecting people globally with limited drug treatment. Thus, natural products are currently of interest as promising candidates because of their cost-effectiveness and multi-target abilities. Diplazium esculentum (Retz.) Sw., an edible fern, inhibited acetylcholinesterase in vitro, inferring that it might be a promising candidate for AD treatment by supporting cholinergic neurons. However, evidence demonstrating anti-AD properties of this edible plant via inhibiting of neurotoxic peptides production, amyloid beta (Aβ), both in vitro and in vivo is lacking. Thus, the anti-AD properties of D. esculentum extract both in vitro and in Drosophila models of Aβ-mediated toxicity were elucidated. Findings showed that an ethanolic extract exhibited high phenolics and flavonoids, contributing to antioxidant and inhibitory activities against AD-related enzymes. Notably, the extract acted as a BACE-1 blocker and reduced amyloid beta 42 (Aβ42) peptides in Drosophila models, resulting in improved locomotor behaviors. Information gained from this study suggested that D. esculentum showed potential for AD amelioration and prevention. Further investigations in vertebrates or humans are required to determine the effective doses of D. esculentum against AD, particularly via amyloidogenic pathway.

Friday, March 11th - Evolution

Krinsky, B. H., Arthur, R. K., Xia, S., Sosa, D., Arsala, D., White, K. P. and Long, M. (2021). Rapid Cis-Trans Coevolution Driven by a Novel Gene Retroposed from a Eukaryotic Conserved CCR4-NOT Component in Drosophila. Genes (Basel) 13(1). PubMed ID: 35052398
Summary:
Young, or newly evolved, genes arise ubiquitously across the tree of life, and they can rapidly acquire novel functions that influence a diverse array of biological processes. Previous work identified a young regulatory duplicate gene in Drosophila, Zeus that unexpectedly diverged rapidly from its parent, Caf40, an extremely conserved component in the CCR4-NOT machinery in post-transcriptional and post-translational regulation of eukaryotic cells, and took on roles in the male reproductive system. This neofunctionalization was accompanied by differential binding of the Zeus protein to loci throughout the Drosophila melanogaster genome. However, the way in which new DNA-binding proteins acquire and coevolve with their targets in the genome is not understood. In this study, by comparing Zeus ChIP-Seq data from D. melanogaster and D. simulans to the ancestral Caf40 binding events from D. yakuba, a species that diverged before the duplication event, a dynamic pattern was found in which Zeus binding rapidly coevolved with a previously unknown DNA motif, which was termed Caf40 and Zeus-Associated Motif (CAZAM), under the influence of positive selection. Interestingly, while both copies of Zeus acquired targets at male-biased and testis-specific genes, D. melanogaster and D. simulans proteins have specialized binding on different chromosomes, a pattern echoed in the evolution of the associated motif. Using CRISPR-Cas9-mediated gene knockout of Zeus and RNA-Seq, this study found that Zeus regulated the expression of 661 differentially expressed genes (DEGs). The results suggest that the evolution of young regulatory genes can be coupled to substantial rewiring of the transcriptional networks into which they integrate, even over short evolutionary timescales. These results thus uncover dynamic genome-wide evolutionary processes associated with new genes.
Arun, M. G., Agarwala, A., Syed, Z. A., Jigisha, Kashyap, M., Venkatesan, S., Chechi, T. S., Gupta, V. and Prasad, N. G. (2021). Experimental evolution reveals sex-specific dominance for surviving bacterial infection in laboratory populations of Drosophila melanogaster. Evol Lett 5(6): 657-671. PubMed ID: 34919096
Summary:
Males and females are subjected to distinct kinds of selection pressures, often leading to the evolution of sex-specific genetic architecture, an example being sex-specific dominance. Sex-specific dominance reversals (SSDRs), where alleles at sexually antagonistic loci are at least partially dominant in the sex they benefit, have been documented in Atlantic salmon, rainbow trout, and seed beetles. Another interesting feature of many sexually reproducing organisms is the asymmetric inheritance pattern of X chromosomes, which often leads to distinct evolutionary outcomes on X chromosomes compared to autosomes. Immunocompetence is a trait that has been extensively investigated for sexual dimorphism with growing evidence for sex-specific or sexually antagonistic variation. X chromosomes have been shown to harbor substantial immunity-related genetic variation in the fruit fly, Drosophila melanogaster. Using interpopulation crosses and cytogenetic cloning, this study investigated sex-specific dominance and the role of the X chromosome in improved postinfection survivorship of laboratory populations of D. melanogaster selected against pathogenic challenge by Pseudomonas entomophila. No contribution was detected of the X chromosome to the evolved immunocompetence of the selected populations, as well as to within-population variation in immunocompetence. However, strong evidence was found of sex-specific dominance related to surviving bacterial infection. These results indicate that alleles that confer a survival advantage to the selected populations are, on average, partially dominant in females but partially recessive in males. This could also imply an SSDR for overall fitness, given the putative evidence for sexually antagonistic selection affecting immunocompetence in Drosophila melanogaster. This study also highlighted sex-specific dominance as a potential mechanism of sex differences in immunocompetence, with population-level sex differences primarily driven by sex differences in heterozygotes.
Janssen, R., Turetzek, N. and Pechmann, M. (2022). Lack of evidence for conserved parasegmental grooves in arthropods. Dev Genes Evol. PubMed ID: 35038005
Summary:
In the arthropod model species Drosophila melanogaster, a dipteran fly, segmentation of the anterior-posterior body axis is under control of a hierarchic gene cascade. Segmental boundaries that form morphological grooves are established posteriorly within the segmental expression domain of the segment-polarity gene (SPG) engrailed (en). More important for the development of the fly, however, are the parasegmental boundaries that are established at the interface of en expressing cells and anteriorly adjacent wingless (wg) expressing cells. In Drosophila, both segmental and transient parasegmental grooves form. The latter are positioned anterior to the expression of en. Although the function of the SPGs in establishing and maintaining segmental and parasegmental boundaries is highly conserved among arthropods, parasegmental grooves have only been reported for Drosophila, and a spider (Cupiennius salei). This study presents new data on en expression, and re-evaluate published data from four distantly related spiders, including Cupiennius, and a distantly related chelicerate, the harvestman Phalangium opilio. Gene expression analysis of en genes in these animals does not corroborate the presence of parasegmental grooves. Consequently, these data question the general presence of parasegmental grooves in arthropods.
Vance, Z., Niezabitowski, L., Hurst, L. D. and McLysaght, A. (2022). Evidence from Drosophila Supports Higher Duplicability of Faster Evolving Genes. Genome Biol Evol 14(1). PubMed ID: 35018456
Summary:
The faster rate of evolution of duplicated genes relative to singletons has been well documented in multiple lineages. This observation has generally been attributed to a presumed release from constraint following creation of a redundant, duplicate copy. However, it is not obvious that the relationship operates in this direction. An alternative possibility-that the faster rate of evolution predates the duplication event and the observed differences result from a higher propensity to duplicate in fast-evolving genes-has been tested in primates and in insects. However, these studies arrived at different conclusions and clarity is needed on whether these contrasting results relate to differences in methodology or legitimate biological differences between the lineages selected. This study tested whether duplicable genes are faster evolving independent of duplication in the Drosophila lineage; the results support the conclusion that faster evolving genes are more likely to duplicate, in agreement with previous work in primates. These findings indicate that this characteristic of gene duplication is not restricted to a single lineage and has broad implications for the interpretation of the impact of gene duplication. A subset of "singletons" was identified the defy the general trends and appear to be faster evolving. Further investigation implicates homology detection failure and suggests that these may be duplicable genes with unidentifiable paralogs.
Bruckner, A., Badroos, J. M., Learsch, R. W., Yousefelahiyeh, M., Kitchen, S. A. and Parker, J. (2021). Evolutionary assembly of cooperating cell types in an animal chemical defense system. Cell 184(25): 6138-6156. PubMed ID: 34890552
Summary:
How the functions of multicellular organs emerge from the underlying evolution of cell types is poorly understood. This study deconstructed evolution of an organ novelty: a rove beetle gland that secretes a defensive cocktail. This study showed how gland function arose via assembly of two cell types that manufacture distinct compounds. One cell type, comprising a chemical reservoir within the abdomen, produces alkane and ester compounds. This cell type is a hybrid of cuticle cells and ancient pheromone and adipocyte-like cells, executing its function via a mosaic of enzymes from each parental cell type. The second cell type synthesizes benzoquinones using a chimera of conserved cellular energy and cuticle formation pathways. Evolution of each cell type was shaped by coevolution between the two cell types, yielding a potent secretion that confers adaptive value. These findings illustrate how cooperation between cell types arises, generating new, organ-level behaviors.
Bauer DuMont, V. L., White, S. L., Zinshteyn, D. and Aquadro, C. F. (2021). Molecular population genetics of Sex-lethal (Sxl) in the Drosophila melanogaster species group: a locus that genetically interacts with Wolbachia pipientis in Drosophila melanogaster. G3 (Bethesda) 11(8). PubMed ID: 34849818
Summary:
Sex-lethal (Sxl) is the sex determination switch in Drosophila, and also plays a critical role in germ-line stem cell daughter differentiation in Drosophila melanogaster. Three female-sterile alleles at Sxl in D. melanogaster were previously shown to genetically interact to varying degrees with the maternally inherited endosymbiont Wolbachia pipientis. Given this genetic interaction and W. pipientis' ability to manipulate reproduction in Drosophila, a careful study was carried out of both the population genetics (within four Drosophila species) and molecular evolutionary analysis (across 20 Drosophila species) of Sxl. Consistent with earlier studies, this study found that selective constraint has played a prominent role in Sxl's molecular evolution within Drosophila, but patterns were observed that suggest both episodic bursts of protein evolution and recent positive selection at Sxl. The episodic nature of Sxl's protein evolution is discussed in light of its genetic interaction with W. pipientis.

Thursday, March 10th - Signaling

Cao, X., Rojas, M. and Pastor-Pareja, J. C. (2022). Intrinsic and damage-induced JAK/STAT signaling regulate developmental timing by the Drosophila prothoracic gland. Dis Model Mech 15(1). PubMed ID: 34842272
Summary:
Development involves tightly paced, reproducible sequences of events, yet it must adjust to conditions external to it, such as resource availability and organismal damage. A major mediator of damage-induced immune responses in vertebrates and insects is JAK/STAT signaling. At the same time, JAK/STAT activation by the Drosophila Upd cytokines is pleiotropically involved in normal development of multiple organs. Whether inflammatory and developmental JAK/STAT roles intersect is unknown. This study shows that JAK/STAT is active during development of the prothoracic gland (PG), which controls metamorphosis onset through ecdysone production. Reducing JAK/STAT signaling decreased PG size and advanced metamorphosis. Conversely, JAK/STAT hyperactivation by overexpression of pathway components or SUMOylation loss caused PG hypertrophy and metamorphosis delay. Tissue damage and tumors, known to secrete Upd cytokines, also activated JAK/STAT in the PG and delayed metamorphosis, at least in part by inducing expression of the JAK/STAT target Apontic. JAK/STAT damage signaling, therefore, regulates metamorphosis onset by co-opting its developmental role in the PG. These findings in Drosophila provide insights on how systemic effects of damage and cancer can interfere with hormonally controlled development and developmental transitions.
Yang, S., Wu, X., Daoutidou, E. I., Zhang, Y., Shimell, M., Chuang, K. H., Peterson, A. J., O'Connor, M. B. and Zheng, X. (2022). The NDNF-like factor Nord is a Hedgehog-induced extracellular BMP modulator that regulates Drosophila wing patterning and growth. Elife 11. PubMed ID: 35037619
Summary:
Hedgehog (Hh) and bone morphogenetic proteins (BMPs) pattern the developing Drosophila wing by functioning as short- and long-range morphogens, respectively. This study shows that a previously unknown Hh-dependent mechanism fine-tunes the activity of BMPs. Through genome-wide expression profiling of the Drosophila wing imaginal discs, this study identified nord as a novel target gene of the Hh signaling pathway. Nord is related to the vertebrate Neuron Derived Neurotrophic Factor (NDNF) involved in Congenital Hypogonadotropic Hypogonadism and several types of cancer. Loss- and gain-of-function analyses implicate Nord in the regulation of wing growth and proper crossvein patterning. At the molecular level, biochemical evidence ia presented that Nord is a secreted BMP-binding protein and localizes to the extracellular matrix. Nord binds to Decapentaplegic (Dpp) or the heterodimer Dpp-Glass bottom boat (Gbb) to modulate their release and activity. Furthermore, this study demonstrates that Nord is a dosage-depend BMP modulator, where low levels of Nord promote and high levels inhibit BMP signaling. Taken together, it is proposed that Hh-induced Nord expression fine tunes both the range and strength of BMP signaling in the developing Drosophila wing.
Chen, Y., Liu, T., Shen, J. and Zhang, J. (2021). Phenotypical and genetical characterization of the Mad(1-2) allele during Drosophila wing development. Cells Dev 169: 203761. PubMed ID: 34875394
Summary:
Growth and patterning of Drosophila wing depends upon the sequential organizing activities of Hedgehog (Hh) and Decapentaplegic (Dpp) signaling pathways. The Hh signaling directly activates the expression of dpp through the transcription factor Cubitus interruptus (Ci). Dpp itself functions as a long-range morphogen to promote cell proliferation and differentiation through an essential transcription factor encoded by Mad. This study reports that the Mad1-2 allele exhibits phenotypes distinct from classical Dpp pathway mutants in the developing wing. The activity of Dpp signaling is attenuated in Mad1-2 mutant cells. However, activation of Dpp signaling is found in a subset of cells surrounding homozygous Mad1-2 clones when the clones are located at the anterior compartment of wing disc. Further analysis reveals that Mad1-2 mutant cells display high level of Hh signaling activity and accumulate significant amount of Ci. Unexpectedly, whole genome resequencing identifies multiple mutations in the 3'UTR region of Pka-C1 genomic loci in the Mad1-2 stock. Genetic and molecular evidence is provided that the Pka-C1 mutations carried by Mad1-2 likely underlies the observed Hh signaling defects. Therefore, the contribution of Pka-C1 mutation should be taken in consideration when analyzing Mad1-2 phenotypes. The isolation of independent Mad and Pka-C1 alleles from the Mad1-2 stock further supports these conclusions.
Goyal, M., Tomar, A., Madhwal, S. and Mukherjee, T. (2022). Blood progenitor redox homeostasis through olfaction-derived systemic GABA in hematopoietic growth control in Drosophila. Development 149(8). PubMed ID: 34850846
Summary:
The role of reactive oxygen species (ROS) in myeloid development is well established. However, its aberrant generation alters hematopoiesis. Thus, a comprehensive understanding of events controlling ROS homeostasis forms the central focus of this study. It was shown that, in homeostasis, myeloid-like blood progenitor cells of the Drosophila larvae, which reside in a specialized hematopoietic organ termed the lymph gland, use TCA (the Krebs cycle) to generate ROS. However, excessive ROS production leads to lymph gland growth retardation. Therefore, to moderate blood progenitor ROS, Drosophila larvae rely on olfaction and its downstream systemic GABA. GABA internalization and its breakdown into succinate by progenitor cells activates pyruvate dehydrogenase kinase (PDK), which controls inhibitory phosphorylation of pyruvate dehydrogenase (PDH). PDH is the rate-limiting enzyme that connects pyruvate to the TCA cycle and to oxidative phosphorylation. Thus, GABA metabolism via PDK activation maintains TCA activity and blood progenitor ROS homeostasis, and supports normal lymph gland growth. Consequently, animals that fail to smell also fail to sustain TCA activity and ROS homeostasis, which leads to lymph gland growth retardation. Overall, this study describes the requirement of animal odor-sensing and GABA in myeloid ROS regulation and hematopoietic growth control.
Ding, Y., Wang, G., Zhan, M., Sun, X., Deng, Y., Zhao, Y., Liu, B., Liu, Q., Wu, S. and Zhou, Z. (2021). Hippo signaling suppresses tumor cell metastasis via a Yki-Src42A positive feedback loop. Cell Death Dis 12(12): 1126. PubMed ID: 34862372
Summary:
Metastasis is an important cause of death from malignant tumors. It is of great significance to explore the molecular mechanism of metastasis for the development of anti-cancer drugs. This study found that the Hippo pathway hampers tumor cell metastasis in vivo. Silence of hpo or its downstream wts promotes tumor cell migration in a Yki-dependent manner. Furthermore, inhibition of the Hippo pathway promotes tumor cell migration through transcriptional activating src42A, a Drosophila homolog of the SRC oncogene. Yki activates src42A transcription through direct binding its intron region. Intriguingly, Src42A further increases Yki transcriptional activity to form a positive feedback loop. Finally, it was shown that SRC is also a target of YAP and important for YAP to promote the migration of human hepatocellular carcinoma cells. Together, these findings uncover a conserved Yki/YAP-Src42A/SRC positive feedback loop promoting tumor cell migration and provide SRC as a potential therapeutic target for YAP-driven metastatic tumors.
Hu, D. J., Yun, J., Elstrott, J. and Jasper, H. (2021). Non-canonical Wnt signaling promotes directed migration of intestinal stem cells to sites of injury. Nat Commun 12(1): 7150. PubMed ID: 34887411
Summary:
Tissue regeneration after injury requires coordinated regulation of stem cell activation, division, and daughter cell differentiation, processes that are increasingly well understood in many regenerating tissues. How accurate stem cell positioning and localized integration of new cells into the damaged epithelium are achieved, however, remains unclear. This study shows that enteroendocrine cells coordinate stem cell migration towards a wound in the Drosophila intestinal epithelium. In response to injury, enteroendocrine cells release the N-terminal domain of the PTK7 orthologue, Otk, which activates non-canonical Wnt signaling in intestinal stem cells, promoting actin-based protrusion formation and stem cell migration towards a wound. This migratory behavior is closely linked to proliferation, and that it is required for efficient tissue repair during injury. These findings highlight the role of non-canonical Wnt signaling in regeneration of the intestinal epithelium, and identify enteroendocrine cell-released ligands as critical coordinators of intestinal stem cell migration.

Wednesday, March 9th - Synapse and Vesicles

Astacio, H., Vasin, A. and Bykhovskaia, M. (2021). Stochastic Properties of Spontaneous Synaptic Transmission at Individual Active Zones. J Neurosci. PubMed ID: 34969867
Summary:
Employing postsynaptically tethered calcium sensor GCaMP, this study investigated spontaneous synaptic transmission at individual active zones (AZs) at the Drosophila (both sexes) neuromuscular junction. Optical monitoring of GCaMP events coupled with focal electrical recordings of synaptic currents revealed "hot spots" of spontaneous transmission, which corresponded to transient states of elevated activity at selected AZs. The elevated spontaneous activity had two temporal components, one at a timescale of minutes and the other at a sub-second timescale. A three-state model was developed of AZ preparedness for spontaneous transmission, and Monte-Carlo simulations were performed of the release process, which produced an accurate quantitative description of the variability and time-course of spontaneous transmission at individual AZs. To investigate the mechanisms of elevated activity, this study first focused on the protein complexin, which binds the SNARE protein complex and serves to clamp spontaneous fusion. Overexpression of Drosophila complexin largely abolished the high-activity states of AZs, while complexin deletion drastically promoted it. A mutation in the SNARE protein Syntaxin-1A had an effect similar to complexin deficiency, promoting the high-activity state. Next, how presynaptic Ca(2+) transients affect the states of elevated activity at individual AZs was tested. Ca(2+) influx was either blocked or promoted pharmacologically, and also Ca(2+) release from internal stores was promoted. These experiments coupled with computations revealed that Ca(2+) transients can trigger bursts of spontaneous events from individual AZs or AZ clusters at a sub-second timescale. Altogether, these results demonstrated that spontaneous transmission is highly heterogeneous, with transient hot spots being regulated by the SNARE machinery and Ca(2+).
Paul, M. M., Dannhauser, S., Morris, L., ..., Vakonakis, I., Heckmann, M. and Langenhan, T. (2022). The human cognition-enhancing CORD7 mutation increases active zone number and synaptic release. Brain. PubMed ID: 35022694
Summary:
Humans carrying the CORD7 (cone-rod dystrophy 7) mutation possess increased verbal IQ and working memory. This autosomal dominant syndrome is caused by the single-amino acid R844H exchange (human numbering) located in the 310 helix of the C2A domain of RIMS1/RIM1 (Rab3-interacting molecule 1). RIM is an evolutionarily conserved multi-domain protein and essential component of presynaptic active zones, which is centrally involved in fast, Ca2+-triggered neurotransmitter release. How the CORD7 mutation affects synaptic function has remained unclear thus far. This study established Drosophila as a disease model for clarifying the effects of the CORD7 mutation on RIM function and synaptic vesicle release. Using X-ray crystallography, the molecular structure of the Drosophila C2A domain was resolved at 1.92 Å resolution and by comparison to its mammalian homolog ascertained that the location of the CORD7 mutation is structurally conserved in fly RIM. rim alleles were generated encoding the R915H CORD7 exchange or R915E,R916E substitutions (fly numbering) to effect local charge reversal at the 310 helix. The CORD7 mutation exerts a semi-dominant rather than a dominant effect on synaptic transmission resulting in faster, more efficient synaptic release and increased size of the readily releasable pool but decreased sensitivity for the fast calcium chelator BAPTA. In addition, the rim CORD7 allele increased the number of presynaptic active zones but left their nanoscopic organization unperturbed as revealed by super-resolution microscopy of the presynaptic scaffold protein Bruchpilot/ELKS/CAST. It is conclude that the CORD7 mutation leads to tighter release coupling, an increased readily releasable pool size and more release sites thereby promoting more efficient synaptic transmitter release.
Gaspar, C. J., Vieira, L. C., Santos, C. C., Christianson, J. C., Jakubec, D., Strisovsky, K., Adrain, C. and Domingos, P. M. (2022). EMC is required for biogenesis of Xport-A, an essential chaperone of Rhodopsin-1 and the TRP channel. EMBO Rep 23(1): e53210. PubMed ID: 34918864
Summary:
The ER membrane protein complex (EMC) is required for the biogenesis of a subset of tail anchored (TA) and polytopic membrane proteins, including Rhodopsin-1 (Rh1) and the TRP channel. To understand the physiological implications of EMC-dependent membrane protein biogenesis, this study performed a bioinformatic identification of Drosophila TA proteins. From 254 predicted TA proteins, screening in larval eye discs identified two proteins that require EMC for their biogenesis: fan and Xport-A. Fan is required for male fertility in Drosophila, and EMC was also shown to be required for this process. Xport-A is essential for the biogenesis of both Rh1 and TRP, raising the possibility that disruption of Rh1 and TRP biogenesis in EMC mutants is secondary to the Xport-A defect. EMC is required for Xport-A TMD membrane insertion and EMC-independent Xport-A mutants rescue Rh1 and TRP biogenesis in EMC mutants. Finally, this work also reveals a role for Xport-A in a glycosylation-dependent triage mechanism during Rh1 biogenesis in the endoplasmic reticulum.
Graham, L. C., Kline, R. A., Lamont, D. J., Gillingwater, T. H., Mabbott, N. A., Skehel, P. A. and Wishart, T. M. (2021). Temporal Profiling of the Cortical Synaptic Mitochondrial Proteome Identifies Ageing Associated Regulators of Stability. Cells 10(12). PubMed ID: 34943911
Summary:
Synapses are particularly susceptible to the effects of advancing age, and mitochondria have long been implicated as organelles contributing to this compartmental vulnerability. Despite this, the mitochondrial molecular cascades promoting age-dependent synaptic demise remain to be elucidated. This study sought to examine how the synaptic mitochondrial proteome (including strongly mitochondrial associated proteins) was dynamically and temporally regulated throughout ageing to determine whether alterations in the expression of individual candidates can influence synaptic stability/morphology. Proteomic profiling of wild-type mouse cortical synaptic and non-synaptic mitochondria across the lifespan revealed significant age-dependent heterogeneity between mitochondrial subpopulations, with aged organelles exhibiting unique protein expression profiles. Recapitulation of aged synaptic mitochondrial protein expression at the Drosophila neuromuscular junction has the propensity to perturb the synaptic architecture, demonstrating that temporal regulation of the mitochondrial proteome may directly modulate the stability of the synapse in vivo.
Rios-Barrera, L. D. and Leptin, M. (2022). An endosome-associated actin network involved in directed apical plasma membrane growth. J Cell Biol 221(3). PubMed ID: 35061016
Summary:
Membrane trafficking plays many roles in morphogenesis, from bulk membrane provision to targeted delivery of proteins and other cargos. In tracheal terminal cells of the Drosophila respiratory system, transport through late endosomes balances membrane delivery between the basal plasma membrane and the apical membrane, which forms a subcellular tube, but it has been unclear how the direction of growth of the subcellular tube with the overall cell growth is coordinated. This study shows that endosomes also organize F-actin. Actin assembles around late endocytic vesicles in the growth cone of the cell, reaching from the tip of the subcellular tube to the leading filopodia of the basal membrane. Preventing nucleation of endosomal actin disturbs the directionality of tube growth, uncoupling it from the direction of cell elongation. Severing actin in this area affects tube integrity. These findings show a new role for late endosomes in directing morphogenesis by organizing actin, in addition to their known role in membrane and protein trafficking.
Crosby, D., Mikolaj, M. R., Nyenhuis, S. B., Bryce, S., Hinshaw, J. E. and Lee, T. H. (2022). Reconstitution of human atlastin fusion activity reveals autoinhibition by the C terminus. J Cell Biol 221(2). PubMed ID: 34817557
Summary:
ER network formation depends on membrane fusion by the atlastin (ATL) GTPase. In humans, three paralogs are differentially expressed with divergent N- and C-terminal extensions, but their respective roles remain unknown. This is partly because, unlike Drosophila ATL, the fusion activity of human ATLs has not been reconstituted. This study reports successful reconstitution of fusion activity by the human ATLs. Unexpectedly, the major splice isoforms of ATL1 and ATL2 are each autoinhibited, albeit to differing degrees. For the more strongly inhibited ATL2, autoinhibition mapped to a C-terminal α-helix is predicted to be continuous with an amphipathic helix required for fusion. Charge reversal of residues in the inhibitory domain strongly activated its fusion activity, and overexpression of this disinhibited version caused ER collapse. Neurons express an ATL2 splice isoform whose sequence differs in the inhibitory domain, and this form showed full fusion activity. These findings reveal autoinhibition and alternate splicing as regulators of atlastin-mediated ER fusion.

Tuesday, March 8th - Methods

Dorkenwald, S., McKellar, C. E., Macrina, T., Kemnitz, N., Lee, K., Lu, R., Wu, J., Popovych, S., Mitchell, E., Nehoran, B., Jia, Z., Bae, J. A., Mu, S., Ih, D., Castro, M., Ogedengbe, O., Halageri, A., Kuehner, K., Sterling, A. R., Ashwood, Z., Zung, J., Brittain, D., Collman, F., Schneider-Mizell, C., Jordan, C., Silversmith, W., Baker, C., Deutsch, D., Encarnacion-Rivera, L., Kumar, S., Burke, A., Bland, D., Gager, J., Hebditch, J., Koolman, S., Moore, M., Morejohn, S., Silverman, B., Willie, K., Willie, R., Yu, S. C., Murthy, M. and Seung, H. S. (2022). FlyWire: online community for whole-brain connectomics. Nat Methods 19(1): 119-128. PubMed ID: 34949809
Summary:
Due to advances in automated image acquisition and analysis, whole-brain connectomes with 100,000 or more neurons are on the horizon. Proofreading of whole-brain automated reconstructions will require many person-years of effort, due to the huge volumes of data involved. This paper presents FlyWire, an online community for proofreading neural circuits in a Drosophila melanogaster brain and explain how its computational and social structures are organized to scale up to whole-brain connectomics. Browser-based three-dimensional interactive segmentation by collaborative editing of a spatially chunked supervoxel graph makes it possible to distribute proofreading to individuals located virtually anywhere in the world. Information in the edit history is programmatically accessible for a variety of uses such as estimating proofreading accuracy or building incentive systems. An open community accelerates proofreading by recruiting more participants and accelerates scientific discovery by requiring information sharing. It is demonstrated how FlyWire enables circuit analysis by reconstructing and analyzing the connectome of mechanosensory neurons.
Bahuguna, S., Redhai, S., Zhou, J., Wang, T., Port, F. and Boutros, M. (2021). Conditional CRISPR-Cas Genome Editing in Drosophila to Generate Intestinal Tumors. Cells 10(11). PubMed ID: 34831379
Summary:
CRISPR-Cas has revolutionized genetics and extensive efforts have been made to enhance its editing efficiency by developing increasingly more elaborate tools. This study evaluated the CRISPR-Cas9 system in Drosophila melanogaster to assess its ability to induce stem cell-derived tumors in the intestine. Conditional tissue-specific CRISPR knockouts were generated using different Cas9 expression vectors with guide RNAs targeting the BMP, Notch, and JNK pathways in intestinal progenitors such as stem cells (ISCs) and enteroblasts (EBs). Perturbing Notch and BMP signaling increased the proliferation of ISCs/EBs and resulted in the formation of intestinal tumors, albeit with different efficiencies. By assessing both the anterior and posterior regions of the midgut, regional differences were observed in ISC/EB proliferation and tumor formation upon mutagenesis. Surprisingly, high continuous expression of Cas9 in ISCs/EBs blocked age-dependent increase in ISCs/EBs proliferation and when combined with gRNAs targeting tumor suppressors, it prevented tumorigenesis. However, no such effects were seen when temporal parameters of Cas9 were adjusted to regulate its expression levels or with a genetically modified version, which expresses Cas9 at lower levels, suggesting that fine-tuning Cas9 expression is essential to avoid deleterious effects. These findings suggest that modifications to Cas9 expression results in differences in editing efficiency and careful considerations are required when choosing reagents for CRISPR-Cas9 mutagenesis studies. In summary, Drosophila can serve as a powerful model for context-dependent CRISPR-Cas based perturbations and to test genome-editing systems in vivo.
Gamez, S., Chaverra-Rodriguez, D., Buchman, A., Kandul, N. P., Mendez-Sanchez, S. C., Bennett, J. B., Sanchez, C. H., Yang, T., Antoshechkin, I., Duque, J. E., Papathanos, P. A., Marshall, J. M. and Akbari, O. S. (2021). Exploiting a Y chromosome-linked Cas9 for sex selection and gene drive. Nat Commun 12(1): 7202. PubMed ID: 34893590
Summary:
CRISPR-based genetic engineering tools aimed to bias sex ratios, or drive effector genes into animal populations, often integrate the transgenes into autosomal chromosomes. However, in species with heterogametic sex chromsomes (e.g. XY, ZW), sex linkage of endonucleases could be beneficial to drive the expression in a sex-specific manner to produce genetic sexing systems, sex ratio distorters, or even sex-specific gene drives, for example. To explore this possibility, this study developed a transgenic line of Drosophila melanogaster expressing Cas9 from the Y chromosome. The utility of this strain was functionally characterized for both sex selection and gene drive finding it to be quite effective. To explore its utility for population control, mathematical models were build illustrating its dynamics as compared to other state-of-the-art systems designed for both population modification and suppression. Taken together, these results contribute to the development of current CRISPR genetic control tools and demonstrate the utility of using sex-linked Cas9 strains for genetic control of animals.
Goldsmith, S. L., Shimell, M. J., Tauscher, P., Daly, S. M., Shimmi, O., O'Connor, M. B. and Newfeld, S. J. (2022). New resources for the Drosophila 4th chromosome: FRT101F enabled mitotic clones and Bloom syndrome helicase enabled meiotic recombination. G3 (Bethesda). PubMed ID: 35084488
Summary:
Genes on the long arm of the Drosophila melanogaster 4th chromosome are difficult to study because the chromosome lacks mitotic and meiotic recombination. Without recombination numerous standard methods of genetic analysis are impossible. This study reports new resources for the 4th. For mitotic recombination a chromosome was generated with an FRT very near the centromere in 101F, and a derivative carries FRT101F with a distal ubiquitously expressed GAL80 transgene. This pair of chromosomes enables both unmarked and MARCM clones. For meiotic recombination it was demonstrates that a Bloom syndrome helicase and recombination defective double mutant genotype can create recombinant 4th chromosomes via female meiosis. All strains will be available to the community via the Bloomington Drosophila Stock Center. Additional resources for studies of the 4th are in preparation and will also be made available. The goal of the 4th Chromosome Resource Project is to accelerate the genetic analysis of protein coding genes on the 4th, including the 44 genes with no demonstrated function. Studies of these previously inaccessible but largely conserved genes will close longstanding gaps in knowledge of metazoan development and physiology.
di Pietro, F., Herszterg, S., Huang, A., Bosveld, F., Alexandre, C., Sancere, L., Pelletier, S., Joudat, A., Kapoor, V., Vincent, J. P. and Bellaiche, Y. (2021). Rapid and robust optogenetic control of gene expression in Drosophila. Dev Cell 56(24): 3393-3404.e3397. PubMed ID: 34879263Summary:
Deciphering gene function requires the ability to control gene expression in space and time. Binary systems such as the Gal4/UAS provide a powerful means to modulate gene expression and to induce loss or gain of function. This is best exemplified in Drosophila, where the Gal4/UAS system has been critical to discover conserved mechanisms in development, physiology, neurobiology, and metabolism, to cite a few. This study describe a transgenic light-inducible Gal4/UAS system (ShineGal4/UAS) based on Magnet photoswitches. It was shown to allow efficient, rapid, and robust activation of UAS-driven transgenes in different tissues and at various developmental stages in Drosophila. Furthermore, how ShineGal4 enables the generation of gain and loss-of-function phenotypes at animal, organ, and cellular levels is illustrated. Thanks to the large repertoire of UAS-driven transgenes, ShineGal4 enriches the Drosophila genetic toolkit by allowing in vivo control of gene expression with high temporal and spatial resolutions.
Xu, J., Kim, A. R., Cheloha, R. W., Fischer, F. A., Li, J. S. S., Feng, Y., Stoneburner, E., Binari, R., Mohr, S. E., Zirin, J., Ploegh, H. L. and Perrimon, N. (2022). Protein visualization and manipulation in Drosophila through the use of epitope tags recognized by nanobodies. Elife 11. PubMed ID: 35076390
Summary:
Expansion of the available repertoire of reagents for visualization and manipulation of proteins will help understand their function. Short epitope tags linked to proteins of interest and recognized by existing binders such as nanobodies facilitate protein studies by obviating the need to isolate new antibodies directed against them. Nanobodies have several advantages over conventional antibodies, as they can be expressed and used as tools for visualization and manipulation of proteins in vivo. This study characterized two short (<15 aa) NanoTag epitopes, 127D01 and VHH05, and their corresponding high-affinity nanobodies. Their use in Drosophila was demonstrated for in vivo protein detection and re-localization, direct and indirect immunofluorescence, immunoblotting, and immunoprecipitation. It was further shown that CRISPR-mediated gene targeting provides a straightforward approach to tagging endogenous proteins with the NanoTags. Single copies of the NanoTags, regardless of their location, suffice for detection. This versatile and validated toolbox of tags and nanobodies will serve as a resource for a wide array of applications, including functional studies in Drosophila and beyond.

Monday, March 9th - Adult neural development and function

Hao, S., Gestrich, J. Y., Zhang, X., Xu, M., Wang, X., Liu, L. and Wei, H. (2021). Neurotransmitters Affect Larval Development by Regulating the Activity of Prothoracicotropic Hormone-Releasing Neurons in Drosophila melanogaster. Front Neurosci 15: 653858. PubMed ID: 34975366
Summary:
Ecdysone, an essential insect steroid hormone, promotes larval metamorphosis by coordinating growth and maturation. In Drosophila melanogaster, prothoracicotropic hormone (PTTH)-releasing neurons are considered to be the primary promoting factor in ecdysone biosynthesis. Recently, studies have reported that the regulatory mechanisms of PTTH release in Drosophila larvae are controlled by different neuropeptides, including allatostatin A and corazonin. However, it remains unclear whether neurotransmitters provide input to PTTH neurons and control the metamorphosis in Drosophila larvae. This study reports that the neurotransmitters acetylcholine (ACh) affect larval development by modulating the activity of PTTH neurons. By downregulating the expression of different subunits of nicotinic ACh receptors in PTTH neurons, pupal volume was significantly increased, whereas pupariation timing was relatively unchanged. It was also identified that PTTH neurons were excited by ACh application ex vivo in a dose-dependent manner via ionotropic nicotinic ACh receptors. Moreover, in Ca(2+) imaging experiments, relatively low doses of OA caused increased Ca(2+) levels in PTTH neurons, whereas higher doses led to decreased Ca(2+) levels. It was also demonstrated that a low dose of OA was conveyed through OA *betal-type receptors. Additionally, electrophysiological experiments revealed that PTTH neurons produced spontaneous activity in vivo, which provides the possibility of the bidirectional regulation, coming from neurons upstream of PTTH cells in Drosophila larvae. In summary, these findings indicate that several different neurotransmitters are involved in the regulation of larval metamorphosis by altering the activity of PTTH neurons in Drosophila.
Inami, S., Sato, T., Kurata, Y., Suzuki, Y., Kitamoto, T. and Sakai, T. (2021). Consolidation and maintenance of long-term memory involve dual functions of the developmental regulator Apterous in clock neurons and mushroom bodies in the Drosophila brain. PLoS Biol 19(12): e3001459. PubMed ID: 34860826
Summary:
Memory is initially labile but can be consolidated into stable long-term memory (LTM) that is stored in the brain for extended periods. Despite recent progress, the molecular and cellular mechanisms underlying the intriguing neurobiological processes of LTM remain incompletely understood. Using the Drosophila courtship conditioning assay as a memory paradigm, this study showed that the LIM homeodomain (LIM-HD) transcription factor Apterous (Ap), which is known to regulate various developmental events, is required for both the consolidation and maintenance of LTM. Interestingly, Ap is involved in these 2 memory processes through distinct mechanisms in different neuronal subsets in the adult brain. Ap and its cofactor Chip (Chi) are indispensable for LTM maintenance in the Drosophila memory center, the mushroom bodies (MBs). On the other hand, Ap plays a crucial role in memory consolidation in a Chi-independent manner in pigment dispersing factor (Pdf)-containing large ventral-lateral clock neurons (l-LNvs) that modulate behavioral arousal and sleep. Since disrupted neurotransmission and electrical silencing in clock neurons impair memory consolidation, Ap is suggested to contribute to the stabilization of memory by ensuring the excitability of l-LNvs. Indeed, ex vivo imaging revealed that a reduced function of Ap, but not Chi, results in exaggerated Cl- responses to the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) in l-LNvs, indicating that wild-type (WT) Ap maintains high l-LNv excitability by suppressing the GABA response. Consistently, enhancing the excitability of l-LNvs by knocking down GABAA receptors compensates for the impaired memory consolidation in ap null mutants. Overall, these results revealed unique dual functions of the developmental regulator Ap for LTM consolidation in clock neurons and LTM maintenance in MBs.
Ganguly, A., Chandel, A., Turner, H., Wang, S., Liman, E. R. and Montell, C. (2021). Requirement for an Otopetrin-like protein for acid taste in Drosophila. Proc Natl Acad Sci U S A 118(51). PubMed ID: 34911758
Summary:
Receptors for bitter, sugar, and other tastes have been identified in the fruit fly Drosophila melanogaster, while a broadly tuned receptor for the taste of acid has been elusive. Previous work showed that such a receptor was unlikely to be encoded by a gene within one of the two major families of taste receptors in Drosophila, the "gustatory receptors" and "ionotropic receptors." To identify the acid taste receptor, this study tested the contributions of genes encoding proteins distantly related to the mammalian Otopertrin1 (OTOP1) proton channel that functions as a sour receptor in mice. RNA interference (RNAi) knockdown or mutation by CRISPR/Cas9 of one of the genes, Otopetrin-Like A (OtopLA), but not of the others (OtopLB or OtopLC) severely impaired the behavioral rejection to a sweet solution laced with high levels of HCl or carboxylic acids and greatly reduced acid-induced action potentials measured from taste hairs. An isoform of OtopLA that was isolated from the proboscis was sufficient to restore behavioral sensitivity and acid-induced action potential firing in OtopLA mutant flies. At lower concentrations, HCl was attractive to the flies, and this attraction was abolished in the OtopLA mutant. Cell type-specific rescue experiments showed that OtopLA functions in distinct subsets of gustatory receptor neurons for repulsion and attraction to high and low levels of protons, respectively. This work highlights a functional conservation of a sensory receptor in flies and mammals and shows that the same receptor can function in both appetitive and repulsive behaviors.
Hamid, R., Sant, H. S. and Kulkarni, M. N. (2021). Choline Transporter regulates olfactory habituation via a neuronal triad of excitatory, inhibitory and mushroom body neurons. PLoS Genet 17(12): e1009938. PubMed ID: 34914708
Summary:
Choline is an essential component of Acetylcholine (ACh) biosynthesis pathway which requires high-affinity Choline transporter (ChT) for its uptake into the presynaptic terminals of cholinergic neurons. A previous study had reported a predominant expression of ChT in memory processing and storing region of the Drosophila brain called mushroom bodies (MBs). It is unknown how ChT contributes to the functional principles of MB operation. This study demonstrated the role of ChT in Habituation, a non-associative form of learning. Odour driven habituation traces are laid down in ChT dependent manner in antennal lobes (AL), projection neurons (PNs), and MBs. Reduced habituation due to knock-down of ChT in MBs causes hypersensitivity towards odour, suggesting that ChT also regulates incoming stimulus suppression. Importantly, it was show for the first time that ChT is not unique to cholinergic neurons but is also required in inhibitory GABAergic neurons to drive habituation behaviour. These results support a model in which ChT regulates both habituation and incoming stimuli through multiple circuit loci via an interplay between excitatory and inhibitory neurons. Strikingly, the lack of ChT in MBs shows characteristics similar to the major reported features of Autism spectrum disorders (ASD), including attenuated habituation, sensory hypersensitivity as well as defective GABAergic signalling. These data establish the role of ChT in habituation and suggest that its dysfunction may contribute to neuropsychiatric disorders like ASD.
Del Castillo, U., Norkett, R., Lu, W., Serpinskaya, A. and Gelfand, V. I. (2022). Ataxin-2 is essential for cytoskeletal dynamics and neurodevelopment in Drosophila. iScience 25(1): 103536. PubMed ID: 34977501
Summary:
Ataxin-2 (Atx2) is a highly conserved RNA binding protein. Atx2 undergoes polyglutamine expansion leading to amyotrophic lateral sclerosis (ALS) or spinocerebellar ataxia type 2 (SCA2). However, the physiological functions of Atx2 in neurons remain unknown. Using the powerful genetics of Drosophila this study shows that Atx2 is essential for normal neuronal cytoskeletal dynamics and organelle trafficking. Upon neuron-specific Atx2 loss, the microtubule and actin networks were abnormally stabilized and cargo transport was drastically inhibited. Depletion of Atx2 caused multiple morphological defects in the nervous system of third instar larvae. These include reduced brain size, impaired axon development, and decreased dendrite outgrowth. Defects in the nervous system caused loss of the ability to crawl and lethality at the pupal stage. Taken together, these data mark Atx2 as a major regulator of cytoskeletal dynamics and denote Atx2 as an essential gene in neurodevelopment, as well as a neurodegenerative factor.
Feng, K. L., Weng, J. Y., Chen, C. C., Abubaker, M. B., Lin, H. W., Charng, C. C., Lo, C. C., de Belle, J. S., Tully, T., Lien, C. C. and Chiang, A. S. (2021). Neuropeptide F inhibits dopamine neuron interference of long-term memory consolidation in Drosophila. iScience 24(12): 103506. PubMed ID: 34934925
Summary:
Long-term memory (LTM) formation requires consolidation processes to overcome interfering signals that erode memory formation. Olfactory memory in Drosophila involves convergent projection neuron (PN; odor) and dopaminergic neuron (DAN; reinforcement) input to the mushroom body (MB). How post-training DAN activity in the posterior lateral protocerebrum (PPL1) continues to regulate memory consolidation remains unknown. This study addressed this question using targeted transgenes in behavior and electrophysiology experiments to show that (1) persistent post-training activity of PPL1-α2α'2 and PPL1-α3 DANs interferes with aversive LTM formation; (2) neuropeptide F (NPF) signaling blocks this interference in PPL1-α2α'2 and PPL1-α3 DANs after spaced training to enable LTM formation; and (3) training-induced NPF release and neurotransmission from two upstream dorsal-anterior-lateral (DAL2) neurons are required to form LTM. Thus, NPF signals from DAL2 neurons to specific PPL1 DANs disinhibit the memory circuit, ensuring that periodic events are remembered as consolidated LTM.

Friday, March 4th - Cytoskeleton and Junctions

Banerjee, R., Chakraborty, P., Yu, M. C. and Gunawardena, S. (2021). A stop or go switch: glycogen synthase kinase 3β phosphorylation of the kinesin 1 motor domain at Ser314 halts motility without detaching from microtubules. Development 148(24). PubMed ID: 34940839
Summary:
It is more than 25 years since the discovery that kinesin 1 is phosphorylated by several protein kinases. However, fundamental questions still remain as to how specific protein kinase(s) contribute to particular motor functions under physiological conditions. Because, within an whole organism, kinase cascades display considerable crosstalk and play multiple roles in cell homeostasis, deciphering which kinase(s) is/are involved in a particular process has been challenging. Previous work found that GSK3β plays a role in motor function. This study reports that a particular site on kinesin 1 motor domain (KHC), S314, is phosphorylated by GSK3β in vivo. The GSK3β-phosphomimetic-KHCS314D stalled kinesin 1 motility without dissociating from microtubules, indicating that constitutive GSK3β phosphorylation of the motor domain acts as a STOP. In contrast, uncoordinated mitochondrial motility was observed in CRISPR/Cas9-GSK3β non-phosphorylatable-KHCS314A Drosophila larval axons, owing to decreased kinesin 1 attachment to microtubules and/or membranes, and reduced ATPase activity. Together, it is proposed that GSK3β phosphorylation fine-tunes kinesin 1 movement in vivo via differential phosphorylation, unraveling the complex in vivo regulatory mechanisms that exist during axonal motility of cargos attached to multiple kinesin 1 and dynein motors.
Zajac, A. L. and Horne-Badovinac, S. (2022). Kinesin-directed secretion of basement membrane proteins to a subdomain of the basolateral surface in Drosophila epithelial cells. Curr Biol. PubMed ID: 35021047
Summary:
Epithelial tissues are lined with a sheet-like basement membrane (BM) extracellular matrix at their basal surfaces that plays essential roles in adhesion and signaling. BMs also provide mechanical support to guide morphogenesis. Despite their importance, little is known about how epithelial cells secrete and assemble BMs during development. BM proteins are sorted into a basolateral secretory pathway distinct from other basolateral proteins. Because BM proteins self-assemble into networks, and the BM lines only a small portion of the basolateral domain, it was hypothesized that the site of BM protein secretion might be tightly controlled. Using the Drosophila follicular epithelium, this study shows that kinesin-3 and kinesin-1 motors work together to define this secretion site. Similar to all epithelia, the follicle cells have polarized microtubules (MTs) along their apical-basal axes. These cells collectively migrate, and they also have polarized MTs along the migratory axis at their basal surfaces. This study found follicle cell MTs form one interconnected network, which allows kinesins to transport Rab10+ BM secretory vesicles both basally and to the trailing edge of each cell. This positions them near the basal surface and the basal-most region of the lateral domain for exocytosis. When kinesin transport is disrupted, the site of BM protein secretion is expanded, and ectopic BM networks form between cells that impede migration and disrupt tissue architecture. These results show how epithelial cells can define a subdomain on their basolateral surface through MT-based transport and highlight the importance of controlling the exocytic site of network-forming proteins.
Saunders, H. A. J., Johnson-Schlitz, D. M., Jenkins, B. V., Volkert, P. J., Yang, S. Z. and Wildonger, J. (2022). Acetylated alpha-tubulin K394 regulates microtubule stability to shape the growth of axon terminals. Curr Biol. PubMed ID: 35081332
Summary:
Microtubules are essential to neuron shape and function. Acetylation of tubulin has the potential to directly tune the behavior and function of microtubules in cells. Although proteomic studies have identified several acetylation sites in α-tubulin, the effects of acetylation at these sites remains largely unknown. This includes the highly conserved residue lysine 394 (K394), which is located at the α-tubulin dimer interface. Using a fly model, this study showed that α-tubulin K394 is acetylated in the nervous system and is an essential residue. Acetylation-blocking mutation in endogenous α-tubulin, K394R, was found to perturb the synaptic morphogenesis of motoneurons and reduces microtubule stability. Intriguingly, the K394R mutation has opposite effects on the growth of two functionally and morphologically distinct motoneurons, revealing neuron-type-specific responses when microtubule stability is altered. Eliminating the deacetylase HDAC6 increases K394 acetylation, and the over-expression of HDAC6 reduces microtubule stability similar to the K394R mutant. Thus, these findings implicate α-tubulin K394 and its acetylation in the regulation of microtubule stability and suggest that HDAC6 regulates K394 acetylation during synaptic morphogenesis.
Doerflinger, H., Zimyanin, V. and St Johnston, D. (2022). The Drosophila anterior-posterior axis is polarized by asymmetric myosin activation. Curr Biol 32(2): 374-385. PubMed ID: 34856125
Summary:
The Drosophila anterior-posterior axis is specified at mid-oogenesis when the Par-1 kinase is recruited to the posterior cortex of the oocyte, where it polarizes the microtubule cytoskeleton to define where the axis determinants, bicoid and oskar mRNAs, localize. This polarity is established in response to an unknown signal from the follicle cells, but how this occurs is unclear. This study shows that the myosin chaperone Unc-45 and non-muscle myosin II (MyoII) are required upstream of Par-1 in polarity establishment. Furthermore, the myosin regulatory light chain (MRLC) is di-phosphorylated at the oocyte posterior in response to the follicle cell signal, inducing longer pulses of myosin contractility at the posterior that may increase cortical tension. Overexpression of MRLC-T21A that cannot be di-phosphorylated or treatment with the myosin light-chain kinase inhibitor ML-7 abolishes Par-1 localization, indicating that the posterior of MRLC di-phosphorylation is essential for both polarity establishment and maintenance. Thus, asymmetric myosin activation polarizes the anterior-posterior axis by recruiting and maintaining Par-1 at the posterior cortex. This raises an intriguing parallel with anterior-posterior axis formation in C. elegans, where MyoII also acts upstream of the PAR proteins to establish polarity, but to localize the anterior PAR proteins rather than Par-1.
Founounou, N., Farhadifar, R., Collu, G. M., Weber, U., Shelley, M. J. and Mlodzik, M. (2021). Tissue fluidity mediated by adherens junction dynamics promotes planar cell polarity-driven ommatidial rotation. Nat Commun 12(1): 6974. PubMed ID: 34848713
Summary:
The phenomenon of tissue fluidity-cells' ability to rearrange relative to each other in confluent tissues-has been linked to several morphogenetic processes and diseases, yet few molecular regulators of tissue fluidity are known. Ommatidial rotation (OR), directed by planar cell polarity signaling, occurs during Drosophila eye morphogenesis and shares many features with polarized cellular migration in vertebrates. This study utilized in vivo live imaging analysis tools to quantify dynamic cellular morphologies during OR, revealing that OR is driven autonomously by ommatidial cell clusters rotating in successive pulses within a permissive substrate. Through analysis of a rotation-specific nemo mutant, this study demonstrated that precise regulation of junctional E-cadherin levels is critical for modulating the mechanical properties of the tissue to allow rotation to progress. This study defines Nemo as a molecular tool to induce a transition from solid-like tissues to more viscoelastic tissues broadening molecular understanding of tissue fluidity.
Baker, F. C., Neiswender, H., Veeranan-Karmegam, R. and Gonsalvez, G. B. (2021). In vivo proximity biotin ligation identifies the interactome of Egalitarian, a Dynein cargo adaptor. Development 148(22). PubMed ID: 35020877
Summary:
Numerous motors of the Kinesin family contribute to plus-end-directed microtubule transport. However, almost all transport towards the minus-end of microtubules involves Dynein. Understanding the mechanism by which Dynein transports this vast diversity of cargo is the focus of intense research. In selected cases, adaptors that link a particular cargo with Dynein have been identified. However, the sheer diversity of cargo suggests that additional adaptors must exist. This study used the Drosophila egg chamber as a model to address this issue. Within egg chambers, Egalitarian is required for linking mRNA with Dynein. However, in the absence of Egalitarian, Dynein transport into the oocyte is severely compromised. This suggests that additional cargoes might be linked to Dynein in an Egalitarian-dependent manner. This study therefore used proximity biotin ligation to define the interactome of Egalitarian. This approach yielded several novel interacting partners, including P body components and proteins that associate with Dynein in mammalian cells. A nanobody-based proximity biotinylation strategy was devised and validated that can be used to define the interactome of any GFP-tagged protein.

Thursday, March 3rd - Cell Cycle

Ovrebø, J. I., Bradley-Gill, M. R., Zielke, N., Kim, M., Marchetti, M., Bohlen, J., Lewis, M., van Straaten, M., Moon, N. S. and Edgar, B. A. (2022). Translational control of E2f1 regulates the Drosophila cell cycle. Proc Natl Acad Sci U S A 119(4). PubMed ID: 35074910
Summary:
E2F transcription factors are master regulators of the eukaryotic cell cycle. In Drosophila, the sole activating E2F, E2F1, is both required for and sufficient to promote G1→S progression. E2F1 activity is regulated both by binding to RB Family repressors and by posttranscriptional control of E2F1 protein levels by the EGFR and TOR signaling pathways. This study investigated cis-regulatory elements in the E2f1 messenger RNA (mRNA) that enable E2f1 translation to respond to these signals and promote mitotic proliferation of wing imaginal disc and intestinal stem cells. Small upstream open reading frames (uORFs) in the 5' untranslated region (UTR) of the E2f1 mRNA limit its translation, impacting rates of cell proliferation. E2f1 transgenes lacking these 5'UTR uORFs caused TOR-independent expression and excess cell proliferation, suggesting that TOR activity can bypass uORF-mediated translational repression. EGFR signaling also enhanced translation but through a mechanism less dependent on 5'UTR uORFs. Further, a region in the E2f1 mRNA was mapped that contains a translational enhancer, which may also be targeted by TOR signaling. This study reveals translational control mechanisms through which growth signaling regulates cell cycle progression.
Ruchert, J. M., Brady, M. M., McMahan, S., Lacey, K. J., Latta, L. C., Sekelsky, J. and Stoffregen, E. P. (2021). Blm helicase facilitates rapid replication of repetitive DNA sequences in early Drosophila development. Genetics 220(1). PubMed ID: 34849849
Summary:
The absence of functional BLM DNA helicase, a member of the RecQ family of helicases, is responsible for the rare human disorder Bloom Syndrome. In Drosophila, Blm protein is essential during early development. Lack of functional maternal Blm during the syncytial cell cycles of Drosophila embryonic development results in severe nuclear defects and lethality. Amongst the small fraction of embryos from Blm mutant mothers that survive to adulthood, a prominent sex-bias favors the class that inherits less repetitive DNA content, which serves as an endogenous source of replication stress. This selection against repetitive DNA content reflects a role for Blm in facilitating replication through repetitive sequences during the rapid S-phases of syncytial cell cycles. During these syncytial cycles, the progeny sex-bias resulting from the absence of maternal Blm is exacerbated by repetitive DNA sequences and by the slowing of replication fork progression, suggesting that the essential role for Blm during this stage is to manage replication fork stress brought about by impediments to fork progression. Additionally, the data suggest that Blm is only required to manage this replication stress during embryonic development, and likely only during the early, rapid syncytial cell cycles.
Das, S., Caballero, M., Kolesnikova, T., Zhimulev, I., Koren, A. and Nordman, J. (2021). Replication timing analysis in polyploid cells reveals Rif1 uses multiple mechanisms to promote underreplication in Drosophila. Genetics 219(3). PubMed ID: 34740250
Summary:
Regulation of DNA replication and copy number is necessary to promote genome stability and maintain cell and tissue function. DNA replication is regulated temporally in a process known as replication timing (RT). Rap1-interacting factor 1 (Rif1) is a key regulator of RT and has a critical function in copy number control in polyploid cells. Previous work has demonstrated that Rif1 functions with SUUR to inhibit replication fork progression and promote underreplication (UR) of specific genomic regions. How Rif1-dependent control of RT factors into its ability to promote UR is unknown. By applying a computational approach to measure RT in Drosophila polyploid cells, this study showed that SUUR and Rif1 have differential roles in controlling UR and RT. The findings reveal that Rif1 acts to promote late replication, which is necessary for SUUR-dependent underreplication. This work provides new insight into the process of UR and its links to RT.
Ruiz-Losada, M., Gonzalez, R., Peropadre, A., Gil-Galvez, A., Tena, J. J., Baonza, A. and Estella, C. (2021). Coordination between cell proliferation and apoptosis after DNA damage in Drosophila. Cell Death Differ. PubMed ID: 34824391
Summary:
Exposure to genotoxic stress promotes cell cycle arrest and DNA repair or apoptosis. These "life" or "death" cell fate decisions often rely on the activity of the tumor suppressor gene p53. Therefore, the precise regulation of p53 is essential to maintain tissue homeostasis and to prevent cancer development. However, how cell cycle progression has an impact on p53 cell fate decision-making is mostly unknown. This work demonstrates that Drosophila p53 proapoptotic activity can be impacted by the G2/M kinase Cdk1. Cell cycle arrested or endocycle-induced cells were shown to be refractory to ionizing radiation-induced apoptosis. p53 binding to the regulatory elements of the proapoptotic genes was shown; its ability to activate their expression is compromised in experimentally arrested cells. These results indicate that p53 genetically and physically interacts with Cdk1 and that p53 proapoptotic role is regulated by the cell cycle status of the cell. A model is proposed in which cell cycle progression and p53 proapoptotic activity are molecularly connected to coordinate the appropriate response after DNA damage.
Kursel, L. E., Cope, H. D. and Rog, O. (2021). Unconventional conservation reveals structure-function relationships in the synaptonemal complex. Elife 10. PubMed ID: 34787570
Summary:
Functional requirements constrain protein evolution, commonly manifesting in a conserved amino acid sequence. This idea is extended to secondary structural features by tracking their conservation in essential meiotic proteins with highly diverged sequences. The synaptonemal complex (SC) is a ~100-nm-wide ladder-like meiotic structure present in all eukaryotic clades, where it aligns parental chromosomes and regulates exchanges between them. Despite the conserved ultrastructure and functions of the SC, SC proteins are highly divergent within Caenorhabditis. However, SC proteins have highly conserved length and coiled-coil domain structure. The same unconventional conservation signature was found in Drosophila and mammals, and it was used to identify a novel SC protein in Pristionchus pacificus, Ppa-SYP-1. This work suggests that coiled-coils play wide-ranging roles in the structure and function of the SC, and more broadly, that expanding sequence analysis beyond measures of per-site similarity can enhance understanding of protein evolution and function.
Losick, V. P. and Duhaime, L. G. (2021). The endocycle restores tissue tension in the Drosophila abdomen post wound repair. Cell Rep 37(2): 109827. PubMed ID: 34644579
Summary:
Polyploidy frequently arises in response to injury, aging, and disease. Despite its prevalence, major gaps exist in understanding of how polyploid cells alter tissue function. In the adult Drosophila epithelium, wound healing is dependent on the generation of multinucleated polyploid cells resulting in a permanent change in the epithelial architecture. This study shows how the wound-induced polyploid cells affect tissue function by altering epithelial mechanics. The mechanosensor nonmuscle myosin II is activated and upregulated in wound-induced polyploid cells and persists after healing completes. Polyploidy enhances relative epithelial tension, which is dependent on the endocycle and not cell fusion post injury. Remarkably, the enhanced epithelial tension mimics the relative tension of the lateral muscle fibers, which are permanently severed by the injury. As a result, this study found that the wound-induced polyploid cells remodel the epithelium to maintain fly abdominal movements, which may help compensate for lost tissue tension (Losick, 2021).

Wednesday, March 2nd - Embryonic Development

de-Carvalho, J., Tlili, S., Hufnagel, L., Saunders, T. E. and Telley, I. A. (2022). Aster repulsion drives short-ranged ordering in the Drosophila syncytial blastoderm. Development 149(2). PubMed ID: 35001104
Summary:
Biological systems are highly complex, yet notably ordered structures can emerge. During syncytial stage development of the Drosophila melanogaster embryo, nuclei synchronously divide for nine cycles within a single cell, after which most of the nuclei reach the cell cortex. The arrival of nuclei at the cortex occurs with remarkable positional order, which is important for subsequent cellularisation and morphological transformations. Yet, the mechanical principles underlying this lattice-like positional order of nuclei remain untested. Using quantification of nuclei position and division orientation together with embryo explants, this study shows that short-ranged repulsive interactions between microtubule asters ensure the regular distribution and maintenance of nuclear positions in the embryo. Such ordered nuclear positioning still occurs with the loss of actin caps and even the loss of the nuclei themselves; the asters can self-organise with similar distribution to nuclei in the wild-type embryo. The explant assay enabled deduction of the nature of the mechanical interaction between pairs of nuclei. This was used to predict how the nuclear division axis orientation changes upon nucleus removal from the embryo cortex, which was confirmed in vivo with laser ablation. Overall, this study shows that short-ranged microtubule-mediated repulsive interactions between asters are important for ordering in the early Drosophila embryo and minimising positional irregularity.
<Duk, M. A., Gursky, V. V., Samsonova, M. G. and Surkova, S. Y. (2021). Application of Domain- and Genotype-Specific Models to Infer Post-Transcriptional Regulation of Segmentation Gene Expression in Drosophila. Life (Basel) 11(11). PubMed ID: 34833107
Summary:
Unlike transcriptional regulation, the post-transcriptional mechanisms underlying zygotic segmentation gene expression in early Drosophila embryo have been insufficiently investigated. Condition-specific post-transcriptional regulation plays an important role in the development of many organisms. A recent study revealed the domain- and genotype-specific differences between mRNA and the protein expression of Drosophila hb, gt, and eve genes in cleavage cycle 14A. This study used this dataset and the dynamic mathematical model to recapitulate protein expression from the corresponding mRNA patterns. The condition-specific nonuniformity in parameter values is further interpreted in terms of possible post-transcriptional modifications. For hb expression in wild-type embryos, the results predict the position-specific differences in protein production. The protein synthesis rate parameter is significantly higher in hb anterior domain compared to the posterior domain. The parameter sets describing Gt protein dynamics in wild-type embryos and Kr mutants are genotype-specific. The spatial discrepancy between gt mRNA and protein posterior expression in Kr mutants is well reproduced by the whole axis model, thus rejecting the involvement of post-transcriptional mechanisms. These models fail to describe the full dynamics of eve expression, presumably due to its complex shape and the variable time delays between mRNA and protein patterns, which likely require a more complex model. Overall, this modeling approach enables the prediction of regulatory scenarios underlying the condition-specific differences between mRNA and protein expression in early embryo.
Cunningham, N. H. J., Bouhlel, I. B. and Conduit, P. T. (2022). Daughter centrioles assemble preferentially towards the nuclear envelope in Drosophila syncytial embryos. Open Biol 12(1): 210343. PubMed ID: 35042404
Summary:
Centrosomes are important organizers of microtubules within animal cells. They comprise a pair of centrioles surrounded by the pericentriolar material, which nucleates and organizes the microtubules. To maintain centrosome numbers, centrioles must duplicate once and only once per cell cycle. During S-phase, a single new 'daughter' centriole is built orthogonally on one side of each radially symmetric 'mother' centriole. Mis-regulation of duplication can result in the simultaneous formation of multiple daughter centrioles around a single mother centriole, leading to centrosome amplification, a hallmark of cancer. It remains unclear how a single duplication site is established. It also remains unknown whether this site is pre-defined or randomly positioned around the mother centriole. This study shows that within Drosophila syncytial embryos daughter centrioles preferentially assemble on the side of the mother facing the nuclear envelope, to which the centrosomes are closely attached. This positional preference is established early during duplication and remains stable throughout daughter centriole assembly, but is lost in centrosomes forced to lose their connection to the nuclear envelope. This shows that non-centrosomal cues influence centriole duplication and raises the possibility that these external cues could help establish a single duplication site.
Hamilton, W. C., Stolarska, M. A. and Ismat, A. (2022). Simulation and in vivo experimentation predict AdamTS-A location of function during caudal visceral mesoderm migration in Drosophila. Dev Dyn. PubMed ID: 35023238
Summary:
Caudal visceral mesoderm (CVM) cells migrate as a loose collective along the trunk visceral mesoderm (TVM) and are surrounded by extracellular matrix (ECM). This study examined how one extracellular protease, AdamTS-A, facilitates CVM migration. A comparison of mathematical simulation to experimental results suggests that location of AdamTS-A action in CVM cells is on the sides of the cell not in contact with the TVM, predominantly at the CVM-ECM interface. CVM migration from a top-down view showed CVM cells migrating along the outside of the TVM substrate in the absence of AdamTS-A. Moreover, overexpression of AdamTS-A resulted in similar, but milder, mis-migration of the CVM. These results contrast with the salivary gland where AdamTS-A is proposed to cleave connections at the trailing edge of migrating cells. Subcellular localization of GFP-tagged AdamTS-A suggests that this protease is not limited to functioning at the trailing edge of CVM cells. In conclusion, using both in vivo experimentation and mathematical simulations, this study has demonstrated that AdamTS-A cleaves connections between CVM cells and the ECM on all sides not attached to the TVM. Clearly, AdamTS-A has a more expansive role around the entire cell in cleaving cell-ECM attachments in cells migrating as a loose collective.
Carmon, S., Jonas, F., Barkai, N., Schejter, E. D. and Shilo, B. Z. (2021). Generation and timing of graded responses to morphogen gradients. Development 148(24). PubMed ID: 34918740
Summary:
Morphogen gradients are known to subdivide a naive cell field into distinct zones of gene expression. This study examined whether morphogens can also induce a graded response within such domains. To this end, the role was explored. of the Dorsal protein nuclear gradient along the dorsoventral axis in defining the graded pattern of actomyosin constriction that initiates gastrulation in early Drosophila embryos. Two complementary mechanisms for graded accumulation of mRNAs of crucial zygotic Dorsal target genes were identified. First, activation of target-gene expression expands over time from the ventral-most region of high nuclear Dorsal to lateral regions, where the levels are lower, as a result of a Dorsal-dependent activation probability of transcription sites. Thus, sites that are activated earlier will exhibit more mRNA accumulation. Second, once the sites are activated, the rate of RNA Polymerase II loading is also dependent on Dorsal levels. Morphological restrictions require that translation of the graded mRNA be delayed until completion of embryonic cell formation. Such timing is achieved by large introns, which provide a delay in production of the mature mRNAs. Spatio-temporal regulation of key zygotic genes therefore shapes the pattern of gastrulation.
Colonnetta, M. M., Abrahante, J. E., Schedl, P., Gohl, D. M. and Deshpande, G. (2021). CLAMP regulates zygotic genome activation in Drosophila embryos. Genetics 219(2). PubMed ID: 34849887
Summary:
Embryonic patterning is critically dependent on zygotic genome activation (ZGA). In Drosophila melanogaster embryos, the pioneer factor Zelda directs ZGA, possibly in conjunction with other factors. This study explored the novel involvement of Chromatin-Linked Adapter for MSL Proteins (CLAMP) during ZGA. CLAMP binds thousands of sites genome-wide throughout early embryogenesis. Interestingly, CLAMP relocates to target promoter sequences across the genome when ZGA is initiated. Although there is a considerable overlap between CLAMP and Zelda binding sites, the proteins display distinct temporal dynamics. To assess whether CLAMP occupancy affects gene expression, transcriptomes of embryos zygotically compromised for either clamp or zelda were examined, and it was found that transcript levels of many zygotically activated genes are similarly affected. Importantly, compromising either clamp or zelda disrupted the expression of critical segmentation and sex determination genes bound by CLAMP (and Zelda). Furthermore, clamp knockdown embryos recapitulate other phenotypes observed in Zelda-depleted embryos, including nuclear division defects, centrosome aberrations, and a disorganized actomyosin network. Based on these data, it is proposed that CLAMP acts in concert with Zelda to regulate early zygotic transcription.

Tuesday, March 1st - Adult Development

Christesen, D., Yang, Y. T., Chen, W., Batterham, P. and Perry, T. (2021). Loss of the Dbeta1 nicotinic acetylcholine receptor subunit disrupts bursicon-driven wing expansion and diminishes adult viability in Drosophila melanogaster. Genetics 219(1). PubMed ID: 34849910
Summary:
Cholinergic signaling dominates the insect central nervous system, contributing to numerous fundamental pathways and behavioral circuits. However, the diverse roles different cholinergic receptors may play are only beginning to be understood. Historically, insect nicotinic acetylcholine receptors have received attention due to several subunits being key insecticide targets. More recently, there has been a focus on teasing apart the roles of these receptors, and their constituent subunits, in native signaling pathways. In this study, CRISPR-Cas9 genome editing was used to generate germline and somatic deletions of the Dβ1 nicotinic acetylcholine receptor subunit and investigate the consequences of loss of function in Drosophila melanogaster. Severe impacts on movement, male courtship, longevity, and wing expansion were found. Loss of Dβ1 was also associated with a reduction in transcript levels for the wing expansion hormone bursicon. Neuron-specific somatic deletion of Dβ1 in bursicon-producing neurons (CCAP-GAL4) was sufficient to disrupt wing expansion. Furthermore, CCAP-GAL4-specific expression of Dβ1 in a germline deletion background was sufficient to rescue the wing phenotype, pinpointing CCAP neurons as the neuronal subset requiring Dβ1 for the wing expansion pathway. Dβ1 is a known target of multiple commercially important insecticides, and the fitness costs exposed in this study explain why field-isolated target-site resistance has only been reported for amino acid replacements and not loss of function. This work reveals the importance of Dβ1-containing nicotinic acetylcholine receptors in CCAP neurons for robust bursicon-driven wing expansion.
Rose, M., Domsch, K., Bartle-Schultheis, J., Reim, I. and Schaub, C. (2022). Twist regulates Yorkie activity to guide lineage reprogramming of syncytial alary muscles. Cell Rep 38(4): 110295. PubMed ID: 35081347
Summary:
Genesis of syncytial muscles is typically considered as a paradigm for an irreversible developmental process. Notably, transdifferentiation of syncytial muscles is naturally occurring during Drosophila development. The ventral longitudinal heart-associated musculature (VLM) arises by a unique mechanism that revokes differentiation states of so-called alary muscles and comprises at least two distinct steps: syncytial muscle cell fragmentation into single myoblasts and successive reprogramming into founder cells that orchestrate de novo fiber formation of the VLM lineage. This study provides evidence that the mesodermal master regulator twist plays a key role during this reprogramming process. Acting downstream of Drosophila Tbx1 (Org-1), Twist is regulating the activity of the Hippo pathway effector Yorkie and is required for the initiation of syncytial muscle dedifferentiation and fragmentation. Subsequently, fibroblast growth factor receptor (FGFR)-Ras-mitogen-activated protein kinase (MAPK) signaling in resulting mononucleated myoblasts maintains Twist expression, thereby stabilizing nuclear Yorkie activity and inducing their lineage switch into founder cells of the VLM./a> that is critical for regulating myosin activity, leads to structural defects. It was further shown that Rbfox1 directly binds the 3'-UTR of target transcripts, regulates the expression level of myogenic transcription factors myocyte enhancer factor 2 and Salm, and both modulates expression of and genetically interacts with the CELF family RNA-binding protein Bruno1 (Bru1). Rbfox1 and Bru1 co-regulate fiber type-specific alternative splicing of structural genes, indicating that regulatory interactions between FOX and CELF family RNA-binding proteins are conserved in fly muscle. Rbfox1 thus affects muscle development by regulating fiber type-specific splicing and expression dynamics of identity genes and structural proteins.
Rice, G. R., David, J. R., Gompel, N., Yassin, A. and Rebeiz, M. (2021). Resolving between novelty and homology in the rapidly evolving phallus of Drosophila. J Exp Zool B Mol Dev Evol. PubMed ID: 34958528
Summary:
The genitalia present some of the most rapidly evolving anatomical structures in the animal kingdom, possessing a variety of parts that can distinguish recently diverged species. In the Drosophila melanogaster group, the phallus is adorned with several processes, pointed outgrowths, that are similar in size and shape between species. However, the complex three-dimensional nature of the phallus can obscure the exact connection points of each process. Previous descriptions based upon adult morphology have primarily assigned phallic processes by their approximate positions in the phallus and have remained largely agnostic regarding their homology relationships. In the absence of clearly identified homology, it can be challenging to model when each structure first evolved. This study employed a comparative developmental analysis of these processes in eight members of the melanogaster species group to precisely identify the tissue from which each process forms. The results indicate that adult phallic processes arise from three pupal primordia in all species. In some cases the same primordia generate homologous structures whereas in other cases, different primordia produce phenotypically similar but remarkably non-homologous structures. This suggests that the same gene regulatory network may have been redeployed to different primordia to induce phenotypically similar traits. These results highlight how traits diversify and can be redeployed, even at short evolutionary scales.
Zou, Y. L., Ding, X., Zhang, L., Xu, L. F., Liang, S. B., Hu, H., Dai, F. Y. and Tong, X. L. (2022). Bmmp influences wing morphology by regulating anterior-posterior and proximal-distal axis development. Insect Sci. PubMed ID: 34986276
Summary:
Insect wings are subject to strong selective pressure, resulting in the evolution of remarkably diverse wing morphologies that largely determine flight capacity. However, the genetic basis and regulatory mechanisms underlying wing size and shape development are not well understood. The silkworm Bombyx mori micropterous (mp) mutant exhibits shortened wing length and enlarged vein spacings, albeit without changes in total wing area. Thus, the mp mutant comprises a valuable genetic resource for studying wing development. This study used molecular mapping to identify the gene responsible for the mp phenotype and designated it Bmmp. Phenotype-causing mutations were identified as indels and single nucleotide polymorphisms in non-coding regions. These mutations resulted in decreased Bmmp mRNA levels and changes in transcript isoform composition. Bmmp null mutants were generated by CRISPR/Cas9 and exhibited changed wing shape, similar to mp mutants, and significantly smaller total wing area. By examining the expression of genes critical to wing development in wildtype and Bmmp null mutants, it was found that Bmmp exerts its function by coordinately modulating anterior-posterior and proximal-distal axis development. A Drosophila mp mutant was studied; Bmmp was found to be functionally conserved in Drosophila. The Drosophila mp mutant strain (see CG7102) exhibits curly wings of reduced size and a complete loss of flight capacity. These results increase understanding of the mechanisms underpinning insect wing development and reveal potential targets for pest control.
Weasner, B. M. and Kumar, J. P. (2022). The timing of cell fate decisions is crucial for initiating pattern formation in the Drosophila eye. Development 149(2). PubMed ID: 35072208
Summary:
The eye-antennal disc of Drosophila is composed of three cell layers: a columnar epithelium called the disc proper (DP); an overlying sheet of squamous cells called the peripodial epithelium (PE); and a strip of cuboidal cells that joins the other two cellular sheets to each other and comprises the outer margin (M) of the disc. The M cells play an important role in patterning the eye because it is here that the Hedgehog (Hh), Decapentaplegic (Dpp) and JAK/STAT pathways function to initiate pattern formation. Dpp signaling is lost from the margin of eyes absent (eya) mutant discs and, as a result, the initiation of retinal patterning is blocked. Based on these observations, Eya has been proposed to control the initiation of the morphogenetic furrow via regulation of Dpp signaling within the M. This study shows that the failure in pattern formation surprisingly results from M cells prematurely adopting a head epidermis fate. This switch in fate normally takes place during pupal development after the eye has been patterned. These results suggest that the timing of cell fate decisions is essential for correct eye development.
Duan, Y., Zhu, W., Zhao, X., Merzendorfer, H., Chen, J., Zou, X. and Yang, Q. (2022). Choline transporter-like protein 2 interacts with chitin synthase 1 and is involved in insect cuticle development. Insect Biochem Mol Biol 141: 103718. PubMed ID: 34982980
Summary:
Chitin is an aminopolysaccharide present in insects as a major structural component of the cuticle. However, current knowledge on the chitin biosynthetic machinery, especially its constituents and mechanism, is limited. Using three independent binding assays, including co-immunoprecipitation, split-ubiquitin membrane yeast two-hybrid assay, and pull-down assay, this study demonstrated that choline transporter-like protein 2 (Ctl2) interacts with krotzkopf verkehrt (kkv) in Drosophila melanogaster. The global knockdown of Ctl2 by RNA interference (RNAi) induced lethality at the larval stage. Tissue-specific RNAi to silence Ctl2 in the tracheal system and in the epidermis of the flies resulted in lethality at the first larval instar. The knockdown of Ctl2 in wings led to shrunken wings containing accumulated fluid. Calcofluor White staining demonstrated reduced chitin content in the first longitudinal vein of Ctl2 knockdown wings. The pro-cuticle, which was thinner compared to wildtype, exhibited a reduced number of chitin laminar layers. Phylogenetic analyses revealed orthologues of Ctl2 in different insect orders with highly conserved domains. These findings provide new insights into cuticle formation, wherein Ctl2 plays an important role as a chitin-synthase interacting protein.

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