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Current papers in developmental biology and gene function


Friday, July 31st, 2020 - Adult development - Adult Neural Development and Function

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Kirkland, N. J., Yuen, A. C., Tozluoglu, M., Hui, N., Paluch, E. K. and Mao, Y. (2020). Tissue Mechanics Regulate Mitotic Nuclear Dynamics during Epithelial Development. Curr Biol. PubMed ID: 32413305
Cell divisions are essential for tissue growth. In pseudostratified epithelia, where nuclei are staggered across the tissue, each nucleus migrates apically before undergoing mitosis. Successful apical nuclear migration is critical for planar-orientated cell divisions in densely packed epithelia. Most previous investigations have focused on the local cellular mechanisms controlling nuclear migration. Inter-species and inter-organ comparisons of different pseudostratified epithelia suggest global tissue architecture may influence nuclear dynamics, but the underlying mechanisms remain elusive. This study used the developing Drosophila wing disc to systematically investigate, in a single epithelial type, how changes in tissue architecture during growth influence mitotic nuclear migration. Distinct nuclear dynamics were observed at discrete developmental stages, as epithelial morphology changes. Genetic and physical perturbations were used to show a direct effect of cell density on mitotic nuclear positioning. Rho kinase and Diaphanous, which facilitate mitotic cell rounding in confined cell conditions, are essential for efficient apical nuclear movement. Perturbation of Diaphanous causes increasing defects in apical nuclear migration as the tissue grows and cell density increases, and these defects can be reversed by acute physical reduction of cell density. These findings reveal how the mechanical environment imposed on cells within a tissue alters the molecular and cellular mechanisms adopted by single cells for mitosis.
Liao, S., Post, S., Lehmann, P., Veenstra, J. A., Tatar, M. and Nassel, D. R. (2020). Regulatory Roles of Drosophila Insulin-Like Peptide 1 (DILP1) in Metabolism Differ in Pupal and Adult Stages. Front Endocrinol (Lausanne) 11: 180. PubMed ID: 32373064
The insulin/IGF-signaling pathway is central in control of nutrient-dependent growth during development, and in adult physiology and longevity. The functional role of DILP1 is far from understood. dilp1/DILP1 is transiently expressed mainly during the pupal stage and the first days of adult life. The role of dilp1 in the pupa, as well as in the first week of adult life, was studied, and some comparisons were made to dilp6 that displays a similar pupal expression profile, but is expressed in fat body rather than brain neurosecretory cells. Mutation of dilp1 diminishes organismal weight during pupal development, whereas overexpression increases it, similar to dilp6 manipulations. No growth effects of dilp1 or dilp6 manipulations were detected during larval development. It was next shown that dilp1 and dilp6 increase metabolic rate in the late pupa and promote lipids as the primary source of catabolic energy. Effects of dilp1 manipulations can also be seen in the adult fly. In newly eclosed female flies, survival during starvation is strongly diminished in dilp1 mutants, but not in dilp2 and dilp1/dilp2 mutants, whereas in older flies, only the double mutants display reduced starvation resistance. Starvation resistance is not affected in male dilp1 mutant flies, suggesting a sex dimorphism in dilp1 function. Overexpression of dilp1 also decreases survival during starvation in female flies and increases egg laying and decreases egg to pupal viability. In conclusion, dilp1 and dilp6 overexpression promotes metabolism and growth of adult tissues during the pupal stage, likely by utilization of stored lipids. Some of the effects of the dilp1 manipulations may carry over from the pupa to affect physiology in young adults, but the data also suggest that dilp1 signaling is important in metabolism and stress resistance in the adult stage.
Christensen, C. F., Koyama, T., Nagy, S., Danielsen, E. T., Texada, M. J., Halberg, K. A. and Rewitz, K. (2020). Ecdysone-dependent feedback regulation of prothoracicotropic hormone controls the timing of developmental maturation. Development 147(14). PubMed ID: 32631830
The activation of a neuroendocrine system that induces a surge in steroid production is a conserved initiator of the juvenile-to-adult transition in many animals. The trigger for maturation is the secretion of brain-derived neuropeptides, yet the mechanisms controlling the timely onset of this event remain ill-defined. This study shows that a regulatory feedback circuit controlling the Drosophila neuropeptide Prothoracicotropic hormone (PTTH) triggers maturation onset. The Ecdysone Receptor (EcR) in the PTTH-expressing neurons (PTTHn) was identified as a regulator of developmental maturation onset. Loss of EcR in these PTTHn impairs PTTH signaling, which delays maturation. The steroid ecdysone dose-dependently affects Ptth transcription, promoting its expression at lower concentrations and inhibiting it at higher concentrations. These findings indicate the existence of a feedback circuit in which rising ecdysone levels trigger, via EcR activity in the PTTHn, the PTTH surge that generates the maturation-inducing ecdysone peak toward the end of larval development. Because steroid feedback is also known to control the vertebrate maturation-inducing hypothalamic-pituitary-gonadal axis, these findings suggest an overall conservation of the feedback-regulatory neuroendocrine circuitry that controls the timing of maturation initiation.
Mohr, T. and Fischer, S. (2020). Ultrastructural evidence for the origin of the subretinal pigment shield in the compound eye of Drosophila melanogaster. J Morphol. PubMed ID: 32395883
Little morphological information is available about subretinal pigment shields in insect compound eyes, especially ultrastructural information. The latter is however needed in order to detect possible smallest projections that emanate from pigment-granule-bearing cells and pass through the basal matrix (BM), but that are not visible in light micrographs. Previous work on the subretinal pigment shield in Drosophila melanogaster suggests that the pigment cell population located below the BM is closely associated with secondary and tertiary pigment cells. Whether these cells stay in connection throughout life with the subretinal regions via thin projections that pass through the fenestrae of the BM, or whether the subretinal parts later become separated during eye development remained so far unknown. This investigation of the periphery of the BM by three-dimensional reconstruction based on serial-sectioning transmission electron microscopy has revealed that the secondary and tertiary pigment cells possess thin projections that pass through the fenestrae of the BM and thus connect the cellular regions above and below the BM in the adult compound eye. The subretinal pigment shield of D. melanogaster is therefore of retinal origin and is not composed of additional subretinal pigment cells. The maintained bond allows the active displacement of pigment granules below the BM during the process of dark and light adaptation of the compound eye.
Scott, R. L., Diao, F., Silva, V., Park, S., Luan, H., Ewer, J. and White, B. H. (2020). Non-canonical Eclosion Hormone-Expressing Cells Regulate Drosophila Ecdysis. iScience 23(5): 101108. PubMed ID: 32408174
Eclosion hormone (EH) was originally identified as a brain-derived hormone capable of inducing the behavioral sequences required for molting across insect species. However, its role in this process (called ecdysis) has since been confounded by discrepancies in the effects of genetic and cellular manipulations of EH function in Drosophila. Although knock-out of the Eh gene results in severe ecdysis-associated deficits accompanied by nearly complete larval lethality, ablation of the only neurons known to express EH (i.e. V(m) neurons) is only partially lethal and surviving adults emerge, albeit abnormally. Using new tools for sensitively detecting Eh gene expression, this study shows that EH is more widely expressed than previously thought, both within the nervous system and in somatic tissues, including trachea. Ablating all Eh-expressing cells has effects that closely match those of Eh gene knock-out; developmentally suppressing them severely disrupts eclosion. The results thus clarify and extend the scope of EH action.
Tiwari, S. K., Toshniwal, A. G., Mandal, S. and Mandal, L. (2020). Fatty acid beta-oxidation is required for the differentiation of larval hematopoietic progenitors in Drosophila. Elife 9. PubMed ID: 32530419
Cell-intrinsic and extrinsic signals regulate the state and fate of stem and progenitor cells. Recent advances in metabolomics illustrate that various metabolic pathways are also important in regulating stem cell fate. However, understanding of the metabolic control of the state and fate of progenitor cells is in its infancy. Using Drosophila hematopoietic organ: lymph gland, this study demonstrated that Fatty Acid Oxidation (FAO) is essential for the differentiation of blood cell progenitors. In the absence of FAO, the progenitors are unable to differentiate and exhibit altered histone acetylation. Interestingly, acetate supplementation rescues both histone acetylation and the differentiation defects. It was further shown that the CPT1/whd (withered), the rate-limiting enzyme of FAO, is transcriptionally regulated by Jun-Kinase (JNK), which has been previously implicated in progenitor differentiation. This study thus reveals how the cellular signaling machinery integrates with the metabolic cue to facilitate the differentiation program.

Thursday, July 30th - Disease Models

Wormser, O., Levy, Y., Bakhrat, A., Bonaccorsi, S., Graziadio, L., Gatti, M., AbuMadigham, A., McKenney, R. J., Okada, K., El Riati, S., Har-Vardi, I., Huleihel, M., Levitas, E., Birk, O. S. and Abdu, U. (2020). Absence of SCAPER causes male infertility in humans and Drosophila by modulating microtubule dynamics during meiosis. J Med Genet. PubMed ID: 32527956
Mutation in S-phase cyclin A-associated protein in the endoplasmic reticulum (SCAPER) have been found across ethnicities and have been shown to cause variable penetrance of an array of pathological traits, including intellectual disability, retinitis pigmentosa and ciliopathies. This study generated and analysed short spindle 3 (ssp3) (SCAPER orthologue) Drosophila CAS9-knockout lines. In vitro microtubule (MT) binding was assayed by total internal reflection fluorescence microscopy. Patients homozygous for a SCAPER mutation lack SCAPER expression in spermatogonia (SPG) and are azoospermic due to early defects in spermatogenesis, leading to the complete absence of meiotic cells. Interestingly, Drosophila null mutants for the ubiquitously expressed ssp3 gene are viable and female fertile but male sterile. It was further shown that male sterility in ssp3 null mutants is due to failure in both chromosome segregation and cytokinesis. In cells undergoing male meiosis, the MTs emanating from the centrosomes do not appear to interact properly with the chromosomes, which remain dispersed within dividing spermatocytes (SPCs). In addition, mutant SPCs are unable to assemble a normal central spindle and undergo cytokinesis. Consistent with these results, an in vitro assay demonstrated that both SCAPER and ssp3 directly bind MTs. These results show that SCAPER null mutations block the entry into meiosis of SPG, causing azoospermia. Null mutations in ssp3 specifically disrupt MT dynamics during male meiosis, leading to sterility. Moreover, both SCAPER and Ssp3 bind MTs in vitro. These results raise the intriguing possibility of a common feature between human and Drosophila meiosis.
Zheng, W., Ocorr, K. and Tatar, M. (2020). Extra-cellular matrix induced by steroids and aging through a G-protein coupled receptor in a Drosophila model of renal fibrosis. Dis Model Mech. PubMed ID: 32461236
Aldosterone is produced by the mammalian adrenal cortex to modulate blood pressure and fluid balance, however excessive, prolonged aldosterone promotes fibrosis and kidney failure. How aldosterone triggers disease may involve actions independent of its canonical mineralocorticoid receptor. This study presents a Drosophila model of renal pathology caused by excess extra-cellular matrix formation, stimulated by exogenous aldosterone and by insect ecdysone. Chronic administration of aldosterone or ecdysone induces expression and accumulation of collagen-like Pericardin at adult nephrocytes - podocyte-like cells that filter circulating hemolymph. Excess Pericardin deposition disrupts nephrocyte (glomerular) filtration and causes proteinuria in Drosophila, hallmarks of mammalian kidney failure. Steroid-induced Pericardin production arises from cardiomyocytes associated with nephrocytes, potentially reflecting an analogous role of mammalian myofibroblasts in fibrotic disease. Remarkably, the canonical ecdysteroid nuclear hormone receptor, Ecdysone Receptor EcR, is not required for aldosterone or ecdysone to stimulate Pericardin production or associated renal pathology. Instead, these hormones require a cardiomyocyte-associated G-protein coupled receptor, Dopamine-EcR (DopEcR), a membrane-associated receptor previously characterized in the fly brain as affecting behavior. DopEcR in the brain is known to affect behavior through interactions with the Drosophila epidermal growth factor receptor, dEGFR. This study finds the steroids ecdysone and aldosterone require dEGFR in cardiomyocytes to induce fibrosis of the cardiac-renal system. As well, endogenous ecdysone that becomes elevated with age is found to foster age-associated fibrosis, and to require both cardiomyocyte DopEcR and dEGFR. This Drosophila renal disease model reveals a novel signaling pathway through which steroids may modulate mammalian fibrosis through potential orthologs of DopEcR.
Zhao, M., Kao, C. S., Arndt, C., Tran, D. D., Cho, W. I., Maksimovic, K., Chen, X. X. L., Khan, M., Zhu, H., Qiao, J., Peng, K., Hong, J., Xu, J., Kim, D., Kim, J. R., Lee, J., van Bruggen, R., Yoon, W. H. and Park, J. (2020). Knockdown of genes involved in axonal transport enhances the toxicity of human neuromuscular disease-linked MATR3 mutations in Drosophila. FEBS Lett. PubMed ID: 32515490
Mutations in the nuclear matrix protein Matrin 3 (MATR3) have been identified in amyotrophic lateral sclerosis (ALS) and myopathy. To investigate the mechanisms underlying MATR3 mutations in neuromuscular diseases and efficiently screen for modifiers of MATR3 toxicity, transgenic MATR3 flies were generated. The findings indicate that expression of wildtype or mutant MATR3 in motor neurons reduces climbing ability and lifespan of flies, while their expression in indirect flight muscles results in abnormal wing positioning and muscle degeneration. In both motor neurons and indirect flight muscles, mutant MATR3 expression results in more severe phenotypes than wildtype MATR3, demonstrating that the disease-linked mutations confer pathogenicity. A targeted candidate screen was conducted for modifiers of the MATR3 abnormal wing phenotype, and multiple enhancers involved in axonal transport were identfied. Knockdown of these genes enhanced protein levels and insolubility of mutant MATR3. These results suggest that accumulation of mutant MATR3 contributes to toxicity and implicate axonal transport dysfunction in disease pathogenesis.
Woolums, B. M., McCray, B. A., Sung, H., Tabuchi, M., Sullivan, J. M., Ruppell, K. T., Yang, Y., Mamah, C., Aisenberg, W. H., Saavedra-Rivera, P. C., Larin, B. S., Lau, A. R., Robinson, D. N., Xiang, Y., Wu, M. N., Sumner, C. J. and Lloyd, T. E. (2020). TRPV4 disrupts mitochondrial transport and causes axonal degeneration via a CaMKII-dependent elevation of intracellular Ca(2). Nat Commun 11(1): 2679. PubMed ID: 32471994
The cation channel transient receptor potential vanilloid 4 (TRPV4) is one of the few identified ion channels that can directly cause inherited neurodegeneration syndromes, but the molecular mechanisms are unknown. This study shows that in vivo expression of a neuropathy-causing TRPV4 mutant (TRPV4(R269C)) causes dose-dependent neuronal dysfunction and axonal degeneration, which are rescued by genetic or pharmacological blockade of TRPV4 channel activity. TRPV4(R269C) triggers increased intracellular Ca(2+) through a Ca(2+)/calmodulin-dependent protein kinase II (CaMKII)-mediated mechanism, and CaMKII inhibition prevents both increased intracellular Ca(2+) and neurotoxicity in Drosophila and cultured primary mouse neurons. Importantly, TRPV4 activity impairs axonal mitochondrial transport, and TRPV4-mediated neurotoxicity is modulated by the Ca(2+)-binding mitochondrial GTPase Miro. These data highlight an integral role for CaMKII in neuronal TRPV4-associated Ca(2+) responses, the importance of tightly regulated Ca(2+) dynamics for mitochondrial axonal transport, and the therapeutic promise of TRPV4 antagonists for patients with TRPV4-related neurodegenerative diseases.
Yamakawa-Kobayashi, K., Ohhara, Y., Kawashima, T., Ohishi, Y. and Kayashima, Y. (2020). Loss of CNDP causes a shorter lifespan and higher sensitivity to oxidative stress in Drosophila melanogaster. Biomed Res 41(3): 131-138. PubMed ID: 32522930
Increasing oxidative stress seems to be the result of an imbalance between free radical production and antioxidant defenses. During the course of aging, oxidative stress causes tissue/cellular damage, which is implicated in numerous age-related diseases. Carnosinase (CN or CNDP) is dipeptidase, which is associated with carnosine and/or glutathione (GSH) metabolism, those are the most abundant naturally occurring endogenous dipeptide and tripeptides with antioxidant and free radical scavenger properties. This study generated Drosophila cndp (dcndp) mutant flies using the CRISPR/Cas9 system to study the roles of dcndp in vivo. dcndp mutant flies exhibit shorter lifespan and increased sensitivity to paraquat or hydrogen peroxide induced oxidative stress. These results suggest that dcndp maintains homeostatic conditions, protecting cells and tissues against the harmful effects of oxidative stress in the course of aging.
Yamaguchi, A., Ishikawa, K. I., Inoshita, T., Shiba-Fukushima, K., Saiki, S., Hatano, T., Mori, A., Oji, Y., Okuzumi, A., Li, Y., Funayama, M., Imai, Y., Hattori, N. and Akamatsu, W. (2020).. Identifying Therapeutic Agents for Amelioration of Mitochondrial Clearance Disorder in Neurons of Familial Parkinson Disease. Stem Cell Reports 14(6): 1060-1075. PubMed ID: 32470327
Parkinson disease (PD) is a neurodegenerative disorder caused by the progressive loss of midbrain dopaminergic neurons, and mitochondrial dysfunction is involved in its pathogenesis. This study aimed to establish an imaging-based, semi-automatic, high-throughput system for the quantitative detection of disease-specific phenotypes in dopaminergic neurons from induced pluripotent stem cells (iPSCs) derived from patients with familial PD having Parkin or PINK1 mutations, which exhibit abnormal mitochondrial homeostasis. The proposed system recapitulates the deficiency of mitochondrial clearance, ROS accumulation, and increasing apoptosis in these familial PD-derived neurons. 320 compounds were screened for their ability to ameliorate multiple phenotypes, and four candidate drugs were identified. Some of these drugs improved the locomotion defects and reduced ATP production caused by PINK1 inactivation in Drosophila and were effective for idiopathic PD-derived neurons with impaired mitochondrial clearance. These findings suggest that the proposed high-throughput system has potential for identifying effective drugs for familial and idiopathic PD.

Wednesday, July 29th - Signaling

Vandehoef, C., Molaei, M. and Karpac, J. (2020). Dietary Adaptation of Microbiota in Drosophila Requires NF-kappaB-Dependent Control of the Translational Regulator 4E-BP. Cell Rep 31(10): 107736. PubMed ID: 32521261
Dietary nutrients shape complex interactions between hosts and their commensal gut bacteria, further promoting flexibility in host-microbiota associations that can drive nutritional symbiosis. However, it remains less clear if diet-dependent host signaling mechanisms also influence these associations. Using Drosophila, this study shows that nuclear factor κB (NF-κB)/Relish, an innate immune transcription factor emerging as a signaling node linking nutrient-immune-metabolic interactions, is vital to adapt gut microbiota species composition to host diet macronutrient composition. Relish was found to be required within midgut enterocytes to amplify host-Lactobacillus associations, an important bacterial mediator of nutritional symbiosis, and thus modulate microbiota composition in response to dietary adaptation. Relish limits diet-dependent transcriptional inducibility of the cap-dependent translation inhibitor 4E-BP/Thor to control microbiota composition. Furthermore, maintaining cap-dependent translation in response to dietary adaptation is critical to amplify host-Lactobacillus associations. These results highlight that NF-κB-dependent host signaling mechanisms, in coordination with host translation control, shape diet-microbiota interactions.
Waghmare, I., Wang, X. and Page-McCaw, A. (2020). Dally-like protein sequesters multiple Wnt ligands in the Drosophila germarium. Dev Biol. PubMed ID: 32473955
Cells in multicellular organisms rely on secreted ligands for development and morphogenesis. Several mechanisms modulate the availability and distribution of secreted ligands, determining their ability to signal locally and at long range from their source. One of these mechanisms is Dally-like protein (Dlp), a cell-surface glypican that exhibits biphasic functions in Drosophila wing discs, promoting Wg signaling at long-range from Wg source cells and inhibiting Wg signaling near source cells. In the germarium at the tip of the ovary, Dlp promotes long-range distribution of Wg from cap cells to follicle stem cells. However, the germarium also expresses other Wnts - Wnt2, Wnt4, and Wnt6 - that function locally in escort cells to promote oogenesis. Whether and how local functions of these Wnts are regulated remains unknown. This study shows that the dlp overexpression phenotype is multifaceted and phenocopies multiple Wnt loss-of-function phenotypes. Each aspect of dlp overexpression phenotype is suppressed by co-expression of individual Wnts, and the suppression pattern exhibited by each Wnt suggests that Wnts have functional specificity in the germarium. Further, dlp knockdown phenocopies Wnt gain-of-function phenotypes. Together these data show that Dlp inhibits the functions of each Wnt. All four Wnts co-immunoprecipitate with Dlp in S2R+ cells, suggesting that in the germarium, Dlp sequesters Wnts to inhibit local paracrine Wnt signaling. These results indicate that Dlp modulates the availability of multiple extracellular Wnts for local paracrine Wnt signaling in the germarium.
Zhang, F., Pirooznia, M. and Xu, H. (2020). Mitochondria regulate intestinal stem cell proliferation and epithelial homeostasis through FOXO. Mol Biol Cell: mbcE19100560. PubMed ID: 32374658
A metabolic transition from glycolysis to oxidative phosphorylation often associates with differentiation of many types of stem cells. However, the link between mitochondrial respiration and stem cells' behavior is not fully understood. This study genetically disrupted electron transport chain (ETC) complexes in the intestinal stem cells (ISCs) of Drosophila. ISCs carrying impaired ETC proliferated much more slowly than normal, produced very few enteroblasts, which failed to further differentiate into enterocytes. One of the main impediments to ISC proliferation and lineage specification appeared to be abnormally elevated forkhead box O (FOXO) signaling in the ETC-deficient ISCs, as genetically suppressing the signaling pathway partially restored the number of enterocytes. Contrary to common belief, reactive oxygen species (ROS) accumulation did not appear to mediate the ETC mutant phenotype. These results demonstrate that mitochondrial respiration is essential for Drosophila ISC proliferation and lineage specification in vivo and acts at least partially by repressing endogenous FOXO signaling.
Vishal, K., Lovato, T. L., Bragg, C., Chechenova, M. B. and Cripps, R. M. (2020). FGF signaling promotes myoblast proliferation through activation of wingless signaling. Dev Biol. PubMed ID: 32445643
Indirect flight muscles (IFMs) are the largest muscles in Drosophila and are made up of hundreds of myonuclei. The generation of these giant muscles requires a large pool of wing disc associated adult muscle precursors (AMPs), however the factors that control proliferation to form this myoblast pool are incompletely known. This study examined the role of fibroblast growth factor (FGF) signaling in the proliferation of wing disc associated myoblasts. The components of FGF signaling are expressed in myoblasts and surrounding epithelial cells of the wing disc. Next, this study showed that attenuation of FGF signaling results in a diminished myoblast pool. This reduction in the pool size is due to decreased myoblast proliferation. By contrast, activating the FGF signaling pathway increases the myoblast pool size and restores the proliferative capacity of FGF knockdown flies. Finally, the results demonstrate that the FGF receptor Heartless acts through up-regulating β-catenin/Armadillo signaling to promote myoblast proliferation. These studies identify a novel role for FGF signaling during IFM formation and uncover the mechanism through which FGF coordinates with Wingless signaling to promote myoblast proliferation.
Wang, C., Shui, K., Ma, S., Lin, S., Zhang, Y., Wen, B., Deng, W., Xu, H., Hu, H., Guo, A., Xue, Y. and Zhang, L. (2020). Integrated omics in Drosophila uncover a circadian kinome. Nat Commun 11(1): 2710. PubMed ID: 32483184
Most organisms on the earth exhibit circadian rhythms in behavior and physiology, which are driven by endogenous clocks. Phosphorylation plays a central role in timing the clock, but how this contributes to overt rhythms is unclear. Phosphoproteomics in conjunction with transcriptomic and proteomic profiling was conducted using fly heads. By developing a pipeline for integrating multi-omics data, 789 (~17%) phosphorylation sites with circadian oscillations were identified. It is predicted that 27 potential circadian kinases participate in phosphorylating these sites, including 7 previously known to function in the clock. The remaining 20 kinases were screened for effects on circadian rhythms, and an additional 3 were found to be involved in regulating locomotor rhythm. A signal web was re-constructed that includes the 10 circadian kinases, and GASKET was identified as a potentially important regulator. Taken together, this study uncovered a circadian kinome that potentially shapes the temporal pattern of the entire circadian molecular landscapes.
Zhang, B., Binks, T. and Burke, R. (2020). The E3 ubiquitin ligase Slimb/beta-TrCP is required for normal copper homeostasis in Drosophila. Biochim Biophys Acta Mol Cell Res: 118768. PubMed ID: 32502619
The Drosophila Slimb (Slmb) gene encodes a Skp1-Cul1-F-box (SCP) E3 ubiquitin ligase orthologous to the human β-TrCP/BTRC protein. Slmb and/or BTRC play regulatory roles in numerous biological processes by ubiquitinating several substrate proteins which are then targeted for proteasomal degradation. This study demonstrates an additional role for Slmb in maintaining cellular copper homeostasis. In the thorax, midgut and eye, Slmb knockdown causes copper deficiency phenotypes which can be rescued by increasing cellular copper levels via decreased efflux or increased uptake. Furthermore, Slmb knockdown results in decreased levels of the copper transporters Ctr1A and ATP7, indicating Slmb is required to regulate copper homeostasis. Evidence is presented that the transcription factor Cap-n-Collar (Nrf2 in mammals), a known substrate of Slmb/BTRC, mediates Slmb's regulatory effect on Ctr1A in a post-transcriptional manner.

Tuesday, July 28th - Adult Physiology

Wilson, K. A., Beck, J. N., Nelson, C. S., Hilsabeck, T. A., Promislow, D., Brem, R. B. and Kapahi, P. (2020). GWAS for Lifespan and Decline in Climbing Ability in Flies upon Dietary Restriction Reveal decima as a Mediator of Insulin-like Peptide Production. Curr Biol. PubMed ID: 32502405
Dietary restriction (DR) is the most robust means to extend lifespan and delay age-related diseases across species. An underlying assumption in the aging field is that DR enhances both lifespan and physical activity through similar mechanisms, but this has not been rigorously tested in different genetic backgrounds. Furthermore, nutrient response genes responsible for lifespan extension or age-related decline in functionality remain underexplored in natural populations. To address this, nutrient-dependent changes were measured in lifespan and age-related decline in climbing ability in the Drosophila Genetic Reference Panel fly strains. On average, DR extended lifespan and delayed decline in climbing ability, but there was a lack of correlation between these traits across individual strains, suggesting that distinct genetic factors modulate these traits independently and that genotype determines response to diet. Only 50% of strains showed positive response to DR for both lifespan and climbing ability, 14% showed a negative response for one trait but not both, and 35% showed no change in one or both traits. Through GWAS, a number of genes were uncovered previously not known to be diet responsive nor to influence lifespan or climbing ability. decima/CG34351 was validated as a gene that alters lifespan and daedalus/CG33690 as one that influences age-related decline in climbing ability. decima was found to influences insulin-like peptide transcription in the GABA receptor neurons downstream of short neuropeptide F precursor (sNPF) signaling. Modulating these genes produced independent effects on lifespan and physical activity decline, which suggests that these age-related traits can be regulated through distinct mechanisms.
Yurkevych, I. S., Gray, L. J., Gospodaryov, D. V., Burdylyuk, N. I., Storey, K. B., Simpson, S. J. and Lushchak, O. (2020). Development of fly tolerance to consuming a high-protein diet requires physiological, metabolic and transcriptional changes. Biogerontology. PubMed ID: 32468146
Mortality in insects consuming high-protein-and-low-carbohydrate diets resembles a type III lifespan curve with increased mortality at an early age and few survivors that live a relatively long lifespan. A Drosophila line was selected for ability to live for a long time on an imbalanced high-protein-low-carbohydrate diet by carrying out five rounds of breeding to select for the most long-lived survivors. Adaptation to this diet in the selected line was studied at the biochemical, physiological and transcriptomic levels. The selected line of flies consumed less of the imbalanced food but also accumulated more storage metabolites: glycogen, triacylglycerides, and trehalose. Selected flies also had a higher activity of alanine transaminase and a higher urea content. Adaptation of the selected line on the transcriptomic level was characterized by down-regulation of genes encoding serine endopeptidases (Jon25i, Jon25ii, betaTry, and others) but up-regulation of genes encoding proteins related to the immune system, such as antimicrobial peptides, Turandot-family humoral factors, hexamerin isoforms, and vitellogenin. These sets of down- and up-regulated genes were similar to those observed in fruit flies with suppressed juvenile hormone signaling. These data show that the physiological adaptation of fruit flies to a high-protein-low-carbohydrate diet occurs via intuitive pathways, namely a decrease in food consumption, conversion of amino acids into ketoacids to compensate for the lack of carbohydrate, and accumulation of storage metabolites to eliminate the negative effects of excess amino acids. Nevertheless, transcriptomic adaptation occurs in a counter-intuitive way, likely via an influence of gut microbiota on food digestion.
Winwood-Smith, H. S., White, C. R. and Franklin, C. E. (2020). Flight activity and glycogen depletion on a low-carbohydrate diet. J Exp Biol. PubMed ID: 32532863
Glycogen is a critical store for locomotion. Depleted glycogen stores are associated with increased fatigue during exercise. The reduced effectiveness of low-carbohydrate diets for weight loss over longer time periods may arise because such diets reduce glycogen stores and thereby physical activity energy expenditure. To explore the effect of a low-carbohydrate diet on activity and glycogen utilisation, adult Drosophila melanogaster were fed a standard or low-carbohydrate diet for nine days, and patterns of flight activity and rates of glycogen depletion were measured. It was hypothesised that flight activity and rates of glycogen depletion would be reduced on a low-carbohydrate diet. Flight activity was elevated in the low-carbohydrate group but glycogen depletion rates were unchanged. It is concluded that increased activity is likely a foraging response to carbohydrate deficiency and it is speculated that the previously demonstrated metabolic depression that occurs on a low-carbohydrate diet in this species may allow for increased flight activity without increased glycogen depletion.
Yap, Z. Y., Strucinska, K., Matsuzaki, S., Lee, S., Si, Y., Humphries, K., Tarnopolsky, M. A. and Yoon, W. H. (2020). A biallelic pathogenic variant in the OGDH gene results in a neurological disorder with features of a mitochondrial disease. J Inherit Metab Dis. PubMed ID: 32383294
2-oxoglutarate dehydrogenase (OGDH) is a rate-liming enzyme in the mitochondrial TCA cycle. α-ketoglutarate dehydrogenase deficiency has been reported in association with developmental delay, movement disorders and metabolic decompensation. Two individuals were identified carrying a homozygous missense variant c.959A > G (p.N320S) in the OGDH gene. These individuals presented with global developmental delay, elevated lactate, ataxia and seizure. Transfection of human OGDH cDNA in HEK293 cells carrying p.N320S also produced significantly lower protein levels compared to those with wild-type cDNA. Loss of Drosophila Ogdh (dOgdh) caused early developmental lethality, rescued by expressing wild-type dOgdh or human OGDH cDNA. In contrast, expression of the mutant OGDH (OGDHN320S) or dOgdh carrying homologous mutations to human OGDH p.N320S variant failed to rescue lethality of dOgdh null mutants. Knockdown of dOgdh in the nervous system resulted in locomotion defects which were rescued by dOgdhWT expression. Collectively, the results indicate that c.959A > G variant in OGDH leads to an amino acid change causing a severe loss of OGDH protein function. This study establishes in the first time a genetic link between an OGDH gene mutation and OGDH deficiency.
Philipsen, M., Gu, C. and Ewing, A. G. (2020). Imaging Mass Spectrometry Shows That Zinc Deficiency Leads to Lipids Changes in Drosophila Brain Similar to Cognitive Impairing Drugs. Chembiochem. PubMed ID: 32402134
Several diseases and disorders have been suggested to be associated with zinc deficiency, especially learning and memory impairments. To have better understanding about the connection between lipid changes and cognitive impairments, this study investigated the effects of zinc chelated diet on certain brain lipids of Drosophila melanogaster using time-of-flight secondary ion mass spectrometry (ToF-SIMS). The data revealed that there are increases in the levels of phosphatidyl choline and phosphatidylinositol in the central brains of the zinc deficient flies compared to the control flies. In contrast, the abundance of phosphatidylethanolamine in the brains of the zinc deficient flies is lower. These data are consistent with that of cognitive diminishing drugs providing insight into the biological and molecular effects of zinc deficiency on the major brain lipids and opening a new treatment target for cognitive deficit in zinc deficiency.
Yon, M., Decoville, M., Sarou-Kanian, V., Fayon, F. and Birman, S. (2020). Spatially-resolved metabolic profiling of living Drosophila in neurodegenerative conditions using (1)H magic angle spinning NMR. Sci Rep 10(1): 9516. PubMed ID: 32528106
Drosophila flies are versatile animal models for the study of gene mutations in neuronal pathologies. Their small size allows performing in vivo Magic Angle Spinning (MAS) experiments to obtain high-resolution (1)H nuclear magnetic resonance (NMR) spectra. This study used spatially-resolved (1)H high-resolution MAS NMR to investigate in vivo metabolite contents in different segments of the fly body. A comparative study of metabolic changes was performed for three neurodegenerative disorders: two cell-specific neuronal and glial models of Huntington disease (HD) and a model of glutamate excitotoxicity. It is shown that these pathologies are characterized by specific and sometimes anatomically localized variations in metabolite concentrations. In two cases, the modifications of (1)H MAS NMR spectra localized in fly heads were significant enough to allow the creation of a predictive model

Monday July 27th - Early Embryonic Development

Omura, C. S. and Lott, S. E. (2020). The conserved regulatory basis of mRNA contributions to the early Drosophila embryo differs between the maternal and zygotic genomes. PLoS Genet 16(3): e1008645. PubMed ID: 32226006
The gene products that drive early development are critical for setting up developmental trajectories in all animals. The earliest stages of development are fueled by maternally provided mRNAs until the zygote can take over transcription of its own genome. In early development, both maternally deposited and zygotically transcribed gene products have been well characterized in model systems. Previously, it was demonstrated that across the genus Drosophila, maternal and zygotic mRNAs are largely conserved but also showed a surprising amount of change across species, with more differences evolving at the zygotic stage than the maternal stage. This study used comparative methods to elucidate the regulatory mechanisms underlying maternal deposition and zygotic transcription across species. Through motif analysis, considerable conservation of regulatory mechanisms was discovered associated with maternal transcription, as compared to zygotic transcription. It was also found that the regulatory mechanisms active in the maternal and zygotic genomes are quite different. For maternally deposited genes, many signals were uncovered that are consistent with transcriptional regulation at the level of chromatin state through factors enriched in the ovary, rather than precisely controlled gene-specific factors. For genes expressed only by the zygotic genome, evidence was found for previously identified regulators such as Zelda and GAGA-factor, with multiple analyses pointing toward gene-specific regulation. The observed mechanisms of regulation are consistent with what is known about regulation in these two genomes: during oogenesis, the maternal genome is optimized to quickly produce a large volume of transcripts to provide to the oocyte; after zygotic genome activation, mechanisms are employed to activate transcription of specific genes in a spatiotemporally precise manner. Thus the genetic architecture of the maternal and zygotic genomes, and the specific requirements for the transcripts present at each stage of embryogenesis, determine the regulatory mechanisms responsible for transcripts present at these stages.
Lau, L. Y., Reverter, A., Hudson, N. J., Naval-Sanchez, M., Fortes, M. R. S. and Alexandre, P. A. (2020). Dynamics of Gene Co-expression Networks in Time-Series Data: A Case Study in Drosophila melanogaster Embryogenesis. Front Genet 11: 517. PubMed ID: 32528531
Co-expression networks tightly coordinate the spatiotemporal patterns of gene expression unfolding during development. Due to the dynamic nature of developmental processes simply overlaying gene expression patterns onto static representations of co-expression networks may be misleading. This study aimed to formally quantitate topological changes of co-expression networks during embryonic development using a publicly available Drosophila melanogaster transcriptome data set comprising 14 time points. A network approach was deployed that inferred 10 discrete co-expression networks by smoothly sliding along from early to late development using 5 consecutive time points per window. Such an approach allows changing network structure, including the presence of hubs, modules and other topological parameters to be quantitated. To explore the dynamic aspects of gene expression captured by this approach, focus was placed on regulator genes with apparent influence over particular aspects of development. Those key regulators were selected using a differential network algorithm to contrast the first 7 (early) with the last 7 (late) developmental time points. This assigns high scores to genes whose connectivity to abundant differentially expressed target genes has changed dramatically between states. A list of key regulators was produced - some increasing (e.g., Tusp, slbo, Sidpn, DCAF12, and chinmo) and some decreasing (Rfx, bap, Hmx, Awh, and mld) connectivity during development - which reflects their role in different stages of embryogenesis. The networks that were constructed can be explored and interpreted within Cytoscape software and provide a new systems biology approach for the Drosophila research community to better visualize and interpret developmental regulation of gene expression.
Yang, Z., Zhu, H., Kong, K., Wu, X., Chen, J., Li, P., Jiang, J., Zhao, J., Cui, B. and Liu, F. (2020). The dynamic transmission of positional information in stau-mutants during Drosophila embryogenesis. Elife 9. PubMed ID: 32511091
It has been suggested that Staufen (Stau) is key in controlling the variability of the posterior boundary of the Hb anterior domain (x(Hb)). However, its underlying mechanism is elusive. This study quantified the dynamic 3D expression of segmentation genes in Drosophila embryos. With improved control of measurement errors, it was show nx(Hb) of stau- mutants reproducibly moves posteriorly by 10% of the embryo length (EL) to the wild type (WT) position in the nuclear cycle (nc) 14, and its variability at short time windows is comparable as that of the WT. Moreover, for stau- mutants, the upstream Bicoid (Bcd) gradients show equivalent relative intensity noise to that of the WT in nc12-nc14, and the downstream Even-skipped (Eve) and cephalic furrow (CF) show the same positional errors as the WT. These results indicate that threshold-dependent activation and self-organized filtering are not mutually exclusive but could both be implemented in early Drosophila embryogenesis.
Wang, X., Merkel, M., Sutter, L. B., Erdemci-Tandogan, G., Manning, M. L. and Kasza, K. E. (2020). Anisotropy links cell shapes to tissue flow during convergent extension. Proc Natl Acad Sci U S A. PubMed ID: 32467168
The mechanisms that allow or prevent tissue reorganization, especially in the presence of strongly anisotropic forces, remain unclear. This question was studied in the converging and extending Drosophila germband epithelium, which displays planar-polarized myosin II and experiences anisotropic forces from neighboring tissues. In contrast to isotropic tissues, cell shape alone is not sufficient to predict the onset of rapid cell rearrangement. From theoretical considerations and vertex model simulations, it is predicted that in anisotropic tissues, two experimentally accessible metrics of cell patterns-the cell shape index and a cell alignment index-are required to determine whether an anisotropic tissue is in a solid-like or fluid-like state. Changes in cell shape and alignment over time in the Drosophila germband were shown to predict the onset of rapid cell rearrangement in both wild-type and snail twist mutant embryos, where theoretical prediction is further improved when cell packing disorder is also accounted for. These findings suggest that convergent extension is associated with a transition to more fluid-like tissue behavior, which may help accommodate tissue-shape changes during rapid developmental events.
Irizarry, J., McGehee, J., Kim, G., Stein, D. and Stathopoulos, A. (2020). Twist-dependent ratchet functioning downstream from Dorsal revealed using a light-inducible degron. Genes Dev. PubMed ID: 32467225
Graded transcription factors are pivotal regulators of embryonic patterning, but whether their role changes over time is unclear. A light-regulated protein degradation system was used to assay temporal dependence of the transcription factor Dorsal in dorsal-ventral axis patterning of Drosophila embryos. Surprisingly, the high-threshold target gene snail only requires Dorsal input early but not late when Dorsal levels peak. Instead, late snail expression can be supported by action of the Twist transcription factor, specifically, through one enhancer, sna.distal. This study demonstrates that continuous input is not required for some Dorsal targets and downstream responses, such as twist, function as molecular ratchets.
Henn, L., ..., Boros. I. M. (2020). Alternative linker histone permits fast paced nuclear divisions in early Drosophila embryo. Nucleic Acids Res. PubMed ID: 32710625
In most animals, the start of embryogenesis requires specific histones. In Drosophila linker histone variant BigH1 is present in early embryos. To uncover the specific role of this alternative linker histone at early embryogenesis, fly lines were established in which domains of BigH1 have been replaced partially or completely with that of H1. Analysis of the resulting Drosophila lines revealed that at normal temperature somatic H1 can substitute the alternative linker histone, but at low temperature the globular and C-terminal domains of BigH1 are essential for embryogenesis. In the presence of BigH1 nucleosome stability increases and core histone incorporation into nucleosomes is more rapid, while nucleosome spacing is unchanged. Chromatin formation in the presence of BigH1 permits the fast-paced nuclear divisions of the early embryo. A model is proposed that explains how this specific linker histone ensures the rapid nucleosome reassembly required during quick replication cycles at the start of embryogenesis.

Friday, July 24th - Disease Models

Stankovic, D., Claudius, A. K., Schertel, T., Bresser, T. and Uhlirova, M. (2020). Drosophila model to study Retinitis pigmentosa pathology associated with mutations in the core splicing factor Prp8. Dis Model Mech. PubMed ID: 32424050
Retinitis pigmentosa (RP) represents genetically heterogeneous and clinically variable disease characterized by progressive degeneration of photoreceptors resulting in a gradual loss of vision. The autosomal dominant RP type 13 (RP13) has been linked to the malfunction of PRPF8, the essential component of the spliceosome. Over twenty different RP-associated PRPF8 mutations have been identified in human patients. However, the cellular and molecular consequences of their expression in vivo in specific tissue contexts remain largely unknown. This study establish the Drosophila melanogaster model for RP13 by introducing the nine distinct RP mutations into the fly Prp8 ortholog and expressing these mutant proteins in precise spatiotemporal patterns using the Gal4/UAS system. All nine RP-Prp8 mutations negatively impacted developmental timing, albeit to a different extent, when expressed in the endocrine cells producing the primary insect molting hormone. In the developing eye primordium, uncommitted epithelial precursors rather than differentiated photoreceptors appeared sensitive to Prp8 malfunction. Expression of the two most pathogenic variants, Prp8(S>F) and Prp8(H>R), induced apoptosis causing alterations to the adult eye morphology. The affected tissue mounted stress and cytoprotective responses, while genetic programs underlying neuronal function were attenuated. Importantly, the penetrance and expressivity increased under prp8 heterozygosity. In contrast, blocking apoptosis alleviated cell loss but not the redox imbalance. Remarkably, the pathogenicity of the RP-Prp8 mutations in the Drosophila correlates with the severity of clinical phenotypes in patients carrying the equivalent mutations highlighting the suitability of the Drosophila model for in-depth functional studies of the mechanisms underlying RP13 etiology.
Minakawa, E. N., Popiel, H. A., Tada, M., Takahashi, T., Yamane, H., Saitoh, Y., Takahashi, Y., Ozawa, D., Takeda, A., Takeuchi, T., Okamoto, Y., Yamamoto, K., Suzuki, M., Fujita, H., Ito, C., Yagihara, H., Saito, Y., Watase, K., Adachi, H., Katsuno, M., Mochizuki, H., Shiraki, K., Sobue, G., Toda, T., Wada, K., Onodera, O. and Nagai, Y. (2020). Arginine is a disease modifier for polyQ disease models that stabilizes polyQ protein conformation. Brain. PubMed ID: 32436573
The polyglutamine (polyQ) diseases are a group of inherited neurodegenerative diseases that include Huntington's disease, various spinocerebellar ataxias, spinal and bulbar muscular atrophy, and dentatorubral pallidoluysian atrophy. They are caused by the abnormal expansion of a CAG repeat coding for the polyQ stretch in the causative gene of each disease. The expanded polyQ stretches trigger abnormal β-sheet conformational transition and oligomerization followed by aggregation of the polyQ proteins in the affected neurons, leading to neuronal toxicity and neurodegeneration. Disease-modifying therapies that attenuate both symptoms and molecular pathogenesis of polyQ diseases remain an unmet clinical need. This study identified arginine, a chemical chaperone that facilitates proper protein folding, as a novel compound that targets the upstream processes of polyQ protein aggregation by stabilizing the polyQ protein conformation. Representative chemical chaperones were screened using an in vitro polyQ aggregation assay, and arginine was identified as a potent polyQ aggregation inhibitor. In vitro and cellular assays revealed that arginine exerts its anti-aggregation property by inhibiting the toxic β-sheet conformational transition and oligomerization of polyQ proteins before the formation of insoluble aggregates. Arginine exhibited therapeutic effects on neurological symptoms and protein aggregation pathology in Caenorhabditis elegans, Drosophila, and two different mouse models of polyQ diseases. Arginine was also effective in a polyQ mouse model when administered after symptom onset. As arginine has been safely used for urea cycle defects and for mitochondrial myopathy, encephalopathy, lactic acid and stroke syndrome patients, and efficiently crosses the blood-brain barrier, a drug-repositioning approach for arginine would enable prompt clinical application as a promising disease-modifier drug for the polyQ diseases.
Ruzzi, L. R., Schilman, P. E., San Martin, A., Lew, S. E., Gelb, B. D. and Pagani, M. R. (2020). The Phosphatase CSW Controls Life Span by Insulin Signaling and Metabolism Throughout Adult Life in Drosophila. Front Genet 11: 364. PubMed ID: 32457793
Noonan syndrome and related disorders are caused by mutations in genes encoding for proteins of the RAS-ERK1/2 signaling pathway, which affect development by enhanced ERK1/2 activity. However, the mutations' effects throughout adult life are unclear. This study identified that the protein most commonly affected in Noonan syndrome, the phosphatase SHP2, known in Drosophila as corkscrew (CSW), controls life span, triglyceride levels, and metabolism without affecting ERK signaling pathway. This study found that CSW loss-of-function mutations extended life span by interacting with components of the insulin signaling pathway and impairing AKT activity in adult flies. By expressing csw-RNAi in different organs, it was determined that CSW extended life span by acting in organs that regulate energy availability, including gut, fat body and neurons. In contrast to that in control animals, loss of CSW leads to reduced homeostasis in metabolic rate during activity. Clinically relevant gain-of-function csw allele reduced life span, when expressed in fat body, but not in other tissues. However, overexpression of a wild-type allele did not affect life span, showing a specific effect of the gain-of-function allele independently of a gene dosage effect. It is concluded that CSW normally regulates life span and that mutations in SHP2 are expected to have critical effects throughout life by insulin-dependent mechanisms in addition to the well-known RAS-ERK1/2-dependent developmental alterations.
Raimer, A. C., Singh, S. S., Edula, M. R., Paris-Davila, T., Vandadi, V., Spring, A. M. and Matera, A. G. (2020). Temperature-sensitive spinal muscular atrophy-causing point mutations lead to SMN instability, locomotor defects and premature lethality in Drosophila. Dis Model Mech 13(5). PubMed ID: 32501283
Spinal muscular atrophy (SMA) is the leading genetic cause of death in young children, arising from homozygous deletion or mutation of the survival motor neuron 1 (SMN1) gene. SMN protein expressed from a paralogous gene, SMN2, is the primary genetic modifier of SMA; small changes in overall SMN levels cause dramatic changes in disease severity. Thus, deeper insight into mechanisms that regulate SMN protein stability should lead to better therapeutic outcomes. This study shows that SMA patient-derived missense mutations in the Drosophila SMN Tudor domain exhibit a pronounced temperature sensitivity that affects organismal viability, larval locomotor function and adult longevity. These disease-related phenotypes are domain specific and result from decreased SMN stability at elevated temperature. This system was utilized to manipulate SMN levels during various stages of Drosophila development. Owing to a large maternal contribution of mRNA and protein, Smn is not expressed zygotically during embryogenesis. Interestingly, it was found that only baseline levels of SMN are required during larval stages, whereas high levels of the protein are required during pupation. This previously uncharacterized period of elevated SMN expression, during which the majority of adult tissues are formed and differentiated, could be an important and translationally relevant developmental stage in which to study SMN function. Taken together, these findings illustrate a novel in vivo role for the SMN Tudor domain in maintaining SMN homeostasis and highlight the necessity for high SMN levels at crucial developmental time points that are conserved from Drosophila to humans.
Wen, X., An, P., Li, H., Zhou, Z., Sun, Y., Wang, J., Ma, L. and Lu, B. (2020). Tau Accumulation via Reduced Autophagy Mediates GGGGCC Repeat Expansion-Induced Neurodegeneration in Drosophila Model of ALS. Neurosci Bull. PubMed ID: 32500377
Expansions of trinucleotide or hexanucleotide repeats lead to several neurodegenerative disorders, including Huntington disease [caused by expanded CAG repeats (CAGr) in the HTT gene], and amyotrophic lateral sclerosis [ALS, possibly caused by expanded GGGGCC repeats (G4C2r) in the C9ORF72 gene], of which the molecular mechanisms remain unclear. This study demonstrated that lowering the Drosophila homologue of tau protein (dtau) significantly rescued in vivo neurodegeneration, motor performance impairments, and the shortened life-span in Drosophila expressing expanded CAGr or expanded G4C2r. Expression of human tau (htau4R) restored the disease-related phenotypes that had been mitigated by the loss of dtau, suggesting an evolutionarily-conserved role of tau in neurodegeneration. This study further revealed that G4C2r expression increased tau accumulation by inhibiting autophagosome-lysosome fusion, possibly due to lowering the level of BAG3, a regulator of autophagy and tau. Taken together, these results reveal a novel mechanism by which expanded G4C2r causes neurodegeneration via an evolutionarily-conserved mechanism. These findings provide novel autophagy-related mechanistic insights into C9ORF72-ALS and possible entry points to disease treatment.
Sanna, S., Esposito, S., Masala, A., Sini, P., Nieddu, G., Galioto, M., Fais, M., Iaccarino, C., Cestra, G. and Crosio, C. (2020). HDAC1 inhibition ameliorates TDP-43-induced cell death in vitro and in vivo. Cell Death Dis 11(5): 369. PubMed ID: 32409664
TDP-43 pathology is a disease hallmark that characterizes both amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-TDP). TDP-43 undergoes several posttranslational modifications that can change its biological activities and its aggregative propensity, which is a common hallmark of different neurodegenerative conditions. New evidence is provided by the current study pointing at TDP-43 acetylation in ALS cellular models. Using both in vitro and in vivo approaches, it was demonstrated that TDP-43 interacts with histone deacetylase 1 (HDAC1) via RRM1 and RRM2 domains, that are known to contain the two major TDP-43 acetylation sites, K142 and K192. Moreover, this study showed that TDP-43 is a direct transcriptional activator of CHOP promoter and this activity is regulated by acetylation. Finally and most importantly, it was observed both in cell culture and in Drosophila that a HDCA1 reduced level (genomic inactivation or siRNA) or treatment with pan-HDAC inhibitors exert a protective role against WT or pathological mutant TDP-43 toxicity, suggesting TDP-43 acetylation as a new potential therapeutic target. HDAC inhibition efficacy in neurodegeneration has long been debated, but future investigations are warranted in this area. Selection of more specific HDAC inhibitors is still a promising option for neuronal protection especially as HDAC1 appears as a downstream target of both TDP- 43 and FUS, another ALS-related gene.

Thursday, July 23rd - Adult Physiology

von Frieling, J., Faisal, M. N., Sporn, F., Pfefferkorn, R., Nolte, S. S., Sommer, F., Rosenstiel, P. and Roeder, T. (2020). A high-fat diet induces a microbiota-dependent increase in stem cell activity in the Drosophila intestine. PLoS Genet 16(5): e1008789. PubMed ID: 32453733
Over-consumption of high-fat diets (HFDs) is associated with several pathologies. Although the intestine is the organ that comes into direct contact with all diet components, the impact of HFD has mostly been studied in organs that are linked to obesity and obesity related disorders. Drosophila was used as a simple model to disentangle the effects of a HFD on the intestinal structure and physiology from the plethora of other effects caused by this nutritional intervention. A HFD, composed of triglycerides with saturated fatty acids, was shown to trigger activation of intestinal stem cells in the Drosophila midgut. This stem cell activation was transient and dependent on the presence of an intestinal microbiota, as it was completely absent in germ free animals. Moreover, major components of the signal transduction pathway have been elucidated. In this study, JNK (basket) in enterocytes was necessary to trigger synthesis of the cytokine upd3 in these cells. This ligand in turn activated the JAK/STAT pathway in intestinal stem cells. Chronic subjection to a HFD markedly altered both the microbiota composition and the bacterial load. Although HFD-induced stem cell activity was transient, long-lasting changes to the cellular composition, including a substantial increase in the number of enteroendocrine cells, were observed. Taken together, a HFD enhances stem cell activity in the Drosophila gut and this effect is completely reliant on the indigenous microbiota and also dependent on JNK signaling within intestinal enterocytes.
Wen, D. T., Wang, W. Q., Hou, W. Q., Cai, S. X. and Zhai, S. S. (2020). Endurance exercise protects aging Drosophila from high-salt diet (HSD)-induced climbing capacity decline and lifespan decrease by enhancing antioxidant capacity. Biol Open 9(5). PubMed ID: 32414766
A high-salt diet (HSD) is a major cause of many chronic and age-related defects such as myocardial hypertrophy, locomotor impairment and mortality. Exercise training can efficiently prevent and treat many chronic and age-related diseases. However, it remains unclear whether endurance exercise can resist HSD-induced impairment of climbing capacity and longevity in aging individuals. In this study, flies were given exercise training and fed a HSD from 1-week old to 5-weeks old. Overexpression or knockdown of salt, encoding a sodium/solute symporter in the SLC5 family involved in response to salt stress, and dFOXO were accomplished by UAS/Gal4 system. The results showed that a HSD, salt gene overexpression and dFOXO knockdown significantly reduced climbing endurance, climbing index, survival, dFOXO expression and SOD activity level, and increased malondialdehyde level in aging flies. Inversely, in a HSD aging flies, endurance exercise and dFOXO overexpression significantly increased their climbing ability, lifespan and antioxidant capacity, but they did not significantly change the salt gene expression. Overall, current results indicated that a HSD accelerated the age-related decline of climbing capacity and mortality via upregulating salt expression and inhibiting the dFOXO/SOD pathway. Increased dFOXO/SOD pathway activity played a key role in mediating endurance exercise resistance to the low salt tolerance-induced impairment of climbing capacity and longevity in aging Drosophila.
Shimada, S., Kamiya, M., Shigetou, S., Tomiyama, K., Komori, Y., Magara, L., Ihara, M. and Matsuda, K. (2020). The mechanism of loop C-neonicotinoid interactions at insect nicotinic acetylcholine receptor α1 subunit predicts resistance emergence in pests. Sci Rep 10(1): 7529. PubMed ID: 32371996
Neonicotinoids selectively modulate insect nicotinic acetylcholine receptors (insect nAChRs). Studies have shown that serine with ability to form a hydrogen bond in loop C of some insect nAChR α subunits and glutamate with a negative charge at the corresponding position in vertebrate nAChRs may contribute to enhancing and reducing the neonicotinoid actions, respectively. However, there is no clear evidence what loop C properties underpin the target site actions of neonicotinoids. Thus, this study has investigated the effects of S221A and S221Q mutations in loop C of the Drosophila melanogaster Dα1 subunit on the agonist activity of imidacloprid and thiacloprid for Dα1/chicken β2 nAChRs expressed in Xenopus laevis oocytes. The S221A mutation hardly affected either the affinity or efficacy for ACh and imidacloprid, whereas it only slightly reduced the efficacy for thiacloprid on the nAChRs with a higher composition ratio of β2 to Dα1 subunits. The S221Q mutation markedly reduced the efficacy of the neonicotinoids for the nAChRs with a higher composition of the β2 subunit lacking basic residues critical for binding neonicotinoids. Hence, the possibility exists of enhanced neonicotinoid resistance in pest insect species by a mutation of the serine when it occurs in the R81T resistant populations lacking the basic residue in loop D of the β1 subunit.
Simoes, P., Santos, M. A., Carromeu-Santos, A., Quina, A. S., Santos, M. and Matos, M. (2020). Beneficial developmental acclimation in reproductive performance under cold but not heat stress. J Therm Biol 90: 102580. PubMed ID: 32479384
Thermal plasticity can help organisms coping with climate change. This study analysed how laboratory populations of the ectotherm species Drosophila subobscura, originally from two distinct latitudes and evolving for several generations in a stable thermal environment (18°C), respond plastically to new thermal challenges. Adult performance (fecundity traits as a fitness proxy) was measured of the experimental populations when exposed to five thermal regimes, three with the same temperature during development and adulthood (15-15°C, 18-18°C, 25-25°C), and two where flies developed at 18°C and were exposed, during adulthood, to either 15°C or 25°C. This study testd whether (1) flies undergo stress at the two more extreme temperatures; (2) development at a given temperature enhances adult performance at such temperature (i.e. acclimation), and (3) populations with different biogeographical history show plasticity differences. The findings show (1) an optimal performance at 18°C only if flies were subjected to the same temperature as juveniles and adults; (2) the occurrence of developmental acclimation at lower temperatures; (3) detrimental effects of higher developmental temperature on adult performance; and (4) a minor impact of historical background on thermal response. This study indicates that thermal plasticity during development may have a limited role in helping adults cope with warmer - though not colder - temperatures, with a potential negative impact on population persistence under climate change. It also emphasizes the importance of analysing the impact of temperature on all stages of the life cycle to better characterize the thermal limits.
Seong, K. H., Ly, N. H., Katou, Y., Yokota, N., Nakato, R., Murakami, S., Hirayama, A., Fukuda, S., Kang, S., Soga, T., Shirahige, K. and Ishii, S. (2020). Paternal restraint stress affects offspring metabolism via ATF-2 dependent mechanisms in Drosophila melanogaster germ cells. Commun Biol 3(1): 208. PubMed ID: 32367035
Paternal environmental factors can epigenetically influence gene expressions in offspring. This study demonstrates that restraint stress, an experimental model for strong psychological stress, to fathers affects the epigenome, transcriptome, and metabolome of offspring in a MEKK1-dATF2 pathway-dependent manner in Drosophila melanogaster. Genes involved in amino acid metabolism are upregulated by paternal restraint stress, while genes involved in glycolysis and the tricarboxylic acid (TCA) cycle are downregulated. The effects of paternal restraint stress are also confirmed by metabolome analysis. dATF-2 is highly expressed in testicular germ cells, and restraint stress also induces p38 activation in the testes. Restraint stress induces Unpaired 3 (Upd3), a Drosophila homolog of Interleukin 6 (IL-6). Moreover, paternal overexpression of upd3 in somatic cells disrupts heterochromatin in offspring but not in offspring from dATF-2 mutant fathers. These results indicate that paternal restraint stress affects metabolism in offspring via inheritance of dATF-2-dependent epigenetic changes.
Sorge, S., Theelke, J., Yildirim, K., Hertenstein, H., McMullen, E., Muller, S., Altburger, C., Schirmeier, S. and Lohmann, I. (2020). ATF4-Induced Warburg Metabolism Drives Over-Proliferation in Drosophila. Cell Rep 31(7): 107659. PubMed ID: 32433968
The mitochondrial electron transport chain (ETC) enables essential metabolic reactions; nonetheless, the cellular responses to defects in mitochondria and the modulation of signaling pathway outputs are not understood. This study shows that Notch signaling and ETC attenuation via knockdown of COX7a induces massive over-proliferation. The tumor-like growth is caused by a transcriptional response through the eIF2α-kinase PERK and ATF4, which activates the expression of metabolic enzymes, nutrient transporters, and mitochondrial chaperones. This stress adaptation is found to be beneficial for progenitor cell fitness, as it renders cells sensitive to proliferation induced by the Notch signaling pathway. Intriguingly, over-proliferation is not caused by transcriptional cooperation of Notch and ATF4, but it is mediated in part by pH changes resulting from the Warburg metabolism induced by ETC attenuation. These results suggest that ETC function is monitored by the PERK-ATF4 pathway, which can be hijacked by growth-promoting signaling pathways, leading to oncogenic pathway activity.

Wednesday, July 22nd - Gonadogenesis

Rowe, M., Paculis, L., Tapia, F., Xu, Q., Xie, Q., Liu, M., Jevitt, A. and Jia, D. (2020). Analysis of the Temporal Patterning of Notch Downstream Targets during Drosophila melanogaster Egg Chamber Development. Sci Rep 10(1): 7370. PubMed ID: 32355165
Living organisms require complex signaling interactions and proper regulation of these interactions to influence biological processes. Of these complex networks, one of the most distinguished is the Notch pathway. Dysregulation of this pathway often results in defects during organismal development and can be a causative mechanism for initiation and progression of cancer. Despite previous research entailing the importance of this signaling pathway and the organismal processes that it is involved in, less is known concerning the major Notch downstream targets, especially the onset and sequence in which they are modulated during normal development. As timing of regulation may be linked to many biological processes, this study investigated and established a model of temporal patterning of major Notch downstream targets including broad, cut, and hindsight during Drosophila melanogaster egg chamber development. It was confirmed the sequential order of Broad upregulation, Hindsight upregulation, and Cut downregulation. In addition, Notch signaling could be activated at stage 4, one stage earlier than the stage 5, a previously long-held belief. However, further mitotic marker analysis re-stated that mitotic cycle continues until stage 5. Through this study, the effectiveness and reliability of the MATLAB toolbox, designed to systematically identify egg chamber stages based on area size, ratio, and additional morphological characteristics, was once again validated.
Chen, Z., Wang, Z. H., Zhang, G., Bleck, C. K. E., Chung, D. J., Madison, G. P., Lindberg, E., Combs, C., Balaban, R. S. and Xu, H. (2020). Mitochondrial DNA segregation and replication restrict the transmission of detrimental mutation. J Cell Biol 219(7). PubMed ID: 32375181
Although mitochondrial DNA (mtDNA) is prone to accumulate mutations and lacks conventional DNA repair mechanisms, deleterious mutations are exceedingly rare. How the transmission of detrimental mtDNA mutations is restricted through the maternal lineage is debated. This study demonstrated that mitochondrial fission, together with the lack of mtDNA replication, segregate mtDNA into individual organelles in the Drosophila early germarium. After mtDNA segregation, mtDNA transcription begins, which activates respiration. Mitochondria harboring wild-type genomes have functional electron transport chains and propagate more vigorously than mitochondria containing deleterious mutations in hetreoplasmic cells. Therefore, mtDNA expression acts as a stress test for the integrity of mitochondrial genomes and sets the stage for replication competition. These observations support selective inheritance at the organelle level through a series of developmentally orchestrated mitochondrial processes. This study also showed that the Balbiani body has a minor role in mtDNA selective inheritance by supplying healthy mitochondria to the pole plasm. These two mechanisms may act synergistically to secure the transmission of functional mtDNA through Drosophila oogenesis.
Fox, E. F., Lamb, M. C., Mellentine, S. Q. and Tootle, T. L. (2020). Prostaglandins regulate invasive, collective border cell migration. Mol Biol Cell: mbcE19100578. PubMed ID: 32432969
While prostaglandins (PGs), short-range lipid signals, regulate single cell migration, their roles in collective migration remain unclear. To address this, Drosophila border cell migration, an invasive, collective migration that occurs during Stage 9 of oogenesis, was used. Pxt is the Drosophila cyclooxygenase-like enzyme responsible for PG synthesis. Loss of Pxt results in both delayed border cell migration and elongated clusters, whereas somatic Pxt knockdown causes delayed migration and compacted clusters. These findings suggest PGs act in both the border cells and nurse cells, the substrate on which the border cells migrate. As PGs regulate the actin bundler Fascin, and Fascin is required in for on-time migration, this study assessed whether PGs regulate Fascin to promote border cell migration. Co-reduction of Pxt and Fascin results in delayed migration and elongated clusters. The latter may be due to altered cell adhesion, as loss of Pxt or Fascin, or co-reduction of both, decreases integrin levels on the border cell membranes. Conversely, integrin localization is unaffected by somatic knockdown of Pxt. Together these data lead to the model that PG signaling controls Fascin in the border cells to promote migration and in the nurse cells to maintain cluster cohesion.
Tu, R., Duan, B., Song, X. and Xie, T. (2020). Dlp-mediated Hh and Wnt signaling interdependence is critical in the niche for germline stem cell progeny differentiation. Sci Adv 6(20): eaaz0480. PubMed ID: 32426496
Although multiple signaling pathways work synergistically in various niches to control stem cell self-renewal and differentiation, it remains poorly understood how they cooperate with one another molecularly. In the Drosophila ovary, Hh and Wnt pathways function in the niche to promote germline stem cell (GSC) progeny differentiation. This study shows that glypican Dlp-mediated Hh and Wnt signaling interdependence operates in the niche to promote GSC progeny differentiation by preventing BMP signaling. Hh/Wnt-mediated dlp repression is essential for their signaling interdependence in niche cells and for GSC progeny differentiation by preventing BMP signaling. Mechanistically, Hh and Wnt downstream transcription factors directly bind to the same dlp regulatory region and recruit corepressors composed of transcription factor Croc and Egg/H3K9 trimethylase to repress Dlp expression. Therefore, this study reveals a novel mechanism for Hh/Wnt signaling-mediated direct dlp repression and a novel regulatory mechanism for Dlp-mediated Hh/Wnt signaling interdependence in the GSC differentiation niche.
Ota, R. and Kobayashi, S. (2020). Myc plays an important role in Drosophila P-M hybrid dysgenesis to eliminate germline cells with genetic damage. Commun Biol 3(1): 185. PubMed ID: 32322015
Genetic damage in the germline induced by P-element mobilization causes a syndrome known as P-M hybrid dysgenesis (HD), which manifests as elevated mutation frequency and loss of germline cells. This study found that Myc plays an important role in eliminating germline cells in the context of HD. P-element mobilization resulted in downregulation of Myc expression in the germline. Myc knockdown caused germline elimination; conversely, Myc overexpression rescued the germline loss caused by P-element mobilization. Moreover, restoration of fertility by Myc resulted in the production of gametes with elevated mutation frequency and reduced ability to undergo development. These results demonstrate that Myc downregulation mediates elimination of germline cells with accumulated genetic damage, and that failure to remove these cells results in increased production of aberrant gametes. Therefore, it is proposed that elimination of germline cells mediated by Myc downregulation is a quality control mechanism that maintains the genomic integrity of the germline.
Popkova, A., Stone, O. J., Chen, L., Qin, X., Liu, C., Liu, J., Belguise, K., Montell, D. J., Hahn, K. M., Rauzi, M. and Wang, X. (2020). A Cdc42-mediated supracellular network drives polarized forces and Drosophila egg chamber extension. Nat Commun 11(1): 1921. PubMed ID: 32317641
Actomyosin supracellular networks emerge during development and tissue repair. These cytoskeletal structures are able to generate large scale forces that can extensively remodel epithelia driving tissue buckling, closure and extension. How supracellular networks emerge, are controlled and mechanically work still remain elusive. During Drosophila oogenesis, the egg chamber elongates along the anterior-posterior axis. This study shows that a dorsal-ventral polarized supracellular F-actin network, running around the egg chamber on the basal side of follicle cells, emerges from polarized intercellular filopodia that radiate from basal stress fibers and extend penetrating neighboring cell cortexes. Filopodia can be mechanosensitive and function as cell-cell anchoring sites. The small GTPase Cdc42 governs the formation and distribution of intercellular filopodia and stress fibers in follicle cells. Finally, this study shows that a Cdc42-dependent supracellular cytoskeletal network provides a scaffold integrating local oscillatory actomyosin contractions at the tissue scale to drive global polarized forces and tissue elongation.

Tuesday, July 21st - Immune Response

Cammarata-Mouchtouris, A., Nguyen, X. H., Acker, A., Bonnay, F., Goto, A., Orian, A., Fauvarque, M. O., Boutros, M., Reichhart, J. M. and Matt, N. (2020). Hyd ubiquitinates the NF-kappaB co-factor Akirin to operate an effective immune response in Drosophila. PLoS Pathog 16(4): e1008458. PubMed ID: 32339205
The Immune Deficiency (IMD) pathway in Drosophila melanogaster is activated upon microbial challenge with Gram-negative bacteria to trigger the innate immune response. In order to decipher this nuclear factor kappaB (NF-kappaB) signaling pathway, an in vitro RNAi screen was undertaken targeting E3 ubiquitin ligases specifically, and the HECT-type E3 ubiquitin ligase Hyperplastic discs (Hyd) was identified as a new actor in the IMD pathway. Hyd mediated Lys63 (K63)-linked polyubiquitination of the NF-kappaB cofactor Akirin was identified as being required for efficient binding of Akirin to the NF-kappaB transcription factor Relish. This Hyd-dependent interaction was required for the transcription of immunity-related genes that are activated by both Relish and Akirin but was dispensable for the transcription of genes that depend solely on Relish. Therefore Hyd is key in NF-kappaB transcriptional selectivity downstream of the IMD pathway. Drosophila depleted of Akirin or Hyd failed to express the full set of genes encoding immune-induced anti-microbial peptides and succumbed to immune challenges. It was further shown that UBR5, the mammalian homolog of Hyd, was also required downstream of the NF-kappaB pathway for the activation of Interleukin 6 (IL6) transcription by IL-1beta in cultured human cells. These findings link the action of an E3 ubiquitin ligase to the activation of immune effector genes, deepening the understanding of the involvement of ubiquitination in inflammation and identifying a potential target for the control of inflammatory diseases.
Valanne, S., Jarvela-Stolting, M., Harjula, S. E., Myllymaki, H., Salminen, T. S. and Ramet, M. (2020). Osa-Containing Brahma Complex Regulates Innate Immunity and the Expression of Metabolic Genes in Drosophila. J Immunol. PubMed ID: 32198143
Negative regulation of innate immunity is essential to avoid autoinflammation. In Drosophila melanogaster, NF-kappaB signaling-mediated immune responses are negatively regulated at multiple levels. Using a Drosophila RNA interference in vitro screen, this study identified a set of genes inhibiting immune activation. Four of these genes encode members of the chromatin remodeling Osa-containing Brahma (BAP) complex. Silencing additional two genes of the BAP complex was shown to have the same phenotype, confirming its role in immune regulation in vitro. In vivo, the knockdown of osa and brahma was shown to enhance the expression of the Toll pathway-mediated antimicrobial peptides when the flies were challenged with Gram-positive bacteria Micrococcus luteus. In this setting, osa knockdown had a particularly strong effect on immune effectors that are predominantly activated by the Imd pathway. Accordingly, Drosophila NF-kappaB Relish expression was increased by osa silencing. These transcriptional changes were associated with enhanced survival from M. luteus + E. faecalis infection. Besides regulating the expression of immune effector genes, osa RNA interference decreased the expression of a large group of genes involved in metabolism, particularly proteolysis. Of note, the expression of the recently characterized, immune-inducible gene Induced by Infection (IBIN) was diminished in osa knockdown flies. Although IBIN has been shown to modulate metabolism upon infection, the expression of selected Osa-regulated metabolism genes was not rescued by overexpressing IBIN. It is concluded that the BAP complex regulates expression of genes involved in metabolism at least partially independent or downstream of IBIN. Moreover, Osa affects the NF-kappaB-mediated immune response by regulating Drosophila NF-kappaB factor Relish expression.
Chapman, J. R., Dowell, M. A., Chan, R. and Unckless, R. L. (2020). The Genetic Basis of Natural Variation in Drosophila melanogaster Immune Defense against Enterococcus faecalis. Genes (Basel) 11(2). PubMed ID: 32098395
Dissecting the genetic basis of natural variation in disease response in hosts provides insights into the coevolutionary dynamics of host-pathogen interactions. A genome-wide association study of Drosophila melanogaster survival after infection with the Gram-positive entomopathogenic bacterium Enterococcus faecalis is reported. There was considerable variation in defense against E. faecalis infection among inbred lines of the Drosophila Genetics Reference Panel. Single nucleotide polymorphisms were identified associated with six genes with a significant association with survival, none of which were canonical immune genes. To validate the role of these genes in immune defense, their expression was knocked-down using RNAi and survival of infected hosts was followed, confirming a role for the genes krishah and S6k in immune defense. A putative role was identified for the Bomanin gene BomBc1 (also known as IM23), in E. faecalis infection response. This study adds to the growing set of association studies for infection in Drosophila melanogaster and suggests that the genetic causes of variation in immune defense differ for different pathogens.
Iatsenko, I., Marra, A., Boquete, J. P., Pena, J. and Lemaitre, B. (2020). Iron sequestration by transferrin 1 mediates nutritional immunity in Drosophila melanogaster. Proc Natl Acad Sci U S A. PubMed ID: 32188787
This study used Drosophila as an in vivo model to investigate the role of transferrins in host defense. Systemic infections with a variety of pathogens trigger a hypoferremic response in flies, namely, iron withdrawal from the hemolymph and accumulation in the fat body. Notably, this hypoferremia to infection requires NF-kappaB immune pathways, Toll and Imd, revealing that these pathways also mediate nutritional immunity in flies. The iron transporter Transferrin 1 (Tsf1) was shown to be induced by infections downstream of the Toll and Imd pathways and is necessary for iron relocation from the hemolymph to the fat body. Consistent with elevated iron levels in the hemolymph, Tsf1 mutants exhibited increased susceptibility to Pseudomonas bacteria and Mucorales fungi, which could be rescued by chemical chelation of iron. Furthermore, using siderophore-deficient Pseudomonas aeruginosa, it was discover that the siderophore pyoverdine is necessary for pathogenesis in wild-type flies, but it becomes dispensable in Tsf1 mutants due to excessive iron present in the hemolymph of these flies. As such, this study reveals that, similar to mammals, Drosophila uses iron limitation as an immune defense mechanism mediated by conserved iron-transporting proteins transferrins. This in vivo work, together with accumulating in vitro studies, supports the immune role of insect transferrins against infections via an iron withholding strategy.
Tattikota, S. G., Cho, B., Liu, Y., Hu, Y., Barrera, V., Steinbaugh, M. J., Yoon, S. H., Comjean, A., Li, F., Dervis, F., Hung, R. J., Nam, J. W., Ho Sui, S., Shim, J. and Perrimon, N. (2020). A single-cell survey of Drosophila blood. Elife 9. PubMed ID: 32396065
Drosophila blood cells, called hemocytes, are classified into plasmatocytes, crystal cells, and lamellocytes based on the expression of a few marker genes and cell morphologies, which are inadequate to classify the complete hemocyte repertoire. This study used single-cell RNA sequencing (scRNA-seq) to map hemocytes across different inflammatory conditions in larvae. Plasmatocytes were resolved into different states based on the expression of genes involved in cell cycle, antimicrobial response, and metabolism, together with the identification of intermediate states. Further, rare subsets within crystal cells and lamellocytes were discovered that express fibroblast growth factor (FGF) ligand branchless and receptor breathless, respectively. These FGF components were identified as required for mediating effective immune responses against parasitoid wasp eggs, highlighting a novel role for FGF signaling in inter-hemocyte crosstalk. This scRNA-seq analysis reveals the diversity of hemocytes and provides a rich resource of gene expression profiles for a systems-level understanding of their functions.
Fu, Y., Huang, X., Zhang, P., van de Leemput, J. and Han, Z. (2020). Single-cell RNA sequencing identifies novel cell types in Drosophila blood. J Genet Genomics. PubMed ID: 32487456
Drosophila has been extensively used to model the human blood-immune system, as both systems share many developmental and immune response mechanisms. However, while many human blood cell types have been identified, only three were found in flies: plasmatocytes, crystal cells and lamellocytes. To better understand the complexity of fly blood system, single-cell RNA sequencing technology was used to generate comprehensive gene expression profiles for Drosophila circulating blood cells. In addition to the known cell types, two new Drosophila blood cell types were identified: thanacytes and primocytes. Thanacytes, which express many stimulus response genes, are involved in distinct responses to different types of bacteria. Primocytes, which express cell fate commitment and signaling genes, appear to be involved in keeping stem cells in the circulating blood. Furthermore, the data revealed four novel plasmatocyte subtypes (Ppn(+), CAH7(+), Lsp(+) and reservoir plasmatocytes), each with unique molecular identities and distinct predicted functions. Cross-species markers from Drosophila hemocytes to human blood cells were identified. This analysis unveiled a more complex Drosophila blood system and broadened the scope of using Drosophila to model human blood system in development and disease.

Monday, July 20th - Adult neural development and function

Ogueta, M., Hardie, R. C. and Stanewsky, R. (2020). Light Sampling via Throttled Visual Phototransduction Robustly Synchronizes the Drosophila Circadian Clock. Curr Biol. PubMed ID: 32502413
The daily changes of light and dark exemplify a prominent cue for the synchronization of circadian clocks with the environment. The match between external and internal time is crucial for the fitness of organisms, and desynchronization has been linked to numerous physical and mental health problems. Organisms therefore developed complex and not fully understood mechanisms to synchronize their circadian clock to light. In mammals and in Drosophila, both the visual system and non-image-forming photoreceptors contribute to circadian clock resetting. In Drosophila, light-dependent degradation of the clock protein TIMELESS by the blue light photoreceptor Cryptochrome is considered the main mechanism for clock synchronization, although the visual system also contributes. To better understand the visual system contribution, a genetic variant exhibiting extremely slow phototransduction kinetics, yet normal sensitivity, was generated. In this variant, the visual system is able to contribute its full share to circadian clock entrainment, both with regard to behavioral and molecular light synchronization. This function depends on an alternative phospholipase C-β enzyme, encoded by PLC21C, presumably playing a dedicated role in clock resetting. This pathway requires the ubiquitin ligase CULLIN-3, possibly mediating CRY-independent degradation of TIMELESS during light:dark cycles. These results suggest that the PLC21C-mediated contribution to circadian clock entrainment operates on a drastically slower timescale compared with fast, norpA-dependent visual phototransduction. These findings are therefore consistent with the general idea that the visual system samples light over prolonged periods of time (h) in order to reliably synchronize their internal clocks with the external time.
Naidu, V. G., Zhang, Y., Lowe, S., Ray, A., Zhu, H. and Li, X. (2020). Temporal progression of Drosophila medulla neuroblasts generates the transcription factor combination to control T1 neuron morphogenesis. Dev Biol. PubMed ID: 32442418
The Drosophila medulla, part of the visual processing center of the brain, contains more than 70 neural types generated by medulla neuroblasts which sequentially express several TTFs, including Homothorax (Hth), eyeless (Ey), Sloppy paired 1 and 2 (Slp), Dichaete (D) and Tailless (Tll). However, it is not clear how such a small number of TTFs could give rise to diverse combinations of neuronal transcription factors that specify a large number of medulla neuron types. This study reports how temporal patterning specifies one neural type, the T1 neuron. The T1 neuron is the only medulla neuron type that expresses the combination of three transcription factors Ocelliless (Oc or Otd), Sox102F and Ets65A. Using CRISPR-Cas9 system, this study shows that each transcription factor is required for the correct morphogenesis of T1 neurons. Interestingly, Oc, Sox102F and Ets65A initiate expression in neurons beginning at different temporal stages and last in a few subsequent temporal stages. Oc expressing neurons are generated in the Ey, Slp and D stages; Sox102F expressing neurons are produced in the Slp and D stages; while Ets65A is expressed in subsets of medulla neurons born in the D and later stages. The TTF Ey, Slp or D is required to initiate the expression of Oc, Sox102F or Ets65A in neurons, respectively. Thus, the neurons expressing all three transcription factors are born in the D stage and become T1 neurons. In neurons where the three transcription factors do not overlap, each of the three transcription factors can act in combination with other neuronal transcription factors to specify different neural fates. This study shows that this way of expression regulation of neuronal transcription factors by temporal patterning can generate more possible combinations of transcription factors in neural progeny to diversify neural fates.
Sun, R., Delly, J., Sereno, E., Wong, S., Chen, X., Wang, Y., Huang, Y. and Greenspan, R. J. (2020). Anti-instinctive Learning Behavior Revealed by Locomotion-Triggered Mild Heat Stress in Drosophila. Front Behav Neurosci 14: 41. PubMed ID: 32372923
Anti-instinctive learning, an ability to modify an animal's innate behaviors in ways that go against one's innate tendency, can confer great evolutionary advantages to animals and enable them to better adapt to the changing environment. Yet, understanding of anti-instinctive learning and its underlying mechanisms is still limited. This work describes a new anti-instinctive learning behavior of fruit flies. This learning paradigm requires the fruit fly to respond to a recurring, aversive, mild heat stress by modifying its innate locomotion behavior. Experiencing movement-triggered mild heat stress repeatedly significantly reduced walking activity in wild type fruit flies, indicating that fruit flies are capable of anti-instinctive learning. This study also reports that such learning ability is reduced in dopamine 1-like receptor 1 (Dop1R1) null mutant and dopamine 2-like receptor (Dop2R) null mutant flies, suggesting that these two dopamine receptors are involved in mediating anti-instinctive learning in flies.
Stadele, C., Keles, M. F., Mongeau, J. M. and Frye, M. A. (2020). Non-canonical Receptive Field Properties and Neuromodulation of Feature-Detecting Neurons in Flies. Curr Biol. PubMed ID: 32442460
This study investigated 19 types of putative feature selective lobula columnar (LC) neurons in the optic lobe of the fruit fly Drosophila melanogaster to characterize divergent properties of feature selection. LC12 and LC15 were identified as feature detectors. LC15 encodes moving bars, whereas LC12 is selective for the motion of discrete objects, mostly independent of size. Neither is selective for contrast polarity, speed, or direction, highlighting key differences in the underlying algorithms for feature detection and motion vision. The onset of background motion was shown to suppress object responses by LC12 and LC15. Surprisingly, the application of octopamine, which is released during flight, reverses the suppressive influence of background motion, rendering both LCs able to track moving objects superimposed against background motion. These results provide a comparative framework for the function and modulation of feature detectors and new insights into the underlying neuronal mechanisms involved in visual feature detection.
Schuetzenberger, A. and Borst, A. (2020). Seeing Natural Images through the Eye of a Fly with Remote Focusing Two-Photon Microscopy. iScience 23(6): 101170. PubMed ID: 32502966
Visual systems of many animals, including the fruit fly Drosophila, represent the surrounding space as 2D maps, formed by populations of neurons. Advanced genetic tools make the fly visual system especially well accessible. However, in typical in vivo preparations for two-photon calcium imaging, relatively few neurons can be recorded at the same time. This study presents an extension to a conventional two-photon microscope, based on remote focusing, which enables real-time rotation of the imaging plane, and thus flexible alignment to cellular structures, without resolution or speed trade-off. This study simultaneously recorded from over 100 neighboring cells spanning the 2D retinotopic map. The map representation of moving natural images, which is found is comparable to noise predictions, was characterized. This method increases throughput 10-fold and allows visualization of a significant fraction of the fly's visual field. Furthermore, this system can be applied in general for a more flexible investigation of neural circuits.
Scaplen, K. M., Talay, M., Nunez, K. M., Salamon, S., Waterman, A. G., Gang, S., Song, S. L., Barnea, G. and Kaun, K. R. (2020). Circuits that encode and guide alcohol-associated preference. Elife 9. PubMed ID: 32497004
A powerful feature of adaptive memory is its inherent flexibility. Alcohol and other addictive substances can remold neural circuits important for memory to reduce this flexibility. However, the mechanism through which pertinent circuits are selected and shaped remains unclear. This study shows that circuits required for alcohol-associated preference shift from population level dopaminergic activation to select dopamine neurons that predict behavioral choice in Drosophila melanogaster. During memory expression, subsets of dopamine neurons directly and indirectly modulate the activity of interconnected glutamatergic and cholinergic mushroom body output neurons (MBON). Transsynaptic tracing of neurons important for memory expression revealed a convergent center of memory consolidation within the mushroom body (MB) implicated in arousal, and a structure outside the MB implicated in integration of naïve and learned responses. These findings provide a circuit framework through which dopamine neuronal activation shifts from reward delivery to cue onset, and provide insight into the maladaptive nature of memory.

Friday, July 17th - Signaling

Ryan, S. M., Wildman, K., Oceguera-Perez, B., Barbee, S., Mortimer, N. T. and Vrailas-Mortimer, A. D. (2020). Evolutionarily conserved transcription factors drive the oxidative stress response in Drosophila. J Exp Biol. PubMed ID: 32532866
As organisms are constantly exposed to the damaging effects of oxidative stress through both environmental exposure as well as internal metabolic processes, they have evolved a variety of mechanisms to cope with this stress. One such mechanism is the highly conserved p38 MAPK (p38K) pathway, which is known to be to post-translationally activated in response to oxidative stress resulting in the activation of downstream antioxidant targets. However, little is known about the role of p38K transcriptional regulation in response to oxidative stress. Therefore, this study analyzed the p38K gene family across the genus Drosophila to identify conserved regulatory elements. Oxidative stress exposure was found to result in increased p38K protein levels in multiple Drosophila species and is associated with increased oxidative stress resistance. It was also found that the p38Kb genomic locus includes conserved AP-1 and lola-PT transcription factor consensus sites. Accordingly, over-expression of these transcription factors in D. melanogaster is sufficient to induce transcription of p38Kb and enhances resistance to oxidative stress. It was further found that the presence of a putative lola-PT binding site in the p38Kb locus of a given species is predictive of the species' survival in response to oxidative stress. Through this comparative genomics approach, this study has identified biologically relevant putative transcription factor binding sites that regulate the expression of p38Kb and are associated with resistance to oxidative stress. These findings reveal a novel mode of regulation for p38K genes and suggests that transcription may play as important a role in p38K mediated stress responses as post-translational modifications.
Santa-Cruz Mateos, C., Valencia-Exposito, A., Palacios, I. M. and Martin-Bermudo, M. D. (2020). Integrins regulate epithelial cell shape by controlling the architecture and mechanical properties of basal actomyosin networks. PLoS Genet 16(6): e1008717. PubMed ID: 32479493
Forces generated by the actomyosin cytoskeleton are key contributors to many morphogenetic processes. The actomyosin cytoskeleton organises in different types of networks depending on intracellular signals and on cell-cell and cell-extracellular matrix (ECM) interactions. However, actomyosin networks are not static and transitions between them have been proposed to drive morphogenesis. Still, little is known about the mechanisms that regulate the dynamics of actomyosin networks during morphogenesis. This work uses the Drosophila follicular epithelium, real-time imaging, laser ablation and quantitative analysis to study the role of integrins on the regulation of basal actomyosin networks organisation and dynamics and the potential contribution of this role to cell shape. Elimination of integrins from follicle cells was shown to impair F-actin recruitment to basal medial actomyosin stress fibers. The available F-actin redistributes to the so-called whip-like structures, present at tricellular junctions, and into a new type of actin-rich protrusions that emanate from the basal cortex and project towards the medial region. These F-actin protrusions are dynamic and changes in total protrusion area correlate with periodic cycles of basal myosin accumulation and constriction pulses of the cell membrane. Finally, this study found that follicle cells lacking integrin function show increased membrane tension and reduced basal surface. Furthermore, the actin-rich protrusions are responsible for these phenotypes as their elimination in integrin mutant follicle cells rescues both tension and basal surface defects. It is thus proposed that the role of integrins as regulators of stress fibers plays a key role on controlling epithelial cell shape, as integrin disruption promotes reorganisation into other types of actomyosin networks, in a manner that interferes with proper expansion of epithelial basal surfaces.
Sanaki, Y., Nagata, R., Kizawa, D., Leopold, P. and Igaki, T. (2020). Hyperinsulinemia Drives Epithelial Tumorigenesis by Abrogating Cell Competition. Dev Cell 53(4): 379-389. PubMed ID: 32386602
Metabolic diseases such as type 2 diabetes are associated with increased cancer incidence. This study shows that hyperinsulinemia promotes epithelial tumorigenesis by abrogating cell competition. In Drosophila eye imaginal epithelium, oncogenic scribble (scrib) mutant cells are eliminated by cell competition when surrounded by wild-type cells. Through a genetic screen, this study found that flies heterozygous for the insulin receptor substrate chico allow scrib cells to evade cell competition and develop into tumors. Intriguingly, chico is required in the brain's insulin-producing cells (IPCs) to execute cell competition remotely. Mechanistically, chico downregulation in IPCs causes hyperinsulinemia by upregulating a Drosophila insulin Dilp2, which activates insulin-mTOR signaling and thus boosts protein synthesis in scrib cells. A diet-induced increase in insulin levels also triggers scrib tumorigenesis, and pharmacological repression of protein synthesis prevents hyperinsulinemia-induced scrib overgrowth. These findings provide an in vivo mechanistic link between metabolic disease and cancer risk via systemic regulation of cell competition.
Rojas, G., Orellana, I., Rosales-Rojas, R., Garcia-Olivares, J., Comer, J. and Vergara-Jaque, A. (2020). Structural determinants of the dopamine transporter regulation mediated by G proteins. J Chem Inf Model. PubMed ID: 32525311
Dopamine clearance in the brain is controlled by the dopamine transporter (DAT), a protein residing in the plasma membrane, which drives reuptake of extracellular dopamine into presynaptic neurons. Studies have revealed that the βγ subunits of heterotrimeric G proteins modulate DAT function through a physical association with the C-terminal region of the transporter. This study refined the crystal structure of the Drosophila melanogaster DAT (dDAT), modeling de novo the N- and C-terminal domains; subsequently, the full-length dDAT structure was used to generate a comparative model of human DAT (hDAT). Both proteins were assembled with Gβ1γ2 subunits employing protein-protein docking, and subsequent molecular dynamics simulations were run to identify the specific interactions governing the formation of the hDAT:Gβγ and dDAT:Gβγ complexes. A [L/F]R[Q/E]R sequence motif containing the residues R588 in hDAT and R587 in dDAT was found as key to bind the Gβγ subunits through electrostatic interactions with a cluster of negatively charged residues located at the top face of the Gβ subunit. Alterations of DAT function have been associated with multiple devastating neuropathological conditions; therefore, this work represents a step toward better understanding DAT regulation by signaling proteins, allowing therapeutic target regions to be predicted.
Pham, T. L. A., Binh, T. D., Liu, G., Nguyen, T. Q. C., Nguyen, Y. D. H., Sahashi, R., Men, T. T. and Kamei, K. (2020). Role of Serotonin Transporter in Eye Development of Drosophila melanogaster. Int J Mol Sci 21(11). PubMed ID: 32521639
Serotonin transporter (SerT) in the brain is an important neurotransmitter transporter involved in mental health. However, its role in peripheral organs is poorly understood. This study investigated the function of SerT in the development of the compound eye in Drosophila melanogaster. SerT knockdown led to excessive cell death and an increased number of cells in S-phase in the posterior eye imaginal disc. Furthermore, the knockdown of SerT in the eye disc suppressed the activation of Akt, and the introduction of PI3K effectively rescued this phenotype. These results suggested that SerT plays a role in the healthy eye development of D. melanogaster by controlling cell death through the regulation of the PI3K/Akt pathway.
Rambur, A., Lours-Calet, C., Beaudoin, C., Bunay, J., Vialat, M., Mirouse, V., Trousson, A., Renaud, Y., Lobaccaro, J. A., Baron, S., Morel, L. and de Joussineau, C. (2020). Sequential Ras/MAPK and PI3K/AKT/mTOR pathways recruitment drives basal extrusion in the prostate-like gland of Drosophila. Nat Commun 11(1): 2300. PubMed ID: 32385236
One of the most important but less understood step of epithelial tumourigenesis occurs when cells acquire the ability to leave their epithelial compartment. This phenomenon, described as basal epithelial cell extrusion (basal extrusion), represents the first step of tumour invasion. Implication of emblematic signalling pathways such as Ras/MAP Kinase and PI3K/protein kinase B (AKT)/mTOR signalling pathways, is scarcely described in this phenomenon. This paper reports a unique model of basal extrusion in the Drosophila accessory gland. There, it was demonstrated that both Ras/MAPK and PI3K/AKT/mTOR pathways are necessary for basal extrusion. Furthermore, as in prostate cancer, this study shows that these pathways are co-activated. This occurs through set up of Epidermal Growth Factor Receptor (EGFR) and Insulin Receptor (InR) dependent autocrine loops, a phenomenon that, considering human data, could be relevant for prostate cancer.

Thursday, July 16th - RNA

Jin, X., Wu, X., Zhou, L., He, T., Yin, Q. and Liu, S. (2020). 20-hydroxyecdysone-responsive microRNAs of insects. RNA Biol. PubMed ID: 32482109
20-hydroxyecdysone (20-HE) plays essential roles in coordinating developmental transitions of insects through responsive protein-coding genes and microRNAs (miRNAs). The involvement of single miRNAs in the ecdysone-signalling pathways has been extensively explored, but the interplay between ecdysone and the majority of miRNAs still remains largely unknown. By small RNA sequencing, this study systematically investigated the genome-wide responses of miRNAs to 20-HE in the embryogenic cell lines of Bombyx mori and Drosophila melanogaster. Over 60 and 70 20-HE-responsive miRNAs were identified in the BmE cell line and S2 cell line, respectively. The response of miRNAs to ecdysone exhibited a time-dependent pattern, and the response intensity increased with extending exposure to 20-HE. The relationship between ecdysone and the miRNAs was further explored through knockdown of ecdysone-signalling pathway genes. Specifically, ecdysone regulated the cluster miR-275 and miR-305 through the coordination of BmEcR-B and downstream BmE75B, and the interaction between BmEcR and miR-275 cluster was strengthened by the feedback regulation of BmE75B. Ecdysone induced miR-275-3p and miR-305-5p through the ecdysone response effectors (EcREs) at the upstream of the pre-miR-275 cluster. Overall, the results might lead to further understanding of the relationship between ecdysone signaling pathways and small RNAs in the development and metamorphosis of insects.
Patraquim, P., Mumtaz, M. A. S., Pueyo, J. I., Aspden, J. L. and Couso, J. P. (2020). Developmental regulation of canonical and small ORF translation from mRNAs. Genome Biol 21(1): 128. PubMed ID: 32471506
Ribosomal profiling has revealed the translation of thousands of sequences outside annotated protein-coding genes, including small open reading frames of less than 100 codons, and the translational regulation of many genes. This study presents an improved version of Poly-Ribo-Seq and applied it to Drosophila melanogaster embryos. Highly correlated samples were obtained across five embryonic stages, with nearly 500 million putative ribosomal footprints mapped to mRNAs, and they were compared to existing Ribo-Seq and proteomic data. This analysis reveals, for the first time in Drosophila, footprints mapping to codons in a phased pattern, the hallmark of productive translation. A simple binomial probability metric is proposed to ascertain translation probability. The results also reveal reproducible ribosomal binding apparently not resulting in productive translation. This non-productive ribosomal binding seems to be especially prevalent amongst upstream short ORFs located in the 5' mRNA leaders, and amongst canonical ORFs during the activation of the zygotic translatome at the maternal-to zygotic transition. It is suggested that this non-productive ribosomal binding might be due to cis-regulatory ribosomal binding and to defective ribosomal scanning of ORFs outside periods of productive translation. The results are compatible with the main function of upstream short ORFs being to buffer the translation of canonical canonical ORFs and show that, in general, small ORFs in mRNAs display markers compatible with an evolutionary transitory state towards full coding function.
Hong, W., Shi, Y., Xu, B. and Jin, Y. (2020). RNA secondary structures in Dscam1 mutually exclusive splicing: Unique evolutionary signature from the midge. Rna. PubMed ID: 32471818
The Drosophila melanogaster gene Dscam1 potentially generates 38,016 distinct isoforms via mutually exclusive splicing, which are required for both nervous and immune functions. However, the mechanism underlying splicing regulation remains obscure. This study shows apparent evolutionary signatures characteristic of competing RNA secondary structures in exon clusters 6 and 9 of Dscam1 in the two midge species (Belgica antarctica and Clunio marinus). Surprisingly, midge Dscam1 encodes only ~6,000 different isoforms through mutually exclusive splicing. Strikingly, the docking site of the exon 6 cluster is conserved in almost all insects and crustaceans but is specific in the midge; however, the docking site-selector base-pairings are conserved. Moreover, the docking site is complementary to all predicted selector sequences downstream of every variable exon 9 of the midge Dscam1, which is in accordance with the broad spectrum of their isoform expression. This suggests that these cis-elements mainly function through the formation of long-range base-pairings. This study provides a vital insight into the evolution and mechanism of Dscam1 alternative splicing.
Lee, S., Hong, J. S., Lim, D. H. and Lee, Y. S. (2020). Roles for Drosophila cap1 2'-O-ribose methyltransferase in the small RNA silencing pathway associated with Argonaute 2. Insect Biochem Mol Biol: 103415. PubMed ID: 32504809
Cap1 2'-O-ribose methyltransferase (CMTR1) modifies RNA transcripts containing the 7-methylguanosine cap via 2'-O-ribose methylation of the first transcribed nucleotide, yielding cap1 structures. However, the role of CMTR1 in small RNA-mediated gene silencing remains unknown. This study identified and characterized a Drosophila CMTR1 gene (dCMTR1) mutation. The catalytic activity of dCMTR1 was involved in the biogenesis of small interfering RNAs (siRNAs) but not microRNAs. Additionally, dCMTR1 interacted with R2D2, a key component for the assembly of the RNA-induced silencing complex (RISC) containing Argonaute 2 (Ago2). Consistent with this finding, loss of dCMTR1 function impaired RISC assembly by inhibiting the unwinding of Ago2-bound siRNA duplexes, thus preventing the retention of the guide strand. Moreover, dCMTR1 is unlikely to modify siRNAs during RISC assembly. Collectively, these data indicate that dCMTR1 is a positive regulator of the small RNA pathway associated with Ago2 with roles in both siRNA biogenesis and RISC assembly.
Muller, M., Schauer, T., Krause, S., Villa, R., Thomae, A. W. and Becker, P. B. (2020). Two-step mechanism for selective incorporation of lncRNA into a chromatin modifier. Nucleic Acids Res. PubMed ID: 32510132
The MLE DExH helicase and the roX lncRNAs are essential components of the chromatin modifying Dosage Compensation Complex (DCC) in Drosophila. To explore the mechanism of ribonucleoprotein complex assembly, vitRIP, an unbiased, transcriptome-wide in vitro assay was developed that reveals RNA binding specificity. MLE has intrinsic specificity for U-/A-rich sequences and tandem stem-loop structures and binds many RNAs beyond roX in vitro. The selectivity of the helicase for physiological substrates is further enhanced by the core DCC. Unwinding of roX2 by MLE induces a highly selective RNA binding surface in the unstructured C-terminus of the MSL2 subunit and triggers-specific association of MLE and roX2 with the core DCC. The exquisite selectivity of roX2 incorporation into the DCC thus originates from intimate cooperation between the helicase and the core DCC involving two distinct RNA selection principles and their mutual refinement.
Maccallini, P., Bavasso, F., Scatolini, L., Bucciarelli, E., Noviello, G., Lisi, V., Palumbo, V., D'Angeli, S., Cacchione, S., Cenci, G., Ciapponi, L., Wakefield, J. G., Gatti, M. and Raffa, G. D. (2020). Intimate functional interactions between TGS1 and the Smn complex revealed by an analysis of the Drosophila eye development. PLoS Genet 16(5): e1008815. PubMed ID: 32453722
Trimethylguanosine synthase 1 (TGS1) is a conserved enzyme that mediates formation of the trimethylguanosine cap on several RNAs, including snRNAs and telomerase RNA. Previous studies have shown that TGS1 binds the Survival Motor Neuron (SMN) protein, whose deficiency causes spinal muscular atrophy (SMA). This study analyzed the roles of the Drosophila orthologs of the human TGS1 and SMN genes. The Drosophila TGS1 protein (dTgs1) physically interacts with all subunits of the Drosophila Smn complex (Smn, Gem2, Gem3, Gem4 and Gem5), and a human TGS1 transgene rescues the mutant phenotype caused by dTgs1 loss. dTgs1 and Smn are required for viability of retinal progenitor cells, and downregulation of these genes leads to a reduced eye size. Importantly, overexpression of dTgs1 partially rescues the eye defects caused by Smn depletion, and vice versa. These results suggest that the Drosophila eye model can be exploited for screens aimed at the identification of genes and drugs that modify the phenotypes elicited by Tgs1 and Smn deficiency. These modifiers could help to understand the molecular mechanisms underlying SMA pathogenesis and devise new therapies for this genetic disease (Maccallini, 2020).

Wednesday, July 14th - Adult Physiology

Hector, T. E., Sgro, C. M. and Hall, M. D. (2020). The influence of immune activation on thermal tolerance along a latitudinal cline. J Evol Biol. PubMed ID: 32506574
Global change is shifting both temperature patterns and the geographic distribution of pathogens, and infection has already been shown to substantially reduce host thermal performance, potentially placing populations at greater risk that previously thought. But what about individuals that are able to successfully clear an infection? While the direct damage a pathogen causes will likely lead to reductions in host's thermal tolerance, the response to infection often shares many underlying pathways with the general stress response, potentially acting as a buffer against subsequent thermal stress. By exposing Drosophila melanogaster to heat-killed bacterial pathogens, this study investigated how activation of a host's immune system can modify any response to both heat and cold temperature stress. In a single focal population, it was found that immune activation can improve a host's knockdown times during heat shock, potentially offsetting some of the damage that would subsequently arise as an infection progresses. Conversely, immune activation had a detrimental effect on CT(max), and did not influence lower thermal tolerance as measured by chill coma recovery time. However, it was also found that the influence of immune activation on heat knockdown times is not generalizable across an entire cline of locally adapted populations. Instead, immune activation led to signals of local adaptation to temperature being lost, erasing the previous advantage that populations in warmer regions had when challenged with heat stress. These results suggest that activation of the immune system may help buffer individuals against the detrimental impact of infection on thermal tolerance; however, any response will be population specific and potentially not easily predicted across larger geographic scales, and dependent on the form of thermal stress faced by a host.
Harrison, B. R., Wang, L., Gajda, E., Hoffman, E. V., Chung, B. Y., Pletcher, S. D., Raftery, D. and Promislow, D. E. L. (2020). The metabolome as a link in the genotype-phenotype map for peroxide resistance in the fruit fly, Drosophila melanogaster. BMC Genomics 21(1): 341. PubMed ID: 32366330
This study used metabolomics to explore the nature of genetic variation for hydrogen peroxide (H(2)O(2)) resistance in the sequenced inbred Drosophila Genetic Reference Panel (DGRP). First genetic variation was studied for H(2)O(2) resistance in 179 DGRP lines and along with identifying the insulin signaling modulator u-shaped and several regulators of feeding behavior, it is estimated that a substantial amount of phenotypic variation can be explained by a polygenic model of genetic variation. Then a portion of the aqueous metabolome was profiled in subsets of eight 'high resistance' lines and eight 'low resistance' lines. These lines were used to represent collections of genotypes that were either resistant or sensitive to the stressor, effectively modeling a discrete trait. Across the range of genotypes in both populations, flies exhibited surprising consistency in their metabolomic signature of resistance. Importantly, the resistance phenotype of these flies was more easily distinguished by their metabolome profiles than by their genotypes. Furthermore, a metabolic response was found to H(2)O(2) in sensitive, but not in resistant genotypes. Metabolomic data further implicated at least two pathways, glycogen and folate metabolism, as determinants of sensitivity to H(2)O(2). A confounding effect was found of feeding behavior on assays involving supplemented food. This work suggests that the metabolome can be a point of convergence for genetic variation influencing complex traits, and can efficiently elucidate mechanisms underlying trait variation.
Long, D. M., Frame, A. K., Reardon, P. N., Cumming, R. C., Hendrix, D. A., Kretzschmar, D. and Giebultowicz, J. M. (2020). Lactate dehydrogenase expression modulates longevity and neurodegeneration in Drosophila melanogaster. Aging (Albany NY) 12. PubMed ID: 32484787
Lactate dehydrogenase (LDH) catalyzes the conversion of glycolysis-derived pyruvate to lactate. Lactate has been shown to play key roles in brain energetics and memory formation. However, lactate levels are elevated in aging and Alzheimer's disease patients, and it is not clear whether lactate plays protective or detrimental roles in these contexts. This study shows that Ldh transcript levels are elevated and cycle with diurnal rhythm in the heads of aged flies and this is associated with increased LDH protein, enzyme activity, and lactate concentrations. To understand the biological significance of increased Ldh gene expression, Ldh levels were genetically manipulated in adult neurons or glia. Overexpression of Ldh in both cell types caused a significant reduction in lifespan whereas Ldh down-regulation resulted in lifespan extension. Moreover, pan-neuronal overexpression of Ldh disrupted circadian locomotor activity rhythms and significantly increased brain neurodegeneration. In contrast, reduction of Ldh in neurons delayed age-dependent neurodegeneration. Thus, this unbiased genetic approach identified Ldh and lactate as potential modulators of aging and longevity in flies.
Jorgensen, L. B., Robertson, R. M. and Overgaard, J. (2020). Neural dysfunction correlates with heat coma and CT(max) in Drosophila but does not set the boundaries for heat stress survival. J Exp Biol. PubMed ID: 32434804
When heated, insects lose coordinated movement followed by the onset of heat coma (CT(max)). This study examined the function of the central nervous system (CNS) in five species of Drosophila with different heat tolerances, while they were exposed to either constant high temperature or a gradual increasing temperature (ramp). Tolerant species were able to preserve CNS function at higher temperatures and for longer durations than sensitive species and similar differences were found for the behavioural indices (loss of coordination and onset of heat coma). Furthermore, the timing and temperature (constant and ramp exposure, respectively) for loss of coordination or complete coma coincided with the occurrence of spreading depolarisation (SD) events in the CNS. These SD events disrupt neurological function and silence the CNS suggesting that CNS failure is the primary cause of impaired coordination and heat coma. Heat mortality occurs soon after heat coma in insects and to examine if CNS failure could also be the proximal cause of heat death, selective heating of the head (CNS) and abdomen (visceral tissues) was tested. When comparing the temperature causing 50% mortality (LT(50)) of each body part to that of the whole animal, it was found that the head was not particularly heat sensitive compared to the abdomen. Accordingly, it is unlikely that nervous failure is the principal/proximate cause of heat mortality in Drosophila.
Orthofer, M., Valsesia, A., ..., Gheldof, N. and Penninger, J. M. (2020). Identification of ALK in Thinness. Cell 181(6): 1246-1262. PubMed ID: 32442405
There is considerable inter-individual variability in susceptibility to weight gain despite an equally obesogenic environment in large parts of the world. Whereas many studies have focused on identifying the genetic susceptibility to obesity, a GWAS was performed on metabolically healthy thin individuals (lowest 6(th) percentile of the population-wide BMI spectrum) in a uniquely phenotyped Estonian cohort. Anaplastic lymphoma kinase (ALK) was discovered as a candidate thinness gene. In Drosophila, RNAi mediated knockdown of Alk led to decreased triglyceride levels. In mice, genetic deletion of Alk resulted in thin animals with marked resistance to diet- and leptin-mutation-induced obesity. Mechanistically, it was found that ALK expression in hypothalamic neurons controls energy expenditure via sympathetic control of adipose tissue lipolysis. Genetic and mechanistic experiments identify ALK as a thinness gene, which is involved in the resistance to weight gain.
Gupta, A. and Stocker, H. (2020). FoxO suppresses endoplasmic reticulum stress to inhibit growth of Tsc1-deficient tissues under nutrient restriction. Elife 9. PubMed ID: 32525804
The transcription factor FoxO has been shown to block proliferation and progression in mTORC1-driven tumorigenesis but the picture of the relevant FoxO target genes remains incomplete. This study employed RNA-seq profiling on single clones isolated using laser capture microdissection from Drosophila larval eye imaginal discs to identify FoxO targets that restrict the proliferation of Tsc1-deficient cells under nutrient restriction (NR). Transcriptomics analysis revealed downregulation of endoplasmic reticulum-associated protein degradation pathway components upon foxo knockdown. Induction of ER stress pharmacologically or by suppression of other ER stress response pathway components led to an enhanced overgrowth of Tsc1 knockdown tissue. Increase of ER stress in Tsc1 loss-of-function cells upon foxo knockdown was also confirmed by elevated expression levels of known ER stress markers. These results highlight the role of FoxO in limiting ER stress to regulate Tsc1 mutant overgrowth.

Tuesday July 14th - Adult neural function and development

Chen, Z. and Montell, C. (2020). A Family of Auxiliary Subunits of the TRP Cation Channel Encoded by the Complex inaF Locus. Genetics. PubMed ID: 32434796
TRP channels function in many types of sensory receptor cells. Despite extensive analyses, an open question is whether there exists a family of auxiliary subunits, which could influence localization, trafficking, and function of TRP channels. Using Drosophila melanogaster, this study reveals a previously unknown TRP interacting protein, INAF-C, which is expressed exclusively in the ultraviolet-sensing R7 photoreceptor cells. INAF-C is encoded by an unusual locus comprised of four distinct coding regions, which give rise to four unique single transmembrane containing proteins. With the exception of INAF-B, roles for the other INAF proteins were unknown. This study found that both INAF-B and INAF-C are required for TRP stability and localization in R7 cells. Conversely, loss of just INAF-B greatly reduced TRP from other types of photoreceptor cells, but not R7. The requirements for TRP and INAF are reciprocal, since loss of TRP decreased the concentrations of both INAF-B and INAF-C. INAF-A, which is not normally expressed in photoreceptor cells, can functionally substitute for INAF-B, indicating that it is a third TRP auxiliary protein. Reminiscent of the structural requirements between K(v) channels and KCNE auxiliary subunits, the co-dependencies of TRP and INAF depended on several transmembrane domains in TRP, and the transmembrane domain and the C-terminus of INAF-B. These studies support a model that the inaF locus encodes a family of at least three TRP auxiliary subunits.
Kim, T., Shin, H., Song, B., Won, C., Yoshida, H., Yamaguchi, M., Cho, K. S. and Lee, I. S. (2020). Overexpression of H3K36 methyltransferase NSD in glial cells affects brain development in Drosophila. Glia. PubMed ID: 32531091
NSD1 is a histone methyltransferase that methylates the lysine 36 at histone H3. NSD duplication is associated with short stature, microcephaly, intellectual disability, and behavioral defects in humans. Ectopic overexpression of NSD, an NSD1 homolog in Drosophila, was shown to induce developmental abnormalities via apoptosis. In this study, to investigate the effects of NSD overexpression on Drosophila brain development, the typical NSD expression pattern was examined in larval brains and found that endogenous NSD promoter activity was detected only in subsets of glial cells. Pan-glial, but not pan-neuronal, NSD overexpression induced apoptosis in larval brain cells. However, pan-glial NSD overexpression also induced caspase-3 cleavage in neuronal cells. Among the various glial cell types, NSD overexpression in only astrocytic glia induced apoptosis and abnormal learning defects in the larval stage. Furthermore, NSD overexpression downregulated the expression of various astrocyte-specific genes, including draper (drpr), possibly owing to an indirect effect of NSD overexpression-induced astrocytic apoptosis. Since drpr plays a role in axon pruning during mushroom body (MB) formation in Drosophila astrocytes, the effect was studied of astrocytic NSD overexpression on this process; it was found to disrupted the clearance of γ-neurons in the MB, subsequently inducing arrhythmic locomotor activity of the fly. Thus, these results suggest that aberrant NSD overexpression may cause neurodevelopmental disorders by interfering with crucial functions of astrocytes in the brain, underlining the importance of the tightly controlled astrocytic NSD expression for proper neurodevelopment.
Estacio-Gómez, A., Hassan, A., Walmsley, E., Le, L. W. and Southall, T. D. (2020). Dynamic neurotransmitter specific transcription factor expression profiles during Drosophila development. Biol Open 9(5). PubMed ID: 32493733
The remarkable diversity of neurons in the nervous system is generated during development, when properties such as cell morphology, receptor profiles and neurotransmitter identities are specified. In order to gain a greater understanding of neurotransmitter specification this study profiled the transcription state of cholinergic, GABAergic and glutamatergic neurons in vivo at three developmental time points. 86 differentially expressed transcription factors were identified that are uniquely enriched, or uniquely depleted, in a specific neurotransmitter type. Some transcription factors show a similar profile across development, others only show enrichment or depletion at specific developmental stages. Profiling of Acj6 (cholinergic enriched) and Ets65A (cholinergic depleted) binding sites in vivo reveals that they both directly bind the ChAT locus, in addition to a wide spectrum of other key neuronal differentiation genes. It was also shown that cholinergic enriched transcription factors are expressed in mostly non-overlapping populations in the adult brain, implying the absence of combinatorial regulation of neurotransmitter fate in this context. Furthermore, the data underlines that, similar to Caenorhabditis elegans, there are no simple transcription factor codes for neurotransmitter type specification.
Rimal, S., Sang, J., Dhakal, S. and Lee, Y. (2020). Cucurbitacin B Activates Bitter-Sensing Gustatory Receptor Neurons via Gustatory Receptor 33a in Drosophila melanogaster. Mol Cells. PubMed ID: 32451368
The Gustatory system enables animals to detect toxic bitter chemicals, which is critical for insects to survive food induced toxicity. Cucurbitacin is widely present in plants such as cucumber and gourds that acts as an anti-herbivore chemical and an insecticide. Cucurbitacin has a harmful effect on insect larvae as well. Although various beneficial effects of cucurbitacin such as alleviating hyperglycemia have also been documented, it is not clear what kinds of molecular sensors are required to detect cucurbitacin in nature. Cucurbitacin B, a major bitter component of bitter melon, was applied to induce action potentials from sensilla of a mouth part of the fly, labellum. This study identified that only Gr33a is required for activating bitter-sensing gustatory receptor neurons by cucurbitacin B among available 26 Grs, 23 Irs, 11 Trp mutants, and 26 Gr-RNAi lines. The difference between control and Gr33a mutant was investigated by analyzing binary food choice assay. Toxic effect of Cucurbitacin B was measured over 0.01 mM range. These findings uncover the molecular sensor of cucurbitacin B in Drosophila melanogaster. It is proposed that the discarded shell of Cucurbitaceae can be developed to make a new insecticide.
Mukherjee, C., Kling, T., Russo, B., Miebach, K., Kess, E., Schifferer, M., Pedro, L. D., Weikert, U., Fard, M. K., Kannaiyan, N., Rossner, M., Aicher, M. L., Goebbels, S., Nave, K. A., Kramer-Albers, E. M., Schneider, A. and Simons, M. (2020). Oligodendrocytes Provide Antioxidant Defense Function for Neurons by Secreting Ferritin Heavy Chain. Cell Metab. PubMed ID: 32531201
An evolutionarily conserved function of glia is to provide metabolic and structural support for neurons. To identify molecules generated by glia and with vital functions for neurons, Drosophila melanogaster was used as a screening tool, and subsequently the findings were translated to mice. A cargo receptor operating in the secretory pathway of glia was found to be essential to maintain axonal integrity by regulating iron buffering. Ferritin heavy chain was identified as the critical secretory cargo, required for the protection against iron-mediated ferroptotic axonal damage. In mice, ferritin heavy chain is highly expressed by oligodendrocytes and secreted by employing an unconventional secretion pathway involving extracellular vesicles. Disrupting the release of extracellular vesicles or the expression of ferritin heavy chain in oligodendrocytes causes neuronal loss and oxidative damage in mice. These data point to a role of oligodendrocytes in providing an antioxidant defense system to support neurons against iron-mediated cytotoxicity.
Fernandez, M. P., Pettibone, H. L., Bogart, J. T., Roell, C. J., Davey, C. E., Pranevicius, A., Huynh, K. V., Lennox, S. M., Kostadinov, B. S. and Shafer, O. T. (2020). Sites of Circadian Clock Neuron Plasticity Mediate Sensory Integration and Entrainment. Curr Biol. PubMed ID: 32386535
Networks of circadian timekeeping in the brain display marked daily changes in neuronal morphology. In Drosophila melanogaster, the striking daily structural remodeling of the dorsal medial termini of the small ventral lateral neurons has long been hypothesized to mediate endogenous circadian timekeeping. To test this model, this study has specifically abrogated these sites of daily neuronal remodeling through the reprogramming of neural development and assessed the effects on circadian timekeeping and clock outputs. Remarkably, the loss of these sites has no measurable effects on endogenous circadian timekeeping or on any of the major output functions of the small ventral lateral neurons. Rather, their loss reduces sites of glutamatergic sensory neurotransmission that normally encodes naturalistic time cues from the environment. These results support an alternative model: structural plasticity in critical clock neurons is the basis for proper integration of light and temperature and gates sensory inputs into circadian clock neuron networks.

Monday, July 13th - Evolution

Hjelmen, C. E., Holmes, V. R., Burrus, C. G., Piron, E., Mynes, M., Garrett, M. A., Blackmon, H. and Johnston, J. S. (2020). Thoracic underreplication in Drosophila species estimates a minimum genome size and the dynamics of added DNA. Evolution. PubMed ID: 32438451
Many cells in the thorax of Drosophila were found to stall during replication, a phenomenon known as underreplication. Unlike underreplication in nuclei of salivary and follicle cells, this stall occurs with less than one complete round of replication. This stall point allows precise estimations of early-replicating euchromatin and late-replicating heterochromatin regions, providing a powerful tool to investigate the dynamics of structural change across the genome. This study measured underreplication in 132 species across the Drosophila genus and leveraged these data to propose a model for estimating the rate at which additional DNA is accumulated as heterochromatin and euchromatin and also predict the minimum genome size for Drosophila. According to comparative phylogenetic approaches, the rates of change of heterochromatin differ strikingly between Drosophila subgenera. Although these subgenera differ in karyotype, there were no differences by chromosome number, suggesting other structural changes may influence accumulation of heterochromatin. Measurements were taken for both sexes, allowing the visualization of genome size and heterochromatin changes for the hypothetical path of XY sex chromosome differentiation. Additionally, the model presented in this study estimates a minimum genome size in Sophophora remarkably close to the smallest insect genome measured to date, in a species over 200 million years diverged from Drosophila.
Massey, J. H., Rice, G. R., Firdaus, A. S., Chen, C. Y., Yeh, S. D., Stern, D. L. and Wittkopp, P. J. (2020). Co-evolving wing spots and mating displays are genetically separable traits in Drosophila. Evolution. PubMed ID: 32363590
The evolution of sexual traits often involves correlated changes in morphology and behavior. For example, in Drosophila, divergent mating displays are often accompanied by divergent pigment patterns. To better understand how such traits co-evolve, this study investigated the genetic basis of correlated divergence in wing pigmentation and mating display between the sibling species Drosophila elegans and Drosophila gunungcola. Drosophila elegans males have an area of black pigment on their wings known as a wing spot and appear to display this spot to females by extending their wings laterally during courtship. By contrast, D. gunungcola lost both of these traits. Using Multiplexed Shotgun Genotyping (MSG), an ~440 kb region on the X chromosome was identified that behaves like a genetic switch controlling the presence or absence of male-specific wing spots. This region includes the candidate gene optomotor-blind (omb), which plays a critical role in patterning the Drosophila wing. The genetic basis of divergent wing display is more complex, with at least two loci on the X chromosome and two loci on autosomes contributing to its evolution. Introgressing the X-linked region affecting wing spot development from D. gunungcola into D. elegans reduced pigmentation in the wing spots but did not affect the wing display, indicating that these are genetically separable traits. Consistent with this observation, broader sampling of wild D. gunungcola populations confirmed that the wing spot and wing display are evolving independently: some D. gunungcola males performed wing displays similar to D. elegans despite lacking wing spots. These data suggest that correlated selection pressures rather than physical linkage or pleiotropy are responsible for the coevolution of these morphological and behavioral traits. They also suggest that the change in morphology evolved prior to the change in behavior.
Homem, R. A., Buttery, B., Richardson, E. E., Tan, Y., Field, L. M., Williamson, M. S. and Davies, T. G. E. (2020). Evolutionary trade-offs of insecticide resistance - the fitness costs associated with target-site mutations in the nAChR of Drosophila melanogaster. Mol Ecol. PubMed ID: 32510730
The evolution of resistance to drugs and pesticides poses a major threat to human health and food security. Neonicotinoids are highly effective insecticides used to control agricultural pests. They target the insect nicotinic acetylcholine receptor and mutations of the receptor that confer resistance have been slow to develop, with only one field-evolved mutation being reported to date. This is an arginine to threonine substitution at position 81 of the nAChR_β1 subunit in neonicotinoid resistant aphids. To validate the role of R81T in neonicotinoid resistance and to test whether it may confer any significant fitness costs to insects, CRISPR/Cas9 was used to introduce an analogous mutation in the genome of Drosophila melanogaster. Flies carrying R81T showed an increased tolerance (resistance) to neonicotinoid insecticides, accompanied by a significant reduction in fitness. In comparison, flies carrying a deletion of the whole nAChR_α6 subunit, the target-site of spinosyns, showed an increased tolerance to this class of insecticides but presented almost no fitness deficits.
Lund-Hansen, K. K., Abbott, J. K. and Morrow, E. H. (2020). Feminization of complex traits in Drosophila melanogaster via female-limited X chromosome evolution. Evolution. PubMed ID: 32438467
A handful of studies have investigated sexually antagonistic constraints on achieving sex-specific fitness optima, although exclusively through male-genome-limited evolution experiments. This article has established a female-limited X chromosome evolution experiment, where an X chromosome balancer was used to enforce the inheritance of the X through the matriline, thus removing exposure to male selective constraints. This approach eliminates the effects of sexually antagonistic selection on the X chromosome, permitting evolution toward a single sex-specific optimum. After multiple generations of selection, strong evidence was found that body size and development time had moved toward a female-specific optimum, whereas reproductive fitness and locomotion activity remained unchanged. The changes in body size and development time are consistent with previous results, and suggest that the X chromosome is enriched for sexually antagonistic genetic variation controlling these particular traits. The lack of change in reproductive fitness and locomotion activity could be due to a number of mutually nonexclusive explanations, including a lack of sexually antagonistic variance on the X chromosome for those traits or confounding effects of the use of the balancer chromosome. This study is the first to employ female-genome-limited selection and adds to the understanding of the complexity of sexually antagonistic genetic variation
Kapun, M., Barron, M. G., Staubach, F., Obbard, D. J. and Gonzalez, J. (2020). Genomic analysis of European Drosophila melanogaster populations reveals longitudinal structure, continent-wide selection, and previously unknown DNA viruses. Mol Biol Evol. PubMed ID: 32413142
Genetic variation is the fuel of evolution, with standing genetic variation especially important for short-term evolution and local adaptation. To date, studies of spatio-temporal patterns of genetic variation in natural populations have been challenging, as comprehensive sampling is logistically difficult, and sequencing of entire populations costly. This study addresses these issues using a collaborative approach, sequencing 48 pooled population samples from 32 locations, and the first continent-wide genomic analysis of genetic variation was performed in European Drosophila melanogaster. These analyses uncover longitudinal population structure, provide evidence for continent-wide selective sweeps, identify candidate genes for local climate adaptation, and document clines in chromosomal inversion and transposable element frequencies. Variation among populations in the composition of the fly microbiome was characterized, and five new DNA viruses were identified in these samples.
Jaksic, A. M., Karner, J., Nolte, V., Hsu, S. K., Barghi, N., Mallard, F., Otte, K. A., Svecnjak, L., Senti, K. A. and Schlotterer, C. (2020). Neuronal function and dopamine signaling evolve at high temperature in Drosophila. Mol Biol Evol. PubMed ID: 32402077
Neuronal activity is temperature-sensitive and affects behavioral traits important for individual fitness, such as locomotion and courtship. Yet not enough is known about the evolutionary response of neuronal phenotypes in new temperature environments. This study used long-term experimental evolution of Drosophila simulans populations exposed to novel temperature regimes. A direct relationship was demonstrated between thermal selective pressure and the evolution of neuronally expressed molecular and behavioral phenotypes. Several essential neuronal genes evolve lower expression at high temperatures and higher expression at low temperatures, with dopaminergic neurons standing out by displaying the most consistent expression change across independent replicates. The link between evolved gene expression and behavioral changes was functionally validated by pharmacological intervention in the experimentally evolved D. simulans populations as well as by genetically triggered expression changes of key genes in D. melanogaster. Since natural temperature clines confirm these results for Drosophila and Anopheles populations, it is concluded that neuronal dopamine evolution is a key factor for temperature adaptation.

Friday, July 10th - Signaling

Mahadeveraju, S., Jung, Y. H. and Erickson, J. W. (2020). Evidence that Runt acts as a counter-repressor of Groucho during Drosophila melanogaster primary sex determination. G3 (Bethesda). PubMed ID: 32457096
Runx proteins are bifunctional transcription factors that both repress and activate transcription in animal cells. Typically, Runx proteins work in concert with other transcriptional regulators, including co-activators and co-repressors to mediate their biological effects. In Drosophila melanogaster the archetypal Runx protein, Runt, functions in numerous processes including segmentation, neurogenesis and sex determination. During primary sex determination Runt acts as one of four X-linked signal element (XSE) proteins that direct female-specific activation of the establishment promoter (Pe) of the master regulatory gene Sex-lethal (Sxl). Successful activation of SxlPe requires that the XSE proteins overcome the repressive effects of maternally deposited Groucho (Gro), a potent co-repressor of the Gro/TLE family. Runx proteins, including Runt, contain a C-terminal peptide, VWRPY, known to bind to Gro/TLE proteins to mediate transcriptional repression. This study shows that Runt's VWRPY co-repressor-interaction domain is needed for Runt to activate SxlPe Deletion of the Gro-interaction domain eliminates Runt-ability to activate SxlPe, whereas replacement with a higher affinity, VWRPW, sequence promotes Runt-mediated transcription. This suggests that Runt may activate SxlPe by antagonizing Gro function, a conclusion consistent with earlier findings that Runt is needed for Sxl expression only in embryonic regions with high Gro activity. Surprisingly it was found that Runt is not required for the initial activation of SxlPe Instead, Runt is needed to keep SxlPe active during the subsequent period of high-level Sxl transcription suggesting that Runt helps amplify the difference between female and male XSE signals by counter-repressing Gro in female, but not in male, embryos.
Li, X., Liu, M., Ren, X., Loncle, N., Wang, Q., Hemba-Waduge, R. U., Yu, S. H., Boube, M., Bourbon, H. G., Ni, J. Q. and Ji, J. Y. (2020). The Mediator CDK8-Cyclin C complex modulates Dpp signaling in Drosophila by stimulating Mad-dependent transcription. PLoS Genet 16(5): e1008832. PubMed ID: 32463833
Dysregulation of CDK8 (Cyclin-Dependent Kinase 8) and its regulatory partner CycC (Cyclin C), two subunits of the conserved Mediator (MED) complex, have been linked to diverse human diseases such as cancer. To identify upstream regulators or downstream effectors of CDK8, a dominant modifier genetic screen was performed in Drosophila based on the defects in vein patterning caused by specific depletion or overexpression of CDK8 or CycC in developing wing imaginal discs. 26 genomic loci were identified whose haploinsufficiency can modify these CDK8- or CycC-specific phenotypes. Further analysis of two overlapping deficiency lines and mutant alleles led to identification of genetic interactions between the CDK8-CycC pair and the components of the Decapentaplegic (Dpp, the Drosophila homolog of TGFβ, or Transforming Growth Factor-β) signaling pathway. It was observed that CDK8-CycC positively regulates transcription activated by Mad (Mothers against dpp), the primary transcription factor downstream of the Dpp/TGFβ signaling pathway. CDK8 can directly interact with Mad in vitro through the linker region between the DNA-binding MH1 (Mad homology 1) domain and the carboxy terminal MH2 (Mad homology 2) transactivation domain. Besides CDK8 and CycC, further analyses of other subunits of the MED complex have revealed six additional subunits that are required for Mad-dependent transcription in the wing discs: Med12, Med13, Med15, Med23, Med24, and Med31. Furthermore, this analyses confirmed the positive roles of CDK9 and Yorkie in regulating Mad-dependent gene expression in vivo. These results suggest that CDK8 and CycC, together with a few other subunits of the MED complex, may coordinate with other transcription cofactors in regulating Mad-dependent transcription during wing development in Drosophila.
Khoury, M. J. and Bilder, D. (2020). Distinct activities of Scrib module proteins organize epithelial polarity. Proc Natl Acad Sci U S A 117(21): 11531-11540. PubMed ID: 32414916
A polarized architecture is central to both epithelial structure and function. In many cells, polarity involves mutual antagonism between the Par complex and the Scribble (Scrib) module. While molecular mechanisms underlying Par-mediated apical determination are well-understood, how Scrib module proteins specify the basolateral domain remains unknown. This study demonstrates dependent and independent activities of Scrib, Discs-large (Dlg), and Lethal giant larvae (Lgl) using the Drosophila follicle epithelium. The data support a linear hierarchy for localization, but rule out previously proposed protein-protein interactions as essential for polarization. Cortical recruitment of Scrib does not require palmitoylation or polar phospholipid binding but instead an independent cortically stabilizing activity of Dlg. Scrib and Dlg do not directly antagonize atypical protein kinase C (aPKC), but may instead restrict aPKC localization by enabling the aPKC-inhibiting activity of Lgl. Importantly, while Scrib, Dlg, and Lgl are each required, all three together are not sufficient to antagonize the Par complex. These data demonstrate previously unappreciated diversity of function within the Scrib module and begin to define the elusive molecular functions of Scrib and Dlg.
Liu, Z., Yang, Y., Gu, A., Xu, J., Mao, Y., Lu, H., Hu, W., Lei, Q. Y., Li, Z., Zhang, M., Cai, Y. and Wen, W. (2020). Par complex cluster formation mediated by phase separation. Nat Commun 11(1): 2266. PubMed ID: 32385244
The evolutionarily conserved Par3/Par6/aPKC complex regulates the polarity establishment of diverse cell types and distinct polarity-driven functions. However, how the Par complex is concentrated beneath the membrane to initiate cell polarization remains unclear. This study shows that the Par complex exhibits cell cycle-dependent condensation in Drosophila neuroblasts, driven by liquid-liquid phase separation. The open conformation of Par3 undergoes autonomous phase separation likely due to its NTD-mediated oligomerization. Par6, via C-terminal tail binding to Par3 PDZ3, can be enriched to Par3 condensates and in return dramatically promote Par3 phase separation. aPKC can also be concentrated to the Par3N/Par6 condensates as a client. Interestingly, activated aPKC can disperse the Par3/Par6 condensates via phosphorylation of Par3. Perturbations of Par3/Par6 phase separation impair the establishment of apical-basal polarity during neuroblast asymmetric divisions and lead to defective lineage development. It is proposed that phase separation may be a common mechanism for localized cortical condensation of cell polarity complexes.
Moulton, M. J., Humphreys, G. B., Kim, A. and Letsou, A. (2020). O-GlcNAcylation Dampens Dpp/BMP Signaling to Ensure Proper Drosophila Embryonic Development. Dev Cell 53(3): 330-343.e333. PubMed ID: 32369743
BMP (bone morphogenetic protein) signaling activity is precisely controlled by both pathway agonists and antagonists. This study identify a previously unrecognized BMP signaling antagonist. The Drosophila BMP type I receptor Sax (Saxophone) functions as a Dpp (Decapentaplegic) receptor in Drosophila embryos, but that its activity is normally inhibited by the O-linked glycosyltransferase Sxc (Super sex combs). In wild-type embryos, Sax activity is inhibited, and the BMP type I receptor Tkv (Thickveins) is the sole conduit for Dpp. In contrast, in sxc mutants, the Dpp signal is transduced by both Tkv and Sax, and elevated Dpp signaling results in embryonic lethality. This study also demonstrates that Sxc O-glycosylates Sax and observe elevated Dpp signaling in response to maternal restriction of dietary sugar. These findings link fertility to nutritive environment and point to Sax signaling as the nutrient-sensitive branch of BMP signaling.
Li, Y., Sun, X., Gao, D., Ding, Y., Liu, J., Chen, J., Luo, J., Zhang, J., Liu, Q. and Zhou, Z. (2020). Dual functions of Rack1 in regulating Hedgehog pathway. Cell Death Differ. PubMed ID: 32467643
Hedgehog (Hh) pathway plays multiple roles in many physiological processes and its dysregulation leads to congenital disorders and cancers. Hh regulates the cellular localization of Smoothened (Smo) and the stability of Cubitus interruptus (Ci) to fine-tune the signal outputs. However, the underlying mechanisms are still unclear. This study shows that the scaffold protein Rack1 plays dual roles in Hh signaling. In the absence of Hh, Rack1 promotes Ci and Cos2 to form a Ci-Rack1-Cos2 complex, culminating in Slimb-mediated Ci proteolysis. In the presence of Hh, Rack1 dissociates from Ci-Rack1-Cos2 complex and forms a trimeric complex with Smo and Usp8, leading to Smo deubiquitination and cell surface accumulation. Furthermore, this study finds the regulation of Rack1 on Hh pathway is conserved from Drosophila to mammalian cells. These findings demonstrate that Rack1 plays dual roles during Hh signal transduction and provide Rack1 as a potential drug target for Hh-related diseases.

Thursday, June 9th - Disease Models

Manivannan, S. N., Darouich, S., Masmoudi, A., Gordon, D., Zender, G., Han, Z., Fitzgerald-Butt, S., White, P., McBride, K. L., Kharrat, M. and Garg, V. (2020). Novel frameshift variant in MYL2 reveals molecular differences between dominant and recessive forms of hypertrophic cardiomyopathy. PLoS Genet 16(5): e1008639. PubMed ID: 32453731
Hypertrophic cardiomyopathy (HCM) is characterized by thickening of the ventricular muscle without dilation and is often associated with dominant pathogenic variants in cardiac sarcomeric protein genes. This study reports a family with two infants diagnosed with infantile-onset HCM and mitral valve dysplasia that led to death before one year of age. Using exome sequencing, one of the affected children was discovered to have a homozygous frameshift variant in Myosin light chain 2, which alters the last 20 amino acids of the protein and is predicted to impact the most C-terminal of the three EF-hand domains in MYL2. The parents are unaffected heterozygous carriers of the variant and the variant is absent in control cohorts from gnomAD. The absence of the phenotype in carriers and the infantile presentation of severe HCM is in contrast to HCM associated with dominant MYL2 variants. Immunohistochemical analysis of the ventricular muscle of the deceased patient with the MYL2-fs variant showed a marked reduction of MYL2 expression compared to an unaffected control. In vitro overexpression studies further indicate that the MYL2-fs variant is actively degraded. In contrast, an HCM-associated missense variant and three other MYL2 stop-gain variants that result in loss of the EF domains are stably expressed but show impaired localization. The degradation of the MYL2-fs can be rescued by inhibiting the cell's proteasome function supporting a post-translational effect of the variant. In vivo rescue experiments with a Drosophila MYL2-homolog (Mlc2) knockdown model indicate that neither the MYL2-fs nor the MYL2:p.Gly162Arg variant supports normal cardiac function. The tools that have been generated for this study provide a rapid screening platform for functional assessment of variants of unknown significance in MYL2. This study supports an autosomal recessive model of inheritance for MYL2 loss-of-function variants in infantile HCM and highlights the variant-specific molecular differences found in MYL2-associated cardiomyopathy.
Leila Abtahi, S., Masoudi, R. and Haddadi, M. (2020). The Distinctive Role of Tau and Amyloid beta in Mitochondrial Dysfunction through Alteration in Mfn2 and Drp1 mRNA Levels: A Comparative Study in Drosophila melanogaster. Gene: 144854. PubMed ID: 32525045
Alzheimer's disease (AD) is one of the most common forms of neurodegenerative diseases. Aggregation of Aβ42 and hyperphosphorylated tau are two major hallmarks of AD. Whether different forms of tau (soluble or hyperphosphorylated) or are the main culprit in the events observed in AD is still under investigation. This study examined the effect of wild-type, prone to hyperphosphorylation and hyperphosphorylated tau, and also Aβ42 peptide on the brain antioxidant defense system and two mitochondrial genes, Marf (homologous to human MFN2) and Drp1 involved in mitochondrial dynamics in transgenic Drosophila melanogaster. AD is an age associated disease. Therefore, the activity of 3 antioxidant agents, CAT, SOD, and GSH levels and the mRNA levels of Marf and Drp1 was assessed in different time points of flies' life cycle. Reduction in cognitive function and antioxidant activity was observed in all lines and time points. The most and the least effect on the eye phenotype was exerted by hyperphosphorylated tau and Aβ42, respectively. In addition, the most remarkable alteration in Marf and Drp1 mRNA level was observed in transgenic flies expressing hyperphosphorylated tau when pan neuronal expression of transgenes was applied. However, when the disease causing gene expression was confined to the mushroom body, Marf and Drp1 mRNA level alteration was more prominent in tau(WT) and tau(E14) transgenic flies, respectively. This may suggest a role for propagation of tau(WT) compared to hyperphosphorylated tau or Aβ42. In conclusion, in spite of antioxidant deficiency caused by different types of tau and Aβ42, it seems that tau exerts more toxic effect on the eye phenotype and mitochondrial genes regulation (Marf and Drp1). Moreover, different mechanisms seem to be involved in mitochondrial gene dysregulation when various forms of tau are expressed.
Hou, Y., Wu, Z., Zhang, Y., Chen, H., Hu, J., Guo, Y., Peng, Y. and Wei, Q. (2020). Functional Analysis of Hydrolethalus Syndrome Protein HYLS1 in Ciliogenesis and Spermatogenesis in Drosophila. Front Cell Dev Biol 8: 301. PubMed ID: 32509774
Cilia and flagella are conserved subcellular organelles, which arise from centrioles and play critical roles in development and reproduction of eukaryotes. Dysfunction of cilia leads to life-threatening ciliopathies. HYLS1 is an evolutionarily conserved centriole protein, which is critical for ciliogenesis, and its mutation causes ciliopathy-hydrolethalus syndrome. However, the molecular function of HYLS1 remains elusive. This study investigated the function of HYLS1/CG42231 in cilia formation using the Drosophila model. Drosophila HYLS1 is a conserved centriole and basal body protein. Deletion of HYLS1 led to sensory cilia dysfunction and spermatogenesis abnormality. Importantly, this study found that Drosophila HYLS1 is essential for giant centriole/basal body elongation in spermatocytes and is required for spermatocyte centriole to efficiently recruit pericentriolar material and for spermatids to assemble the proximal centriole-like structure (the precursor of the second centriole for zygote division). Hence, by taking advantage of the giant centriole/basal body of Drosophila spermatocyte, this study uncovered previously uncharacterized roles of HYLS1 in centriole elongation and assembly.
Huang, K., Miao, T., Chang, K., Kim, J., Kang, P., Jiang, Q., Simmonds, A. J., Di Cara, F. and Bai, H. (2020). Impaired peroxisomal import in Drosophila oenocytes causes cardiac dysfunction by inducing upd3 as a peroxikine. Nat Commun 11(1): 2943. PubMed ID: 32523050
Aging is characterized by a chronic, low-grade inflammation, which is a major risk factor for cardiovascular diseases. It remains poorly understood whether pro-inflammatory factors released from non-cardiac tissues contribute to the non-autonomous regulation of age-related cardiac dysfunction. This study reports that age-dependent induction of cytokine unpaired 3 (upd3) in Drosophila oenocytes (hepatocyte-like cells) is the primary non-autonomous mechanism for cardiac aging. upd3 is significantly up-regulated in aged oenocytes. Oenocyte-specific knockdown of upd3 is sufficient to block aging-induced cardiac arrhythmia. It was further shown that the age-dependent induction of upd3 is triggered by impaired peroxisomal import and elevated JNK signaling in aged oenocytes. Hormonal factors induced by peroxisome dysfunction are referred to as peroxikines. Intriguingly, oenocyte-specific overexpression of Pex5, the key peroxisomal import receptor, blocks age-related upd3 induction and alleviates cardiac arrhythmicity. Thus, these studies identify an important role of hepatocyte-specific peroxisomal import in mediating non-autonomous regulation of cardiac aging.
Johnson, S. L., Iannucci, J., Seeram, N. P. and Grammas, P. (2020). Inhibiting thrombin improves motor function and decreases oxidative stress in the LRRK2 transgenic Drosophila melanogaster model of Parkinson's disease. Biochem Biophys Res Commun 527(2): 532-538. PubMed ID: 32423817
Parkinson's disease (PD) is a complex neurodegenerative disease characterized by the presence of tremors, loss of dopaminergic neurons and accumulation of α-synuclein. While there is no single direct cause of PD, genetic mutations, exposure to pesticides, diet and traumatic brain injury have been identified as risk factors. Increasing evidence suggests that oxidative stress and neuroinflammation contribute to the pathogenesis of neuronal injury in neurodegenerative diseases such as PD and Alzheimer's disease (AD). Previous work documented that the multifunctional inflammatory mediator thrombin contributes to oxidative stress and neuroinflammation in AD. This study explored the role of thrombin in a transgenic PD model, the LRRK2 mutant Drosophila melanogaster. Transgenic flies were treated with the direct thrombin inhibitor dabigatran for 7 days and locomotor activity and indices of oxidative stress evaluated. The data show that dabigatran treatment significantly improved climbing activity, a measurement of locomotor ability, in male but had no effect on locomotor performance in female flies. Dabigatran treatment had no effect on tyrosine hydroxylase levels. Analysis of oxidative stress in male flies showed that dabigatran was able to significantly lower reactive oxygen species levels. Furthermore, Western blot analysis showed that the pro-oxidant proteins iNOS and NOX4 are elevated in LRRK2 male flies compared to wildtype and that treatment with dabigatran reduced expression of these proteins. These results indicate that dabigatran treatment could improve motor function in PD by reducing oxidative stress. These data suggest that targeting thrombin may improve oxidative stress related pathologies in PD.
Kim, J. H., Singh, M., Pan, G., Lopez, A., Zito, N., Bosse, B. and Ye, B. (2020). Frameshift mutations of YPEL3 alter the sensory circuit function in Drosophila. Dis Model Mech 13(6). PubMed ID: 32461240
A frameshift mutation in Yippee-like (YPEL) 3 was recently found from a rare human disorder with peripheral neurological conditions including hypotonia and areflexia. The YPEL gene family is highly conserved from yeast to human, but its members' functions are poorly defined. Moreover, the pathogenicity of the human YPEL3 variant is completely unknown. This study generated a Drosophila model of human YPEL3 variant and a genetic null allele of Drosophila homolog of YPEL3 (referred to as dYPEL3/CG15309). Gene-trap analysis suggests that dYPEL3 is predominantly expressed in subsets of neurons, including larval nociceptors. Analysis of chemical nociception induced by allyl-isothiocyanate (AITC), a natural chemical stimulant, revealed reduced nociceptive responses in both dYPEL3 frameshift and null mutants. Subsequent circuit analysis showed reduced activation of second-order neurons (SONs) in the pathway without affecting nociceptor activation upon AITC treatment. Although the gross axonal and dendritic development of nociceptors was unaffected, the synaptic contact between nociceptors and SONs was decreased by the dYPEL3 mutations. Furthermore, expressing dYPEL3 in larval nociceptors rescued the behavioral deficit in dYPEL3 frameshift mutants, suggesting a presynaptic origin of the deficit. Together, these findings suggest that the frameshift mutation results in YPEL3 loss of function and may cause neurological conditions by weakening synaptic connections through presynaptic mechanisms.

Wednesday, July 8th - Stem Cells

Tamamouna, V., Panagi, M., Theophanous, A., Demosthenous, M., Michail, M., Papadopoulou, M., Teloni, S., Pitsouli, C. and Apidianakis, Y. (2020). Evidence of two types of balance between stem cell mitosis and enterocyte nucleus growth in the Drosophila midgut. Development 147(11). PubMed ID: 32513656
Systemic and stem cell niche-emanating cytokines and growth factors can promote regeneration, through mitosis. High mitosis, however, predisposes for all types of cancer and, thus, a trade-off exists between regeneration capacity and tissue homeostasis. The role of tissue-intrinsic regenerative signaling was studied in stem cell mitosis of adult Drosophila midgut of different genetic backgrounds. Evidence is provided of two naturally occurring types of balance between mitosis and enterocyte nucleus growth: one based mostly on stem cell mitosis producing new cells and the other based mostly on the degree of young enterocyte nucleus size increase. Mitosis promotes intestinal host defense to infection, but predisposes for dysplasia in the form of stem cell-like clusters. Enterocyte nucleus growth also promotes host defense, without the drawback of promoting dysplasia. Through quantitative genetics, eiger was identified as an autocrine and paracrine inducer of stem cell mitosis. eiger expression in immature epithelial cells tilts the balance towards mitosis and dysplasia via a positive-feedback loop of highly mitotic stem cells sustaining more small nucleus enterocytes, which in turn supply more Eiger.
Strilbytska, O. M., Semaniuk, U. V., Storey, K. B., Yurkevych, I. S. and Lushchak, O. (2020). Insulin Signaling in Intestinal Stem and Progenitor Cells as an Important Determinant of Physiological and Metabolic Traits in Drosophila. Cells 9(4). PubMed ID: 32225024
The insulin-IGF-1 signaling (IIS) pathway is conserved throughout multicellular organisms and regulates many traits, including aging, reproduction, feeding, metabolism, stress resistance, and growth. This study presents evidence of a survival-sustaining role for IIS in a subset of gut cells in Drosophila melanogaster, namely the intestinal stem cells (ISCs) and progenitor cells. Using RNAi to knockdown the insulin receptor, inhibition of IIS in ISCs was found to statistically shortened the lifespan of experimental flies compared with non-knockdown controls, and also shortened their survival under starvation or malnutrition conditions. These flies also showed decreased reproduction and feeding, and had lower amounts of glycogen and glucose in the body. In addition, increased expression was observed for the Drosophila transcripts for the insulin-like peptides dilp2, dilp5, and dilp6. This may reflect increased insulin signaling in peripheral tissues supported by up-regulation of the target of the brain insulin gene (tobi). In contrast, activation of IIS (via knockdown of the insulin pathway inhibitor PTEN) in intestinal stem and progenitor cells decreased fly resistance to malnutrition, potentially by affecting adipokinetic hormone signaling. Finally, Pten knockdown to enhance IIS also activated JAK-STAT signaling in gut tissue by up-regulation of upd2, upd3, and soc36 genes, as well as genes encoding the EGF receptor ligands spitz and vein. These results clearly demonstrate that manipulating insulin levels may be used to modulate various fly traits, which are important determinants of organismal survival.
Na, H. J., Akan, I., Abramowitz, L. K. and Hanover, J. A. (2020). Nutrient-Driven O-GlcNAcylation Controls DNA Damage Repair Signaling and Stem/Progenitor Cell Homeostasis. Cell Rep 31(6): 107632. PubMed ID: 32402277
Stem/progenitor cells exhibit high proliferation rates, elevated nutrient uptake, altered metabolic flux, and stress-induced genome instability. O-GlcNAcylation is an essential post-translational modification mediated by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), which act in a nutrient- and stress-responsive manner. The precise role of O-GlcNAc in adult stem cells and the relationship between O-GlcNAc and the DNA damage response (DDR) is poorly understood. This study shows that hyper-O-GlcNacylation leads to elevated insulin signaling, hyperproliferation, and DDR activation that mimic the glucose- and oxidative-stress-induced response. A feedback mechanism was discovered involving key downstream effectors of DDR, ATM, ATR, and CHK1/2 that regulates OGT stability to promote O-GlcNAcylation and elevate DDR. This O-GlcNAc-dependent regulatory pathway is critical for maintaining gut homeostasis in Drosophila and the DDR in mouse embryonic stem cells (ESCs) and mouse embryonic fibroblasts (MEFs). These findings reveal a conserved mechanistic link among O-GlcNAc cycling, stem cell self-renewal, and DDR with profound implications for stem-cell-derived diseases including cancer.
Dattoli, A. A., Carty, B. L., Kochendoerfer, A. M., Morgan, C., Walshe, A. E. and Dunleavy, E. M. (2020). Asymmetric assembly of centromeres epigenetically regulates stem cell fate. J Cell Biol 219(4). PubMed ID: 32328637
Centromeres are epigenetically defined by CENP-A-containing chromatin and are essential for cell division. Previous studies suggest asymmetric inheritance of centromeric proteins upon stem cell division; however, the mechanism and implications of selective chromosome segregation remain unexplored. This study shows that Drosophila female germline stem cells (GSCs) and neuroblasts assemble centromeres after replication and before segregation. Specifically, CENP-A deposition is promoted by CYCLIN A, while excessive CENP-A deposition is prevented by CYCLIN B, through the HASPIN kinase. Furthermore, chromosomes inherited by GSCs incorporate more CENP-A, making stronger kinetochores that capture more spindle microtubules and bias segregation. Importantly, symmetric incorporation of CENP-A on sister chromatids via HASPIN knockdown or overexpression of CENP-A, either alone or together with its assembly factor CAL1, drives stem cell self-renewal. Finally, continued CENP-A assembly in differentiated cells is nonessential for egg development. This work shows that centromere assembly epigenetically drives GSC maintenance and occurs before oocyte meiosis.
Wang, C. and Spradling, A. C. (2020). An abundant quiescent stem cell population in Drosophila Malpighian tubules protects principal cells from kidney stones. Elife 9. PubMed ID: 32175841
Adult Drosophila Malpighian tubules have low rates of cell turnover but are vulnerable to damage caused by stones, like their mammalian counterparts, kidneys. This study shows that Drosophila renal stem cells (RSCs) in the ureter and lower tubules comprise a unique, unipotent regenerative compartment. RSCs respond only to loss of nearby principal cells (PCs), cells critical for maintaining ionic balance. Large polyploid PCs are outnumbered by RSCs, which replace each lost cell with multiple PCs of lower ploidy. Notably, RSCs do not replenish principal cells or stellate cells in the upper tubules. RSCs generate daughters by asymmetric Notch signaling, yet RSCs remain quiescent (cell cycle-arrested) without damage. Nevertheless, the capacity for RSC-mediated repair extends the lifespan of flies carrying kidney stones. It is proposed that abundant, RSC-like stem cells exist in other tissues with low rates of turnover where they may have been mistaken for differentiated tissue cells.
Jin, Z., Chen, J., Huang, H., Wang, J., Lv, J., Yu, M., Guo, X., Zhang, Y., Cai, T. and Xi, R. (2020). The Drosophila Ortholog of Mammalian Transcription Factor Sox9 Regulates Intestinal Homeostasis and Regeneration at an Appropriate Level. Cell Rep 31(8): 107683. PubMed ID: 32460025
Balanced stem cell self-renewal and differentiation is essential for maintaining tissue homeostasis, but the underlying mechanisms are poorly understood. This study identified the transcription factor SRY-related HMG-box (Sox) 100B, which is orthologous to mammalian Sox8/9/10, as a common target and central mediator of the EGFR/Ras and JAK/STAT signaling pathways that coordinates intestinal stem cell (ISC) proliferation and differentiation during both normal epithelial homeostasis and stress-induced intestinal repair in Drosophila. The two stress-responsive pathways directly regulate Sox100B transcription via two separate enhancers. Interestingly, an appropriate level of Sox100B is critical for its function, as its depletion inhibits ISC proliferation via cell cycle arrest, while its overexpression also inhibits ISC proliferation by directly suppressing EGFR expression and additionally promotes ISC differentiation by activating a differentiation-promoting regulatory circuitry composed of Sox100B, ">Sox21a, and Pdm1. Thus, this study reveals a Sox family transcription factor that functions as a stress-responsive signaling nexus that ultimately controls tissue homeostasis and regeneration.

Tuesday, July 7th - Cytoskeleton

Dehapiot, B., Clement, R., Alegot, H., Gazso-Gerhat, G., Philippe, J. M. and Lecuit, T. (2020). Assembly of a persistent apical actin network by the formin Frl/Fmnl tunes epithelial cell deformability. Nat Cell Biol. PubMed ID: 32483386
Tissue remodelling during Drosophila embryogenesis is notably driven by epithelial cell contractility. This behaviour arises from the Rho1-Rok-induced pulsatile accumulation of non-muscle myosin II pulling on actin filaments of the medioapical cortex. While recent studies have highlighted the mechanisms governing the emergence of Rho1-Rok-myosin II pulsatility, little is known about how F-actin organization influences this process. This study shows that the medioapical cortex consists of two entangled F-actin subpopulations. One exhibits pulsatile dynamics of actin polymerization in a Rho1-dependent manner. The other forms a persistent and homogeneous network independent of Rho1. The formin Frl (also known as Fmnl) has been identified as a critical nucleator of the persistent network, since modulating its level in mutants or by overexpression decreases or increases the network density. Absence of this network yields sparse connectivity affecting the homogeneous force transmission to the cell boundaries. This reduces the propagation range of contractile forces and results in tissue-scale morphogenetic defects.
Jammrath, J., Reim, I. and Saumweber, H. (2020). Cbl-Associated Protein CAP contributes to correct formation and robust function of the Drosophila heart tube. PLoS One 15(5): e0233719. PubMed ID: 32469960
In Drosophila, the cardiac tube originates from two bilateral rows of dorsally migrating cells. On meeting at the dorsal midline, coordinated changes in cell shape and adhesive properties transform the two sheets of cells into a linear tube. This study characterized the requirement of Cbl-Associated Protein (CAP) in Drosophila heart formation. In embryos, CAP is expressed in late migrating cardioblasts and is located preferentially at their luminal and abluminal periphery. CAP mutations result in irregular cardioblast alignment and imprecisely controlled cardioblast numbers. Furthermore, CAP mutant embryos show a strongly reduced heart lumen and an aberrant shape of lumen forming cardioblasts. Analysis of double heterozygous animals reveals a genetic interaction of CAP with Integrin- and Talin-encoding genes. In post-embryonic stages, CAP closely colocalizes with Integrin near Z-bands and at cell-cell contact sites. CAP mutants exhibit a reduced contractility in larval hearts and show a locally disrupted morphology, which correlates with a reduced pumping efficiency. These observations imply a function of CAP in linking Integrin signaling with the actin cytoskeleton.
Cui, H., Ali, M. Y., Goyal, P., Zhang, K., Loh, J. Y., Trybus, K. M. and Solmaz, S. R. (2020). Coiled-coil registry shifts in the F684I mutant of Bicaudal D result in cargo-independent activation of dynein motility. Traffic. PubMed ID: 32378283
The dynein adaptor Drosophila Bicaudal D (BicD) is auto-inhibited and activates dynein motility only after cargo is bound, but the underlying mechanism is elusive. In contrast, this study shows that the full-length BicD/F684I mutant activates dynein processivity even in the absence of cargo. X-ray structure of the C-terminal domain of the BicD/F684I mutant reveals a coiled-coil registry shift; in the N-terminal region, the two helices of the homodimer are aligned, whereas they are vertically shifted in the wild-type. One chain is partially disordered and this structural flexibility is confirmed by computations, which reveal that the mutant transitions back and forth between the two registries. It is proposed that a coiled-coil registry shift upon cargo binding activates BicD for dynein recruitment. Moreover, the human homolog BicD2/F743I exhibits diminished binding of cargo adaptor Nup358, implying that a coiled-coil registry shift may be a mechanism to modulate cargo selection for BicD2-dependent transport pathways.
Davidson, A. J. and Wood, W. (2020). Macrophages Use Distinct Actin Regulators to Switch Engulfment Strategies and Ensure Phagocytic Plasticity In Vivo. Cell Rep 31(8): 107692. PubMed ID: 32460022
Macrophages must not only be responsive to an array of different stimuli, such as infection and cellular damage, but also perform phagocytosis within the diverse and complex tissue environments found in vivo. This requires a high degree of morphological and therefore cytoskeletal plasticity. This study uses the exceptional genetics and in vivo imaging of Drosophila embryos to study macrophage phagocytic versatility during apoptotic corpse clearance. Macrophage phagocytosis is highly robust, arising from their possession of two distinct modes of engulfment that utilize exclusive suites of actin-regulatory proteins. "Lamellipodial phagocytosis" is Arp2/3-complex-dependent and allows cells to migrate toward and envelop apoptotic corpses. Alternatively, Diaphanous and Ena drive filopodial phagocytosis to reach out and draw in debris. Macrophages switch to "filopodial phagocytosis" to overcome spatial constraint, providing the robust plasticity necessary to ensure that whatever obstacle they encounter in vivo, they fulfil their critical clearance function.
Gartenmann, L., Vicente, C. C., Wainman, A., Novak, Z. A., Sieber, B., Richens, J. H. and Raff, J. W. (2020). Sas-6, Ana2 and Sas-4 self-organise into macromolecular structures that can be used to probe centriole/centrosome assembly. J Cell Sci. PubMed ID: 32409564
Centriole assembly requires a small number of conserved proteins. The precise pathway of centriole assembly has been difficult to study, as the lack of any one of the core assembly proteins-Plk4, Ana2/STIL, Sas-6, Sas-4/CPAP or Asl/Cep152-leads to the absence of centrioles. This study used Sas-6/Ana2 Particles (SAPs) as a new model to probe the pathway of centriole and centrosome assembly. SAPs form in Drosophila eggs/embryos when Sas-6 and Ana2 are overexpressed. SAP assembly requires Sas-4, but not Plk4, while Asl helps to initiate SAP assembly, but is not required for SAP growth. Although not centrioles, SAPs recruit and organize many centriole and centrosome components, nucleate microtubules, organise actin structures and compete with endogenous centrosomes to form mitotic spindle poles. SAPs require Asl/Cep152 to efficiently recruit pericentriolar material (PCM), but Spd-2/Cep192 can promote some PCM assembly independently of Asl/Cep152. These observations provide new insights into the pathways of centriole and centrosome assembly.
Lv, Z., Rosenbaum, J., Mohr, S., Zhang, X., Kong, D., Preiß, H., Kruss, S., Alim, K., Aspelmeier, T. and Großhans, J. (2020). The emergent yo-yo movement of nuclei driven by cytoskeletal remodeling in pseudo-synchronous mitotic cycles. Curr Biol. PubMed ID: 32470369
Many aspects in tissue morphogenesis are attributed to a collective behavior of the participating cells. Yet, the mechanism for emergence of dynamic tissue behavior is not well understood. This study reports that the "yo-yo"-like nuclear movement in the Drosophila syncytial embryo displays emergent features indicative of collective behavior. Following mitosis, the array of nuclei moves away from the wave front by several nuclear diameters only to return to its starting position about 5 min later. Based on experimental manipulations and numerical simulations, this study finds that the ensemble of elongating and isotropically oriented spindles, rather than individual spindles, is the main driving force for anisotropic nuclear movement. ELMO-dependent F-actin restricts the time for the forward movement and ELMO- and dia-dependent F-actin is essential for the return movement. This study provides insights into how the interactions among the cytoskeleton as individual elements lead to collective movement of the nuclear array on a macroscopic scale.

Monday, July 6th - Synapse and vesicles

Ye, H., Ojelade, S. A., Li-Kroeger, D., Zuo, Z., Wang, L., Li, Y., Gu, J. Y., Tepass, U., Rodal, A. A., Bellen, H. J. and Shulman, J. M. (2020). Retromer subunit, VPS29, regulates synaptic transmission and is required for endolysosomal function in the aging brain. Elife 9. PubMed ID: 32286230
Retromer, including Vps35, Vps26, and Vps29, is a protein complex responsible for recycling proteins within the endolysosomal pathway. Although implicated in both Parkinson's and Alzheimer's disease, understanding of retromer function in the adult brain remains limited, in part because Vps35 and Vps26 are essential for development. In Drosophila, this study finds that Vps29 is dispensable for embryogenesis but required for retromer function in aging adults, including for synaptic transmission, survival, and locomotion. Unexpectedly, in Vps29 mutants, Vps35 and Vps26 proteins are normally expressed and associated, but retromer is mislocalized from neuropil to soma with the Rab7 GTPase. Further, Vps29 phenotypes are suppressed by reducing Rab7 or overexpressing the GTPase activating protein, TBC1D5. With aging, retromer insufficiency triggers progressive endolysosomal dysfunction, with ultrastructural evidence of impaired substrate clearance and lysosomal stress. These results reveal the role of Vps29 in retromer localization and function, highlighting requirements for brain homeostasis in aging.
Araki, T., Osaka, J., Kato, Y., Shimozono, M., Kawamura, H., Iwanaga, R., Hakeda-Suzuki, S. and Suzuki, T. (2020). Systematic identification of genes regulating synaptic remodeling in the Drosophila visual system. Genes Genet Syst. PubMed ID: 32493879
In many animals, neural activity contributes to the adaptive refinement of synaptic properties. However, the molecular mechanisms underlying such activity-dependent synaptic remodeling remain largely unknown. In the synapses of Drosophila melanogaster, the presynaptic active zone (AZ) forms a T-shaped presynaptic density comprising AZ proteins, including Bruchpilot (Brp). A previous study found that the signal from a fusion protein molecular marker consisting of Brp and mCherry becomes diffuse under continuous light over three days (LL), reflecting disassembly of the AZ, while remaining punctate under continuous darkness. To identify the molecular players controlling this synaptic remodeling, the fusion protein molecular marker was used, and RNAi screening was performed against 208 neuron-related transmembrane genes that are highly expressed in the Drosophila visual system. Second analyses using the STaR (synaptic tagging with recombination) technique, which showed a decrease in synapse number under the LL condition, and subsequent mutant and overexpression analysis confirmed that five genes are involved in the activity-dependent AZ disassembly. This work demonstrates the feasibility of identifying genes involved in activity-dependent synaptic remodeling in Drosophila, and also provides unexpected insight into the molecular mechanisms involved in cholesterol metabolism and biosynthesis of the insect molting hormone ecdysone.
Chen, P. L., Huang, K. T., Cheng, C. Y., Li, J. C., Chan, H. Y., Lin, T. Y., Su, M. P., Yang, W. Y., Chang, H. C., Wang, H. D. and Chen, C. H. (2020). Vesicular transport mediates the uptake of cytoplasmic proteins into mitochondria in Drosophila melanogaster. Nat Commun 11(1): 2592. PubMed ID: 32444642
Mitochondrial aging, which results in mitochondrial dysfunction, is strongly linked to many age-related diseases. Aging is associated with mitochondrial enlargement and transport of cytosolic proteins into mitochondria. The underlying homeostatic mechanisms that regulate mitochondrial morphology and function, and their breakdown during aging, remain unclear. This study identified a mitochondrial protein trafficking pathway in Drosophila melanogaster involving the mitochondria-associated protein Dosmit. Dosmit induces mitochondrial enlargement and the formation of double-membraned vesicles containing cytosolic protein within mitochondria. The rate of vesicle formation increases with age. Vesicles originate from the outer mitochondrial membrane as observed by tracking Tom20 localization, and the process is mediated by the mitochondria-associated Rab32 protein. Dosmit expression level is closely linked to the rate of ubiquitinated protein aggregation, which are themselves associated with age-related diseases. The mitochondrial protein trafficking route mediated by Dosmit offers a promising target for future age-related mitochondrial disease therapies.
Dehnen, L., Janz, M., Verma, J. K., Psathaki, O. E., Langemeyer, L., Frohlich, F., Heinisch, J. J., Meyer, H., Ungermann, C. and Paululat, A. (2020). A trimeric metazoan Rab7 GEF complex is crucial for endocytosis and scavenger function. J Cell Sci. PubMed ID: 32499409
Endosome biogenesis in eukaryotic cells is critical for nutrient uptake and plasma membrane integrity. Early endosomes initially contain Rab5, which is replaced by Rab7 on late endosomes prior to their fusion with lysosomes. Recruitment of Rab7 to endosomes requires the Mon1-Ccz1 guanosine exchange factor (GEF). This study shows that full function of the Drosophila Mon1-Ccz1 complex requires a third stoichiometric subunit, termed Bulli (CG8270). Bulli localises to Rab7 positive endosomes, in agreement with its function in the GEF complex. Using Drosophila nephrocytes as a model system, this study observed that absence of Bulli results in (i) reduced endocytosis, (ii) Rab5 accumulation within non-acidified enlarged endosomes, and (iii) defective Rab7 localisation and (iv) impaired endosomal maturation. Moreover, longevity of animals lacking bulli is affected. Both Mon1-Ccz1 dimer and a Bulli-containing trimer display Rab7 GEF activity. In summary, this suggests a key role of Bulli in Rab5 to Rab7 transition during endosomal maturation rather than a direct influence on the GEF activity of Mon1-Ccz1.
Ho, C. H. and Treisman, J. E. (2020). Specific Isoforms of the Guanine-Nucleotide Exchange Factor dPix Couple Neuromuscular Synapse Growth to Muscle Growth. Dev Cell. PubMed ID: 32516570
Developmental growth requires coordination between the growth rates of individual tissues and organs. This study examined how Drosophila neuromuscular synapses grow to match the size of their target muscles. Changes in muscle growth driven by autonomous modulation of insulin receptor signaling produce corresponding changes in synapse size, with each muscle affecting only its presynaptic motor neuron branches. This scaling growth is mechanistically distinct from synaptic plasticity driven by neuronal activity and requires increased postsynaptic differentiation induced by insulin receptor signaling in muscle. This study identified the guanine-nucleotide exchange factor dPix as an effector of insulin receptor signaling. Alternatively spliced dPix isoforms that contain a specific exon are necessary and sufficient for postsynaptic differentiation and scaling growth, and their mRNA levels are regulated by insulin receptor signaling. These findings define a mechanism by which the same signaling pathway promotes both autonomous muscle growth and non-autonomous synapse growth.
Li, Z., Zhang, Q., Chou, S. W., Newman, Z., Turcotte, R., Natan, R., Dai, Q., Isacoff, E. Y. and Ji, N. (2020). Fast widefield imaging of neuronal structure and function with optical sectioning in vivo. Sci Adv 6(19): eaaz3870. PubMed ID: 32494711
Optical microscopy, owing to its noninvasiveness and subcellular resolution, enables in vivo visualization of neuronal structure and function in the physiological context. Optical-sectioning structured illumination microscopy (OS-SIM) is a widefield fluorescence imaging technique that uses structured illumination patterns to encode in-focus structures and optically sections 3D samples. However, its application to in vivo imaging has been limited. In this study, OS-SIM was optimized for in vivo neural imaging. OS-SIM reconstruction algorithms were modified to improve signal-to-noise ratio and correct motion-induced artifacts in live samples. Incorporating an adaptive optics (AO) module to OS-SIM, it was found that correcting sample-induced optical aberrations was essential for achieving accurate structural and functional characterizations in vivo. With AO OS-SIM, fast, high-resolution in vivo imaging was demonstrated with optical sectioning for structural imaging of mouse cortical neurons and zebrafish larval motor neurons, and functional imaging was demonstrated of quantal synaptic transmission at Drosophila larval neuromuscular junctions.

Thursday July 2nd - Cell cycle

Liu, B., Gregor, I., Muller, H. A. and Grosshans, J. (2020). Fluorescence fluctuation analysis reveals PpV dependent Cdc25 protein dynamics in living embryos. PLoS Genet 16(4): e1008735. PubMed ID: 32251417
The protein phosphatase Cdc25 is a key regulator of the cell cycle by activating Cdk-cyclin complexes. Cdc25 is regulated by its expression levels and post-translational mechanisms. In early Drosophila embryogenesis, Cdc25/Twine drives the fast and synchronous nuclear cycles. A pause in the cell cycle and the remodeling to a more generic cell cycle mode with a gap phase are determined by Twine inactivation and destruction in early interphase 14, in response to zygotic genome activation. Although the pseudokinase Tribbles contributes to the timely degradation of Twine, Twine levels are controlled by additional yet unknown post-translational mechanisms. This study applied a non-invasive method based on fluorescence fluctuation analysis (FFA) to record the absolute concentration profiles of Twine with minute-scale resolution in single living embryos. Employing this assay, it was found that Protein phosphatase V (PpV), the homologue of the catalytic subunit of human PP6, ensures appropriately low Twine protein levels at the onset of interphase 14. PpV controls directly or indirectly the phosphorylation of Twine at multiple serine and threonine residues as revealed by phosphosite mapping. Mutational analysis confirmed that these sites are involved in control of Twine protein dynamics, and cell cycle remodeling is delayed in a fraction of the phosphosite mutant embryos. These data reveal a novel mechanism for control of Twine protein levels and their significance for embryonic cell cycle remodeling.
Qian, W., Li, Z., Song, W., Zhao, T., Wang, W., Peng, J., Wei, L., Xia, Q. and Cheng, D. (2020). A novel transcriptional cascade is involved in Fzr-mediated endoreplication. Nucleic Acids Res. PubMed ID: 32182338
Endoreplication, known as endocycle, is a variant of the cell cycle that differs from mitosis and occurs in specific tissues of different organisms. Endoreplicating cells generally undergo multiple rounds of genome replication without chromosome segregation. Previous studies demonstrated that Drosophila fizzy-related protein (Fzr) and its mammalian homolog Cdh1 function as key regulators of endoreplication entrance by activating the anaphase-promoting complex/cyclosome to initiate the ubiquitination and subsequent degradation of cell cycle factors such as Cyclin B (CycB). However, the molecular mechanism underlying Fzr-mediated endoreplication is not completely understood. This study demonstrated that the transcription factor Myc acts downstream of Fzr during endoreplication in Drosophila salivary gland. Mechanistically, Fzr interacts with chromatin-associated histone H2B to enhance H2B ubiquitination in the Myc promoter and promotes Myc transcription. In addition to negatively regulating CycB transcription, the Fzr-ubiquitinated H2B (H2Bub)-Myc signaling cascade also positively regulates the transcription of the MCM6 gene that is involved in DNA replication by directly binding to specific motifs within their promoters. This study further found that the Fzr-H2Bub-Myc signaling cascade regulating endoreplication progression is conserved between insects and mammalian cells. Altogether, this work uncovers a novel transcriptional cascade that is involved in Fzr-mediated endoreplication.
Morita, S., Ota, R., Hayashi, M. and Kobayashi, S. (2020). Repression of G1/S Transition by Transient Inhibition of miR-10404 Expression in Drosophila Primordial Germ Cells. iScience 23(3): 100950. PubMed ID: 32179474
Cell-cycle quiescence is a common feature of early germline development in many animal species. In Drosophila germline progenitors (pole cells), both G2/M and G1/S transitions are blocked. G2/M transition is repressed by maternal Nanos through suppression of Cyclin B production. However, the molecular mechanism underlying blockage of G1/S transition remains elusive. This study found that repression of miR-10404 expression is required to block G1/S transition in pole cells. Expression of miR-10404, a microRNA encoded within the internal transcribed spacer 1 of rDNA, is repressed in early pole cells by maternal polar granule component. This repression delays the degradation of maternal dacapo mRNA, which encodes an inhibitor of G1/S transition. Moreover, derepression of G1/S transition in pole cells causes defects in their maintenance and their migration into the gonads. These observations reveal the mechanism inhibiting G1/S transition in pole cells and its requirement for proper germline development.
Goupil, A., Nano, M., Letort, G., Gemble, S., Edwards, F., Goundiam, O., Gogendeau, D., Pennetier, C. and Basto, R. (2020). Chromosomes function as a barrier to mitotic spindle bipolarity in polyploid cells. J Cell Biol 219(4). PubMed ID: 32328633
Ploidy variations such as genome doubling are frequent in human tumors and have been associated with genetic instability favoring tumor progression. How polyploid cells deal with increased centrosome numbers and DNA content remains unknown. Using Drosophila neuroblasts and human cancer cells to study mitotic spindle assembly in polyploid cells, this study found that most polyploid cells divide in a multipolar manner. Even if an initial centrosome clustering step can occur at mitotic entry, the establishment of kinetochore-microtubule attachments leads to spatial chromosome configurations, whereby the final coalescence of supernumerary poles into a bipolar array is inhibited. Using in silico approaches and various spindle and DNA perturbations, this study shows that chromosomes act as a physical barrier blocking spindle pole coalescence and bipolarity. Importantly, microtubule stabilization suppressed multipolarity by improving both centrosome clustering and pole coalescence. This work identifies inhibitors of bipolar division in polyploid cells and provides a rationale to understand chromosome instability typical of polyploid cancer cells.
Kleinschnitz, K., Viessmann, N., Jordan, M. and Heidmann, S. K. (2020). Condensin I is required for faithful meiosis in Drosophila males. Chromosoma. PubMed ID: 32314039
The heteropentameric condensin complexes play vital roles in the formation and faithful segregation of mitotic chromosomes in eukaryotes. While the different contributions of the two common condensin complexes, condensin I and condensin II, to chromosome morphology and behavior in mitosis have been thoroughly investigated, much less is known about the specific roles of the two complexes during meiotic divisions. In Drosophila melanogaster, faithful mitotic divisions depend on functional condensin I, but not on condensin II. However, meiotic divisions in Drosophila males require functional condensin II subunits. The role of condensin I during male meiosis in Drosophila has been unresolved. This study shows that condensin I-specific subunits localize to meiotic chromatin in both meiosis I and II during Drosophila spermatogenesis. Live cell imaging reveals defects during meiotic divisions after RNAi-mediated knockdown of condensin I-specific mRNAs. This phenotype correlates with reduced male fertility and an increase in nondisjunction events both in meiosis I and meiosis II. Consistently, a reduction in male fertility was also observed after proteasome-mediated degradation of the condensin I subunit Barren. Taken together, these results demonstrate an essential role of condensin I during male meiosis in Drosophila melanogaster.
Landmann, C., Pierre-Elies, P., Goutte-Gattat, D., Montembault, E., Claverie, M. C. and Royou, A. (2020). The Mre11-Rad50-Nbs1 complex mediates the robust recruitment of Polo to DNA lesions during mitosis. J Cell Sci. PubMed ID: 32487663
The DNA damage sensor, Mre11-Rad50-Nbs1 complex, and Polo kinase are recruited to DNA lesions during mitosis. However, their mechanism of recruitment is elusive. Using live-cell imaging combined with the micro-irradiation of single chromosomes, this study analyzed the dynamics of Polo and Mre11 at DNA lesions during mitosis. The two proteins display distinct kinetics. While Polo kinetics at DSBs are Cdk1-driven, Mre11 promptly but briefly associates with DSBs regardless of the phase of mitosis and re-associates with DSBs in the proceeding interphase. Mechanistically, Polo kinase activity is required for its own recruitment and that of the mitotic proteins BubR1 and Bub3 to DSBs. Moreover, depletion of Rad50 severely impaired Polo kinetics at mitotic DSBs. Conversely, ectopic tethering of Mre11 to chromatin is sufficient to recruit Polo. This study highlights a novel pathway that links the DSB sensor MRN complex and Polo kinase to initiate a prompt, decisive response to the presence of DNA damage during mitosis.

Wednesday, July 1st - Adult Development

Imura, E., Shimada-Niwa, Y., Nishimura, T., Huckesfeld, S., Schlegel, P., Ohhara, Y., Kondo, S., Tanimoto, H., Cardona, A., Pankratz, M. J. and Niwa, R. (2020). The Corazonin-PTTH Neuronal Axis Controls Systemic Body Growth by Regulating Basal Ecdysteroid Biosynthesis in Drosophila melanogaster. Curr Biol 30(11): 2156-2165. PubMed ID: 32386525
Steroid hormones play key roles in development, growth, and reproduction in various animal phyla. The insect steroid hormone, ecdysteroid, coordinates growth and maturation, represented by molting and metamorphosis. In Drosophila melanogaster, the prothoracicotropic hormone (PTTH)-producing neurons stimulate peak levels of ecdysteroid biosynthesis for maturation. Additionally, recent studies on PTTH signaling indicated that basal levels of ecdysteroid negatively affect systemic growth prior to maturation. However, it remains unclear how PTTH signaling is regulated for basal ecdysteroid biosynthesis. This study reports that Corazonin (Crz)-producing neurons regulate basal ecdysteroid biosynthesis by affecting PTTH neurons. Crz belongs to gonadotropin-releasing hormone (GnRH) superfamily, implying an analogous role in growth and maturation. Inhibition of Crz neuronal activity increased pupal size, whereas it hardly affected pupariation timing. This phenotype resulted from enhanced growth rate and a delay in ecdysteroid elevation during the mid-third instar larval (L3) stage. Interestingly, Crz receptor (CrzR) expression in PTTH neurons was higher during the mid- than the late-L3 stage. Silencing of CrzR in PTTH neurons increased pupal size, phenocopying the inhibition of Crz neuronal activity. When Crz neurons were optogenetically activated, a strong calcium response was observed in PTTH neurons during the mid-L3, but not the late-L3, stage. Furthermore, octopamine neurons were found to contact Crz neurons in the subesophageal zone (SEZ), transmitting signals for systemic growth. Together, these results suggest that the Crz-PTTH neuronal axis modulates ecdysteroid biosynthesis in response to octopamine, uncovering a regulatory neuroendocrine system in the developmental transition from growth to maturation.
Jung, J., Yeom, E. and Choi, K. W. (2020). Ciao1 interacts with Crumbs and Xpd to regulate organ growth in Drosophila. Cell Death Dis 11(5): 365. PubMed ID: 32404863
Ciao1 is a component of the cytosolic iron-sulfur cluster assembly (CIA) complex along with MMS19 and MIP18. Xeroderma pigmentosum group D (XPD), a DNA helicase involved in regulation of cell cycle and transcription, is a CIA target for iron-sulfur (Fe/S) modification. In vivo function of Ciao1 and Xpd in developing animals has been rarely studied. This study reveals that Ciao1 interacts with Crumbs (Crb), Galla, and Xpd to regulate organ growth in Drosophila. Abnormal growth of eye by overexpressing Crb intracellular domain (Crb(intra)) is suppressed by reducing the Ciao1 level. Loss of Ciao1 or Xpd causes similar impairment in organ growth. RNAi knockdown of both Ciao1 and Xpd show similar phenotypes as Ciao1 or Xpd RNAi alone, suggesting their function in a pathway. Growth defects caused by Ciao1 RNAi are suppressed by overexpression of Xpd. Ciao1 physically interacts with Crb(intra), Galla, and Xpd, supporting their genetic interactions. Remarkably, Xpd RNAi defects can also be suppressed by Ciao1 overexpression, implying a mutual regulation between the two genes. Ciao1 mutant clones in imaginal discs show decreased levels of Cyclin E (CycE) and death-associated inhibitor of apoptosis 1 (Diap1). Xpd mutant clones share the similar reduction of CycE and Diap1. Consequently, knockdown of Ciao1 and Xpd by RNAi show increased apoptotic cell death. Further, CycE overexpression is sufficient to restore the growth defects from Ciao1 RNAi or Xpd RNAi. Interestingly, Diap1 overexpression in Ciao1 mutant clones induces CycE expression, suggesting that reduced CycE in Ciao1 mutant cells is secondary to loss of Diap1. Taken together, this study reveals new roles of Ciao1 and Xpd in cell survival and growth through regulating Diap1 level during organ development.
Banreti, A. R. and Meier, P. (2020). The NMDA receptor regulates competition of epithelial cells in the Drosophila wing. Nat Commun 11(1): 2228. PubMed ID: 32376880
Cell competition is an emerging principle that eliminates suboptimal or potentially dangerous cells. For 'unfit' cells to be detected, their competitive status needs to be compared to the collective fitness of cells within a tissue. This study reports that the NMDA receptor controls cell competition of epithelial cells and Myc supercompetitors in the Drosophila wing disc. While clonal depletion of the NMDA receptor subunit NR2 results in their rapid elimination via the TNF/Eiger>JNK signalling pathway, local over-expression of NR2 causes NR2 cells to acquire supercompetitor-like behaviour that enables them to overtake the tissue through clonal expansion that causes, but also relies on, the killing of surrounding cells. Consistently, NR2 is utilised by Myc clones to provide them with supercompetitor status. Mechanistically, this study found that the JNK>PDK signalling axis in 'loser' cells reprograms their metabolism, driving them to produce and transfer lactate to winners. Preventing lactate transfer from losers to winners abrogates NMDAR-mediated cell competition. These findings demonstrate a functional repurposing of NMDAR in the surveillance of tissue fitness.
Chodankar, A., Sadanandappa, M. K., VijayRaghavan, K. and Ramaswami, M. (2020). Glomerulus-selective regulation of a critical period for interneuron plasticity in the Drosophila antennal lobe. J Neurosci. PubMed ID: 32532889
Several features of the adult nervous systems develop in a "critical period," (CP) during which high levels of plasticity allow neural circuits to be tuned for optimal performance. Through an analysis of long-term olfactory habituation (LTH) in female Drosophila, this study provides new insight into mechanisms by which CPs are regulated in vivo LTH manifests as a persistently reduced behavioural response to an odorant encountered for four continuous days and occurs together with the growth of specific, odorant-responsive glomeruli in the antennal lobe. The CP for behavioral and structural plasticity induced by ethyl butyrate (EB) or carbon dioxide (CO(2)) closes within 48 hours after eclosion. The elaboration of excitatory projection neuron (PN) processes likely contribute to glomerular volume increases: both occur together and similarly require cAMP signalling in the antennal lobe inhibitory local interneurons (iLNs). Further, the CP for structural plasticity could be extended beyond 48 hours if EB- or CO(2)-responsive olfactory sensory neurons (OSNs) are silenced after eclosion; thus, OSN activity is required for closing the CP. Strikingly, silencing of glomerulus-selective OSNs extends the CP for structural plasticity only in respective target glomeruli. This indicates existence of a local, short-range mechanism for regulating CP closure.
Keder, A., Tardieu, C., Malong, L., Filia, A., Kashkenbayeva, A., Newton, F., Georgiades, M., Gale, J. E., Lovett, M., Jarman, A. P. and Albert, J. T. (2020). Homeostatic maintenance and age-related functional decline in the Drosophila ear. Sci Rep 10(1): 7431. PubMed ID: 32366993
Age-related hearing loss (ARHL) is a threat to future human wellbeing. Multiple factors contributing to the terminal auditory decline have been identified; but a unified understanding of ARHL - or the homeostatic maintenance of hearing before its breakdown - is missing. This study presents an in-depth analysis of homeostasis and ageing in the antennal ears of the fruit fly Drosophila melanogaster. Drosophila, just like humans, display ARHL. By focusing on the phase of dynamic stability prior to the eventual hearing loss a set was discovered of evolutionarily conserved homeostasis genes. The transcription factors Onecut (closest human orthologues: ONECUT2, ONECUT3), Optix (SIX3, SIX6), Worniu (SNAI2) and Amos (ATOH1, ATOH7, ATOH8, NEUROD1) emerged as key regulators, acting upstream of core components of the fly's molecular machinery for auditory transduction and amplification. Adult-specific manipulation of homeostatic regulators in the fly's auditory neurons accelerated - or protected against - ARHL.
Chauhan, N., Shrivastava, N. K., Agrawal, N. and Shakarad, M. N. (2020). Wing patterning in faster developing Drosophila is associated with high ecdysone titer and wingless expression. Mech Dev: 103626. PubMed ID: 32526278
'Developmental robustness' is the ability of biological systems to maintain a stable phenotype despite genetic, environmental or physiological perturbations. In holometabolous insects, accurate patterning and development is guaranteed by alignment of final gene expression patterns in tissues at specific developmental stage such as molting and pupariation, irrespective of individual rate of development. In the present study, faster developing Drosophila melanogaster populations were used that show reduction of ~22% in egg to adult development time. Flies from the faster developing population exhibit phenotype constancy, although significantly small in size. The reduction in development time in faster developing flies is possibly due to coordination between higher ecdysteroid release and higher expression of developmental genes. The two together might be ensuring appropriate pattern formation and early exit at each development stage in the populations selected for faster pre-adult development compared to their ancestral controls. This study reports that apart from plasticity in the rate of pattern progression, alteration in the level of gene expression may be responsible for pattern integrity even under reduced development time.
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