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Current papers in developmental biology and gene function


Friday, July 30th - Disease models

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Fellgett, A., Middleton, C. A., Munns, J., Ugbode, C., Jaciuch, D., Wilson, L., Chawla, S. and Elliott, C. J. H. (2021). Multiple Pathways of LRRK2-G2019S/Rab10 Interaction in Dopaminergic Neurons. J Parkinsons Dis. PubMed ID: 34250948
Inherited mutations in the LRRK2 protein are the common causes of Parkinson's disease, but the mechanisms by which increased kinase activity of mutant LRRK2 leads to pathological events remain to be determined. In vitro assays (heterologous cell culture, phospho-protein mass spectrometry) suggest that several Rab proteins might be directly phosphorylated by LRRK2-G2019S. An in vivo screen of Rab expression in dopaminergic neurons in young adult Drosophila demonstrated a strong genetic interaction between LRRK2-G2019S and Rab10. To determine if Rab10 is necessary for LRRK2-induced pathophysiological responses in the neurons that control movement, vision, circadian activity, and memory. These four systems were chosen because they are modulated by dopaminergic neurons in both humans and flies. LRRK2-G2019S was expressed in Drosophila dopaminergic neurons and the effects of Rab10 depletion on Proboscis Extension, retinal neurophysiology, circadian activity pattern ('sleep'), and courtship memory determined in aged flies. Rab10 loss-of-function rescued LRRK2-G2019S induced bradykinesia and retinal signaling deficits. Rab10 knock-down, however, did not rescue the marked sleep phenotype which results from dopaminergic LRRK2-G2019S. Courtship memory is not affected by LRRK2, but is markedly improved by Rab10 depletion. Anatomically, both LRRK2-G2019S and Rab10 are seen in the cytoplasm and at the synaptic endings of dopaminergic neurons. It is concluded that, in Drosophila dopaminergic neurons, Rab10 is involved in some, but not all, LRRK2-induced behavioral deficits. Therefore, variations in Rab expression may contribute to susceptibility of different dopaminergic nuclei to neurodegeneration seen in people with Parkinson's disease.
Brandwine, T., Ifrah, R., Bialistoky, T., Zaguri, R., Rhodes-Mordov, E., Mizrahi-Meissonnier, L., Sharon, D., Katanaev, V. L., Gerlitz, O. and Minke, B. (2021). Knockdown of Dehydrodolichyl Diphosphate Synthase in the Drosophila Retina Leads to a Unique Pattern of Retinal Degeneration. Front Mol Neurosci 14: 693967. PubMed ID: 34290587
Dehydrodolichyl diphosphate synthase (DHDDS) is a ubiquitously expressed enzyme that catalyzes cis-prenyl chain elongation to produce the poly-prenyl backbone of dolichol. Individuals who have biallelic missense mutations in the DHDDS gene are presented with non-syndromic retinitis pigmentosa with unknown underlying mechanism. This study used the Drosophila model to compromise DHDDS ortholog gene (CG10778) in order to look for cellular and molecular mechanisms that, when defective, might be responsible for this retinal disease. The Gal4/UAS system was used to suppress the expression of CG10778 via RNAi-mediated-knockdown in various tissues. Targeted knockdown of CG10778-mRNA in the early embryo using the actin promoter or in the developing wings using the nub promoter resulted in lethality, or wings loss, respectively. Targeted expression of CG10778-RNAi using the glass multiple reporter (GMR)-Gal4 driver (GMR-DHDDS-RNAi) in the larva eye disc and pupal retina resulted in a complex phenotype: (a) TEM retinal sections revealed a unique pattern of retinal-degeneration, where photoreceptors R2 and R5 exhibited a nearly normal structure of their signaling-compartment (rhabdomere), but only at the region of the nucleus, while all other photoreceptors showed retinal degeneration at all regions. (b) Western blot analysis revealed a drastic reduction in rhodopsin levels in GMR-DHDDS-RNAi-flies and TEM sections showed an abnormal accumulation of endoplasmic reticulum (ER). To conclude, compromising DHDDS in the developing retina, while allowing formation of the retina, resulted in a unique pattern of retinal degeneration, characterized by a dramatic reduction in rhodopsin protein level and an abnormal accumulation of ER membranes in the photoreceptors cells, thus indicating that DHDDS is essential for normal retinal formation.
Zhao, J., Xue, J., Zhu, T., He, H., Kang, H., Jiang, X., Huang, W. and Duan, R. (2021). Dysregulated CRMP Mediates Circadian Deficits in a Drosophila Model of Fragile X Syndrome. Neurosci Bull. PubMed ID: 33856646
Fragile X syndrome (FXS) is the leading inherited cause of intellectual disability, resulting from the lack of functional fragile X mental retardation protein (FMRP), an mRNA binding protein mainly serving as a translational regulator. Loss of FMRP leads to dysregulation of target mRNAs. The Drosophila model of FXS show an abnormal circadian rhythm with disruption of the output pathway downstream of the clock network. Yet the FMRP targets involved in circadian regulation have not been identified. This study identified collapsing response mediator protein (CRMP) mRNA as a target of FMRP. Knockdown of pan-neuronal CRMP expression ameliorated the circadian defects and abnormal axonal structures of clock neurons (ventral lateral neurons) in dfmr1 mutant flies. Furthermore, specific reduction of CRMP in the downstream output insulin-producing cells attenuated the aberrant circadian behaviors. Molecular analyses revealed that FMRP binds with CRMP mRNA and negatively regulates its translation. These results indicate that CRMP is an FMRP target and establish an essential role for CRMP in the circadian output in FXS Drosophila.
Zhao, M. J., Yao, X., Wei, P., Zhao, C., Cheng, M., Zhang, D., Xue, W., He, W. T., Xue, W., Zuo, X., Jiang, L. L., Luo, Z., Song, J., Shu, W. J., Yuan, H. Y., Liang, Y., Sun, H., Zhou, Y., Zhou, Y., Zheng, L., Hu, H. Y., Wang, J. and Du, H. N. (2021). O-GlcNAcylation of TDP-43 suppresses proteinopathies and promotes TDP-43's mRNA splicing activity. EMBO Rep: e51649. PubMed ID: 33855783
Pathological TDP-43 aggregation is characteristic of several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-TDP); however, how TDP-43 aggregation and function are regulated remain poorly understood. This study shows that O-GlcNAc transferase OGT-mediated O-GlcNAcylation of TDP-43 suppresses ALS-associated proteinopathies and promotes TDP-43's splicing function. Biochemical and cell-based assays indicate that OGT's catalytic activity suppresses TDP-43 aggregation and hyperphosphorylation, whereas abolishment of TDP-43 O-GlcNAcylation impairs its RNA splicing activity. This study further showed that TDP-43 mutations in the O-GlcNAcylation sites improve locomotion defects of larvae and adult flies and extend adult life spans, following TDP-43 overexpression in Drosophila motor neurons. This study demonstrates that O-GlcNAcylation of TDP-43 promotes proper splicing of many mRNAs, including STMN2, which is required for normal axonal outgrowth and regeneration. These findings suggest that O-GlcNAcylation might be a target for the treatment of TDP-43-linked pathogenesis.
Botero, V., Stanhope, B. A., Brown, E. B., Grenci, E. C., Boto, T., Park, S. J., King, L. B., Murphy, K. R., Colodner, K. J., Walker, J. A., Keene, A. C., Ja, W. W. and Tomchik, S. M. (2021). Neurofibromin regulates metabolic rate via neuronal mechanisms in Drosophila. Nat Commun 12(1): 4285. PubMed ID: 34257279
Neurofibromatosis type 1 is a chronic multisystemic genetic disorder that results from loss of function in the neurofibromin protein. Neurofibromin may regulate metabolism, though the underlying mechanisms remain largely unknown. This study shows that neurofibromin regulates metabolic homeostasis in Drosophila via a discrete neuronal circuit. Loss of neurofibromin increases metabolic rate via a Ras GAP-related domain-dependent mechanism, increases feeding homeostatically, and alters lipid stores and turnover kinetics. The increase in metabolic rate is independent of locomotor activity, and maps to a sparse subset of neurons. Stimulating these neurons increases metabolic rate, linking their dynamic activity state to metabolism over short time scales. These results indicate that neurofibromin regulates metabolic rate via neuronal mechanisms, suggest that cellular and systemic metabolic alterations may represent a pathophysiological mechanism in neurofibromatosis type 1, and provide a platform for investigating the cellular role of neurofibromin in metabolic homeostasis.
Cheng, K. C., Chen, Y. H., Wu, C. L., Lee, W. P., Cheung, C. H. A. and Chiang, H. C. (2021). Rac1 and Akt Exhibit Distinct Roles in Mediating Abeta-Induced Memory Damage and Learning Impairment. Mol Neurobiol. PubMed ID: 34273104
Accumulated β-amyloid (Aβ) in the brain is the hallmark of Alzheimer's disease (AD). Despite Aβ accumulation is known to trigger cellular dysfunctions and learning and memory damage, the detailed molecular mechanism remains elusive. Recent studies have shown that the onset of memory impairment and learning damage in the AD animal is different, suggesting that the underlying mechanism of the development of memory impairment and learning damage may not be the same. In the current study, with the use of Aβ42 transgenic flies as models, this study found that Aβ induces memory damage and learning impairment via differential molecular signaling pathways. In early stage, Aβ activates both Ras and PI3K to regulate Rac1 activity, which affects mostly on memory performance. In later stage, PI3K-Akt is strongly activated by Aβ, which leads to learning damage. Moreover, reduced Akt, but not Rac1, activity promotes cell viability in the Aβ42 transgenic flies, indicating that Akt and Rac1 exhibit differential roles in Aβ regulating toxicity. Taken together, different molecular and cellular mechanisms are involved in Aβ-induced learning damage and memory decline; thus, caution should be taken during the development of therapeutic intervention in the future.

Thursday July 29th - Signaling

Zhang, F., Liu, B., Gao, Y., Long, J. and Zhou, H. (2021). The crystal structure of the FERM and C-terminal domain complex of Drosophila Merlin. Biochem Biophys Res Commun 553: 92-98. PubMed ID: 33765559
NF2/Merlin is an upstream regulator of hippo pathway, and it has two states: an auto-inhibited "closed" state and an active "open" form. Previous studies showed that Drosophila Merlin adopts a more closed conformation. However, the molecular mechanism of conformational regulation remains poorly understood. This study first confirmed the strong interaction between FERM and the C-terminal domain (CTD) of Merlin, and then determined the crystal structure of the FERM/CTD complex, which reveals the structural basis of Merlin adopting a more closed conformation compared to its human cognate NF2. Interestingly, it was found that the conserved lipid-binding site of Merlin might be masked by a linker. Confocal analyses confirmed that all putative lipid-binding site are very important for the membranal location of Merlin. More, it was found that the phosphomimic Thr616Asp mutation weakens the interaction between FERM and CTD of Merlin. Collectively, the crystal structure of the FERM/CTD complex not only provides a mechanistic explanation of functionally dormant conformation of Merlin may also serve as a foundation for revealing the mechanism of conformational regulation of Merlin.
Hodge, S. H., Watts, A., Marley, R., Baines, R. A., Hafen, E. and MacDougall, L. K. (2021). Twitchy, the Drosophila orthologue of the ciliary gating protein FBF1/dyf-19, is required for coordinated locomotion and male fertility. Biol Open. PubMed ID: 34232985
Primary cilia are compartmentalised from the rest of the cell by a ciliary gate comprising transition fibres and a transition zone. The ciliary gate allows the selective import and export of molecules such as transmembrane receptors and transport proteins. Certain motile cilia can also form within the cytosol as exemplified by human and Drosophila sperm. The role of transition fibre proteins has not been well described in the cytoplasmic cilia. Drosophila have both compartmentalized primary cilia, in sensory neurons, and sperm flagella that form within the cytosol. This study describes phenotypes for twitchy the Drosophila orthologue of a transition fibre protein, mammalian FBF1/C. elegans dyf-19. Loss-of-function mutants in twitchy are adult lethal and display a severely uncoordinated phenotype. twitchy flies are too uncoordinated to mate but RNAi-mediated loss of twitchy specifically within the male germline results in coordinated but infertile adults. Examination of sperm from twitchy RNAi-knockdown flies shows that the flagellar axoneme forms, elongates and is post-translationally modified by polyglycylation but the production of motile sperm is impaired. These results indicate that twitchy is required for the function of both sensory cilia that are compartmentalized from the rest of the cell and sperm flagella that are formed within the cytosol of the cell. Twitchy is therefore likely to function as part of a molecular gate in sensory neurons but may have a distinct function in sperm cells.
Biehler, C., Rothenberg, K. E., Jette, A., Gaude, H. M., Fernandez-Gonzalez, R. and Laprise, P. (2021). Pak1 and PP2A antagonize aPKC function to support cortical tension induced by the Crumbs-Yurt complex. Elife 10. PubMed ID: 34212861
The Drosophila polarity protein Crumbs is essential for the establishment and growth of the apical domain in epithelial cells. The protein Yurt limits the ability of Crumbs to promote apical membrane growth, thereby defining proper apical/lateral membrane ratio that is crucial for forming and maintaining complex epithelial structures such as tubes or acini. This study shows that Yurt also increases Myosin-dependent cortical tension downstream of Crumbs. Yurt overexpression thus induces apical constriction in epithelial cells. The kinase aPKC phosphorylates Yurt, thereby dislodging the latter from the apical domain and releasing apical tension. In contrast, the kinase Pak1 promotes Yurt dephosphorylation through activation of the phosphatase PP2A. The Pak1-PP2A module thus opposes aPKC function and supports Yurt-induced apical constriction. Hence, the complex interplay between Yurt, aPKC, Pak1, and PP2A contributes to the functional plasticity of Crumbs. Overall, these data increase understanding of how proteins sustaining epithelial cell polarization and Myosin-dependent cell contractility interact with one another to control epithelial tissue architecture.
Chang, Y. C., Peng, Y. X., Yu, B. H., Chang, H. C., Liang, P. S., Huang, T. Y., Shih, C. J., Chu, L. A. and Sang, T. K. (2021). VCP maintains nuclear size by regulating the DNA damage-associated MDC1-p53-autophagy axis in Drosophila. Nat Commun 12(1): 4258. PubMed ID: 34253734
The maintenance of constant karyoplasmic ratios suggests that nuclear size has physiological significance. Nuclear size anomalies have been linked to malignant transformation, although the mechanism remains unclear. By expressing dominant-negative TER94 mutants in Drosophila photoreceptors, this study shows disruption of VCP (valosin-containing protein, human TER94 ortholog), a ubiquitin-dependent segregase, causes progressive nuclear size increase. Loss of VCP function leads to accumulations of MDC1 (mediator of DNA damage checkpoint protein 1; Drosophila homolog mutator 2), connecting DNA damage or associated responses to enlarged nuclei. TER94 can interact with MDC1 and decreases MDC1 levels, suggesting that MDC1 is a VCP substrate. This evidence indicates that MDC1 accumulation stabilizes p53A (Drosophila homolog: p53), leading to TER94(K2A)-associated nuclear size increase. Together with a previous report that p53A disrupts autophagic flux, it is proposed that the stabilization of p53A in TER94(K2A)-expressing cells likely hinders the removal of nuclear content, resulting in aberrant nuclear size increase.
Barros, C. S. and Bossing, T. (2021). Microtubule disruption upon CNS damage triggers mitotic entry via TNF signaling activation. Cell Rep 36(1): 109325. PubMed ID: 34233183
Repair after traumatic injury often starts with mitotic activation around the lesion edges. Early midline cells in the Drosophila embryonic CNS can enter into division following the traumatic disruption of microtubules. Microtubule disruption activates non-canonical TNF signaling by phosphorylation of TGF-β activated kinase 1 (Tak1) and its target IkappaB kinase (Ik2), culminating in Dorsal/NfkappaB nuclear translocation and Jra/Jun expression. Tak1 and Ik2 are necessary for the damaged-induced divisions. Microtubule disruption caused by Tau accumulation is also reported in Alzheimer's disease (AD). Human Tau expression in Drosophila midline cells is sufficient to induce Tak1 phosphorylation, Dorsal and Jra/Jun expression, and entry into mitosis. Interestingly, activation of Tak1 and Tank binding kinase 1 (Tbk1), the human Ik2 ortholog, and NfkappaB upregulation are observed in AD brains.
Hatori, R., Wood, B. M., Oliveira Barbosa, G. and Kornberg, T. B. (2021). Regulated delivery controls Drosophila Hedgehog, Wingless and Decapentaplegic signaling. Elife 10. PubMed ID: 34292155
Morphogen signaling proteins disperse across tissues to activate signal transduction in target cells. This study investigated dispersion of Hedgehog (Hh), Wnt homolog Wingless (Wg), and Bone morphogenic protein homolog Decapentaplegic (Dpp) in the Drosophila wing imaginal disc. Delivery of Hh, Wg, and Dpp to their respective targets was found to be regulated. <5% of Hh and <25% of Wg are taken up by disc cells and activate signaling. The amount of morphogen that is taken up and initiates signaling did not change when the level of morphogen expression was varied between 50-200% (Hh) or 50-350% (Wg). Similar properties were observed for Dpp. An area of 150 mm x 150 mm was analyzed that includes Hh-responding cells of the disc as well as overlying tracheal cells and myoblasts that are also activated by disc-produced Hh. The extent of signaling in the disc was unaffected by the presence or absence of the tracheal and myoblast cells, suggesting that the mechanism that disperses Hh specifies its destinations to particular cells, and that target cells do not take up Hh from a common pool.

Wednesday, July 28th - Neural development and function

Clements, J., Buhler, K., Winant, M., Vulsteke, V. and Callaerts, P. (2021). Glial and Neuronal Neuroglian, Semaphorin-1a and Plexin A Regulate Morphological and Functional Differentiation of Drosophila Insulin-Producing Cells. Front Endocrinol (Lausanne) 12: 600251. PubMed ID: 34276554
The insulin-producing cells (IPCs), a group of 14 neurons in the Drosophila brain, regulate numerous processes, including energy homeostasis, lifespan, stress response, fecundity, and various behaviors, such as foraging and sleep. Despite their importance, little is known about the development and the factors that regulate morphological and functional differentiation of IPCs. This study describes the use of a new transgenic reporter to characterize the role of the Drosophila L1-CAM homolog Neuroglian (Nrg), and the transmembrane Semaphorin-1a (Sema-1a) and its receptor Plexin A (PlexA) in the differentiation of the insulin-producing neurons. Loss of Nrg results in defasciculation and abnormal neurite branching, including ectopic neurites in the IPC neurons. Cell-type specific RNAi knockdown experiments reveal that Nrg, Sema-1a and PlexA are required in IPCs and glia to control normal morphological differentiation of IPCs albeit with a stronger contribution of Nrg and Sema-1a in glia and of PlexA in the IPCs. These observations provide new insights into the development of the IPC neurons and identify a novel role for Sema-1a in glia. In addition, this study shows that Nrg, Sema-1a and PlexA in glia and IPCs not only regulate morphological but also functional differentiation of the IPCs and that the functional deficits are likely independent of the morphological phenotypes. The requirements of nrg, Sema-1a, and PlexA in IPC development and the expression of their vertebrate counterparts in the hypothalamic-pituitary axis, suggest that these functions may be evolutionarily conserved in the establishment of vertebrate endocrine systems.
Contreras, E. G., Glavic, A., Brand, A. H. and Sierralta, J. (2021). The serine protease homologue, Scarface, is sensitive to nutrient availability and modulates the development of the Drosophila blood brain barrier. J Neurosci. PubMed ID: 34210781
The adaptable transcriptional response to changes in food availability not only ensures animal survival, but also lets progressing with embryonic development. Interestingly, the central nervous system is preferentially protected to periods of malnutrition, a phenomenon known as 'brain sparing'. However, the mechanisms that mediates this response remains poorly understood. To get a better understanding of this, Drosophila melanogaster was used as a model, analysing the transcriptional response of neural stem cells (neuroblasts) and glia of the blood-brain barrier (BBB), from larvae of both sexes, during nutrient restriction using targeted DamID. Differentially expressed genes were found in both neuroblasts and glia of the BBB, although the effect of nutrient deficiency was primarily observed in the BBB. The function of a nutritional sensitive gene expressed in the BBB, the serine protease homologue, scarface (scaf), was characterized. Scaf is expressed in subperineurial glia in the BBB in response to nutrition. Tissue-specific knockdown of scaf increases subperineurial glia endoreplication and proliferation of perineurial glia in the blood-brain barrier. Furthermore, neuroblast proliferation is diminished upon scaf knockdown in subperineurial glia. Interestingly, re-expression of Scaf in subperineurial glia is able to enhance neuroblast proliferation and brain growth of animals in starvation. Finally, this study shows that loss of scaf in the blood-brain barrier increases the sensitivity to drugs in adulthood suggesting a physiological impairment. It is proposed that Scaf integrates the nutrient status to modulate the balance between neurogenesis and growth of the BBB, preserving the proper equilibrium between the size of the barrier and the brain.
Cai, X. T., Li, H., Borch Jensen, M., Maksoud, E., Borneo, J., Liang, Y., Quake, S. R., Luo, L., Haghighi, P. and Jasper, H. (2021). Gut cytokines modulate olfaction through metabolic reprogramming of glia. Nature. PubMed ID: 34290404
Infection-induced aversion against enteropathogens is a conserved sickness behaviour that can promote host survival. The aetiology of this behaviour remains poorly understood, but studies in Drosophila have linked olfactory and gustatory perception to avoidance behaviours against toxic microorganisms. Whether and how enteric infections directly influence sensory perception to induce or modulate such behaviours remains unknown. This study shows that enteropathogen infection in Drosophila can modulate olfaction through metabolic reprogramming of ensheathing glia of the antennal lobe. Infection-induced unpaired cytokine expression in the intestine activates JAK-STAT signalling in ensheathing glia, inducing the expression of glial monocarboxylate transporters and the apolipoprotein glial lazarillo (GLaz), and affecting metabolic coupling of glia and neurons at the antennal lobe. This modulates olfactory discrimination, promotes the avoidance of bacteria-laced food and increases fly survival. Although transient in young flies, gut-induced metabolic reprogramming of ensheathing glia becomes constitutive in old flies owing to age-related intestinal inflammation, which contributes to an age-related decline in olfactory discrimination. These findings identify adaptive glial metabolic reprogramming by gut-derived cytokines as a mechanism that causes lasting changes in a sensory system in ageing flies.
Dhawan, S., Myers, P., Bailey, D. M. D., Ostrovsky, A. D., Evers, J. F. and Landgraf, M. (2021). Reactive Oxygen Species Mediate Activity-Regulated Dendritic Plasticity Through NADPH Oxidase and Aquaporin Regulation. Front Cell Neurosci 15: 641802. PubMed ID: 34290589
Neurons utilize plasticity of dendritic arbors as part of a larger suite of adaptive plasticity mechanisms. This explicitly manifests with motoneurons in the Drosophila embryo and larva, where dendritic arbors are exclusively postsynaptic and are used as homeostatic devices, compensating for changes in synaptic input through adapting their growth and connectivity. Reactive oxygen species (ROS) has been identified as novel plasticity signals instrumental in this form of dendritic adjustment. ROS correlate with levels of neuronal activity and negatively regulate dendritic arbor size. This study investigated NADPH oxidases as potential sources of such activity-regulated ROS and implicate Dual Oxidase (but not Nox), which generates hydrogen peroxide extracellularly. It was further shown that the aquaporins Bib and Drip, but not Prip, are required for activity-regulated ROS-mediated adjustments of dendritic arbor size in motoneurons. These results suggest a model whereby neuronal activity leads to activation of the NADPH oxidase Dual Oxidase, which generates hydrogen peroxide at the extracellular face; aquaporins might then act as conduits that are necessary for these extracellular ROS to be channeled back into the cell where they negatively regulate dendritic arbor size.
Garaulet, D. L., Moro, A. and Lai, E. C. (2021). A double-negative gene regulatory circuit underlies the virgin behavioral state. Cell Rep 36(1): 109335. PubMed ID: 34233178
Virgin females of many species conduct distinctive behaviors, compared with post-mated and/or pregnant individuals. In Drosophila, this post-mating switch is initiated by seminal factors, implying that the default female state is virgin. However, it was recently shown that loss of miR-iab-4/8-mediated repression of the transcription factor Homothorax (Hth) within the abdominal ventral nerve cord (VNC) causes virgins to execute mated behaviors. This study used genomic analysis of mir-iab-4/8 deletion and hth-microRNA (miRNA) binding site mutants (hth[BSmut]) to elucidate doublesex (dsx) as a critical downstream factor. Dsx and Hth proteins are highly complementary in CNS, and Dsx is downregulated in miRNA/hth[BSmut] mutants. Moreover, virgin behavior is highly dose sensitive to developmental dsx function. Strikingly, depletion of Dsx from very restricted abdominal neurons (SAG-1 cells) abrogates female virgin conducts, in favor of mated behaviors. Thus, a double-negative regulatory pathway in the VNC (miR-iab-4/8 ˧ Hth ˧ Dsx) specifies the virgin behavioral state.
Elliott, A. D., Berndt, A., Houpert, M., Roy, S., Scott, R. L., Chow, C. C., Shroff, H. and White, B. H. (2021). Pupal behavior emerges from unstructured muscle activity in response to neuromodulation in Drosophila. Elife 10. PubMed ID: 34236312
Identifying neural substrates of behavior requires defining actions in terms that map onto brain activity. Brain and muscle activity naturally correlate via the output of motor neurons, but apart from simple movements it has been difficult to define behavior in terms of muscle contractions. By mapping the musculature of the pupal fruit fly and comprehensively imaging muscle activation at single cell resolution, this study describes a multiphasic behavioral sequence in Drosophila. This characterization identifies a previously undescribed behavioral phase and permits extraction of major movements by a convolutional neural network. Movements were deconstructed into a syllabary of co-active muscles and specific syllables were identified that are sensitive to neuromodulatory manipulations. Muscle activity shows considerable variability, with sequential increases in stereotypy dependent upon neuromodulation. This work provides a platform for studying whole-animal behavior, quantifying its variability across multiple spatiotemporal scales, and analyzing its neuromodulatory regulation at cellular resolution.

Tuesday, July 27th - RNA

Vinter, D. J., Hoppe, C., Minchington, T. G., Sutcliffe, C. and Ashe, H. L. (2021). Dynamics of hunchback translation in real time and at single mRNA resolution in the Drosophila embryo. Development 148(18). PubMed ID: 33722899
The Hunchback (Hb) transcription factor is critical for anterior-posterior patterning of the Drosophila embryo. Despite the maternal hb mRNA acting as a paradigm for translational regulation, due to its repression in the posterior of the embryo, little is known about the translatability of zygotically transcribed hb mRNAs. This study adapted the SunTag system, developed for imaging translation at single mRNA resolution in tissue culture cells, to the Drosophila embryo to study the translation dynamics of zygotic hb mRNAs. Using single-molecule imaging in fixed and live embryos, evidence is provided for translational repression of zygotic SunTag-hb mRNAs. While the proportion of SunTag-hb mRNAs translated is initially uniform, translation declines from the anterior over time until it becomes restricted to a posterior band in the expression domain. How regulated hb mRNA translation may help establish the sharp Hb expression boundary, which is a model for precision and noise during developmental patterning, is discussed. Overall, the data show how use of the SunTag method on fixed and live embryos is a powerful combination for elucidating spatiotemporal regulation of mRNA translation in Drosophila.
Fabry, M. H., Falconio, F. A., Joud, F., Lythgoe, E. K., Czech, B. and Hannon, G. J. (2021). Maternally inherited piRNAs direct transient heterochromatin formation at active transposons during early Drosophila embryogenesis. Elife 10. PubMed ID: 34236313
The PIWI-interacting RNA (piRNA) pathway controls transposon expression in animal germ cells, thereby ensuring genome stability over generations. In Drosophila, piRNAs are intergenerationally inherited through the maternal lineage, and this has demonstrated importance in the specification of piRNA source loci and in silencing of I- and P-elements in the germ cells of daughters. Maternally inherited Piwi protein enters somatic nuclei in early embryos prior to zygotic genome activation and persists therein for roughly half of the time required to complete embryonic development. To investigate the role of the piRNA pathway in the embryonic soma, a conditionally unstable Piwi protein was created. This enabled maternally deposited Piwi to be cleared from newly laid embryos within 30 minutes and well ahead of the activation of zygotic transcription. Examination of RNA and protein profiles over time, and correlation with patterns of H3K9me3 deposition, suggests a role for maternally deposited Piwi in attenuating zygotic transposon expression in somatic cells of the developing embryo. In particular, robust deposition of piRNAs targeting roo, an element whose expression is mainly restricted to embryonic development, results in the deposition of transient heterochromatic marks at active roo insertions. It is hypothesized that roo, an extremely successful mobile element, may have adopted a lifestyle of expression in the embryonic soma to evade silencing in germ cells.
Munafo, M., Lawless, V. R., Passera, A., MacMillan, S., Bornelov, S., Haussmann, I. U., Soller, M., Hannon, G. J. and Czech, B. (2021). Channel Nuclear Pore Complex subunits are required for transposon silencing in Drosophila. Elife 10. PubMed ID: 33856346
he Nuclear Pore Complex (NPC) is the principal gateway between nucleus and cytoplasm that enables exchange of macromolecular cargo. Composed of multiple copies of ~30 different nucleoporins (Nups), the NPC acts as a selective portal, interacting with factors which individually license passage of specific cargo classes. This study shows that two Nups of the inner channel, Nup54 and Nup58, are essential for transposon silencing via the PIWI-interacting RNA (piRNA) pathway in the Drosophila ovary. In ovarian follicle cells, loss of Nup54 and Nup58 results in compromised piRNA biogenesis exclusively from the flamenco locus, whereas knockdowns of other NPC subunits have widespread consequences. This provides evidence that some nucleoporins can acquire specialised roles in tissue-specific contexts. These findings consolidate the idea that the NPC has functions beyond simply constituting a barrier to nuclear/cytoplasmic exchange, as genomic loci subjected to strong selective pressure can exploit NPC subunits to facilitate their expression.
Aviles-Pagan, E. E., Hara, M. and Orr-Weaver, T. L. (2021). The GNU subunit of PNG kinase, the developmental regulator of mRNA translation, binds BIC-C to localize to RNP granules. Elife 10. PubMed ID: 34250903
Control of mRNA translation is a key mechanism by which the differentiated oocyte transitions to a totipotent embryo. In Drosophila, the PNG kinase complex regulates maternal mRNA translation at the oocyte-to-embryo transition. Previous work showed the GNU activating subunit is crucial in regulating PNG and timing its activity to the window between egg activation and early embryogenesis. This study, finds associations between GNU and proteins of RNP granules and demonstrates that GNU localizes to cytoplasmic RNP granules in the mature oocyte, identifying GNU as a new component of a subset of RNP granules. Furthermore, this study defines roles for the domains of GNU. Interactions between GNU and the granule component BIC-C reveal potential conserved functions for translational regulation in metazoan development. It is proposed that by binding to BIC-C, upon egg activation GNU brings PNG to its initial targets, translational repressors in RNP granules.
Pang, T. L., Ding, Z., Liang, S. B., Li, L., Zhang, B., Zhang, Y., Fan, Y. J. and Xu, Y. Z. (2021). Comprehensive Identification and Alternative Splicing of Microexons in Drosophila. Front Genet 12: 642602. PubMed ID: 33859668
Interrupted exons in the pre-mRNA transcripts are ligated together through RNA splicing, which plays a critical role in the regulation of gene expression. Exons with a length < 30 nt are defined as microexons that are unique in identification. However, microexons, especially those shorter than 8 nt, have not been well studied in many organisms due to difficulties in mapping short segments from sequencing reads. This study analyzed mRNA-seq data from a variety of Drosophila samples with a newly developed bioinformatic tool, ce-TopHat. In addition to the Flybase annotated, 465 new microexons were identified. Differentially alternatively spliced (AS) microexons were investigated between the Drosophila tissues (head, body, and gonad) and genders. Most of the AS microexons were found in the head and two AS microexons were identified in the sex-determination pathway gene fruitless.
Wang, Y., Zhang, L., Ren, H., Ma, L., Guo, J., Mao, D., Lu, Z., Lu, L. and Yan, D. (2021). Role of Hakai in m(6)A modification pathway in Drosophila. Nat Commun 12(1): 2159. PubMed ID: 33846330
N6-methyladenosine (m(6)A), the most abundant internal modification in eukaryotic mRNA, is installed by a multi-component writer complex; however, the exact roles of each component remain poorly understood. This study shows that a potential E3 ubiquitin ligase Hakai colocalizes and interacts with other m(6)A writer components, and Hakai mutants exhibit typical m(6)A pathway defects in Drosophila, such as lowered m(6)A levels in mRNA, aberrant Sxl alternative splicing, wing and behavior defects. Hakai, Vir, Fl(2)d and Flacc form a stable complex, and disruption of either Hakai, Vir or Fl(2)d led to the degradation of the other three components. Furthermore, MeRIP-seq indicates that the effective m(6)A modification is mostly distributed in 5' UTRs in Drosophila, in contrast to the mammalian system. Interestingly, it was demonstrated that m(6)A modification is deposited onto the Sxl mRNA in a sex-specific fashion, which depends on the m(6)A writer. Together, this work not only advances the understanding of mechanism and regulation of the m(6)A writer complex, but also provides insights into how Sxl cooperate with the m(6)A pathway to control its own splicing.

Monday, June 26th - Enhancers and gene regulation

Syed, S., Wilky, H., Raimundo, J., Lim, B. and Amodeo, A. A. (2021). The nuclear to cytoplasmic ratio directly regulates zygotic transcription in Drosophila through multiple modalities. Proc Natl Acad Sci U S A 118(14). PubMed ID: 33790005
Early embryos must rapidly generate large numbers of cells to form an organism. Many species accomplish this through a series of rapid, reductive, and transcriptionally silent cleavage divisions. Previous work has demonstrated that the number of divisions before both cell cycle elongation and zygotic genome activation (ZGA) is regulated by the ratio of nuclear content to cytoplasm (N/C). To understand how the N/C ratio affects the timing of ZGA, the behavior of several previously identified N/C ratio-dependent genes was directly assayed using the MS2-MCP reporter system in living Drosophila embryos with altered ploidy and cell cycle durations. For every gene examined, nascent RNA output per cycle was found to be delayed in haploid embryos. Moreover, the N/C ratio influences transcription through three overlapping modes of action. For some genes (knirps, fushi tarazu, and snail), the effect of ploidy can be primarily attributed to changes in cell cycle duration. However, additional N/C ratio-mediated mechanisms contribute significantly to transcription delays for other genes. For giant and bottleneck, the kinetics of transcription activation are significantly disrupted in haploids, while for frühstart and Krüppel, the N/C ratio controls the probability of transcription initiation. These data demonstrate that the regulatory elements of N/C ratio-dependent genes respond directly to the N/C ratio through multiple modes of regulation.
Aldea, D., Atsuta, Y., Kokalari, B., Schaffner, S. F., Prasasya, R. D., Aharoni, A., Dingwall, H. L., Warder, B. and Kamberov, Y. G. (2021). Repeated mutation of a developmental enhancer contributed to human thermoregulatory evolution. Proc Natl Acad Sci U S A 118(16). PubMed ID: 33850016
Humans sweat to cool their bodies and have by far the highest eccrine sweat gland density among primates. Humans' high eccrine gland density has long been recognized as a hallmark human evolutionary adaptation, but its genetic basis has been unknown. In humans, expression of the Engrailed 1 (EN1; see Drosophila Engrailed) transcription factor correlates with the onset of eccrine gland formation. In mice, regulation of ectodermal En1 expression is a major determinant of natural variation in eccrine gland density between strains, and increased En1 expression promotes the specification of more eccrine glands. This study shows that regulation of EN1 has evolved specifically on the human lineage to promote eccrine gland formation. Using comparative genomics and validation of ectodermal enhancer activity in mice, a human EN1 skin enhancer, hECE18 was identified. Multiple epistatically interacting derived substitutions in the human ECE18 enhancer were shown to increase its activity compared with nonhuman ape orthologs in cultured keratinocytes. Repression of hECE18 in human cultured keratinocytes specifically attenuated EN1 expression, indicating this element positively regulates EN1 in this context. In a humanized enhancer knock-in mouse, hECE18 increased developmental En1 expression in the skin to induce the formation of more eccrine glands. This study uncovers a genetic basis contributing to the evolution of one of the most singular human adaptations and implicates multiple interacting mutations in a single enhancer as a mechanism for human evolutionary change.
Bozhilov, Y. K., Downes, D. J., Telenius, J., Marieke Oudelaar, A., Olivier, E. N., Mountford, J. C., Hughes, J. R., Gibbons, R. J. and Higgs, D. R. (2021). A gain-of-function single nucleotide variant creates a new promoter which acts as an orientation-dependent enhancer-blocker. Nat Commun 12(1): 3806. PubMed ID: 34155213
Many single nucleotide variants (SNVs) associated with human traits and genetic diseases are thought to alter the activity of existing regulatory elements. Some SNVs may also create entirely new regulatory elements which change gene expression, but the mechanism by which they do so is largely unknown. This study shows that a single base change in an otherwise unremarkable region of the human α-globin cluster creates an entirely new promoter and an associated unidirectional transcript. This SNV downregulates α-globin expression causing α-thalassaemia. Of note, the new promoter lying between the α-globin genes and their associated super-enhancer disrupts their interaction in an orientation-dependent manner. Together these observations show how both the order and orientation of the fundamental elements of the genome determine patterns of gene expression and support the concept that active genes may act to disrupt enhancer-promoter interactions in mammals as in Drosophila. Finally, these findings should prompt others to fully evaluate SNVs lying outside of known regulatory elements as causing changes in gene expression by creating new regulatory elements.
Rajpurkar, A. R., Mateo, L. J., Murphy, S. E. and Boettiger, A. N. (2021). Deep learning connects DNA traces to transcription to reveal predictive features beyond enhancer-promoter contact. Nat Commun 12(1): 3423. PubMed ID: 34103507
Chromatin architecture plays an important role in gene regulation. Recent advances in super-resolution microscopy have made it possible to measure chromatin 3D structure and transcription in thousands of single cells. However, leveraging these complex data sets with a computationally unbiased method has been challenging. This study presents a deep learning-based approach to better understand to what degree chromatin structure relates to transcriptional state of individual cells. Furthermore, methods were explored to "unpack the black box" to determine in an unbiased manner which structural features of chromatin regulation are most important for gene expression state. This approach was applied to an Optical Reconstruction of Chromatin Architecture dataset of the Bithorax gene cluster in Drosophila; it was shown to outperforms previous contact-focused methods in predicting expression state from 3D structure. The structural information is distributed across the domain, overlapping and extending beyond domains identified by prior genetic analyses. Individual enhancer-promoter interactions are a minor contributor to predictions of activity.
Schember, I. and Halfon, M. S. (2021). Identification of new Anopheles gambiae transcriptional enhancers using a cross-species prediction approach. Insect Mol Biol. PubMed ID: 33866636
The success of transgenic mosquito vector control approaches relies on well-targeted gene expression, requiring the identification and characterization of a diverse set of mosquito promoters and transcriptional enhancers. However, few enhancers have been characterized in Anopheles gambiae to date. This study employed the SCRMshaw method to predict enhancers in the A. gambiae genome, preferentially targeting vector-relevant tissues such as the salivary glands, midgut and nervous system. A high overall success rate is demonstrated, with at least 8 of 11 (73%) tested sequences validating as enhancers in an in vivo xenotransgenic assay. Four tested sequences drive expression in either the salivary gland or the midgut, making them directly useful for probing the biology of these infection-relevant tissues. The success of this study suggests that computational enhancer prediction should serve as an effective means for identifying A. gambiae enhancers with activity in tissues involved in malaria propagation and transmission.
Ing-Simmons, E., Vaid, R., Bing, X. Y., Levine, M., Mannervik, M. and Vaquerizas, J. M. (2021). Independence of chromatin conformation and gene regulation during Drosophila dorsoventral patterning. Nat Genet 53(4): 487-499. PubMed ID: 33795866
The relationship between chromatin organization and gene regulation remains unclear. While disruption of chromatin domains and domain boundaries can lead to misexpression of developmental genes, acute depletion of regulators of genome organization has a relatively small effect on gene expression. It is therefore uncertain whether gene expression and chromatin state drive chromatin organization or whether changes in chromatin organization facilitate cell-type-specific activation of gene expression. This study used the dorsoventral patterning of the Drosophila melanogaster embryo as a model system; evidence is provided for the independence of chromatin organization and dorsoventral gene expression. Tissue-specific enhancers were defined and linked to expression patterns using single-cell RNA-seq. Surprisingly, despite tissue-specific chromatin states and gene expression, chromatin organization is largely maintained across tissues. The results indicate that tissue-specific chromatin conformation is not necessary for tissue-specific gene expression but rather acts as a scaffold facilitating gene expression when enhancers become active.

Friday, July 23rd - Apoptosis and Autophagy

Manzeger, A., Tagscherer, K., Lorincz, P., Szaker, H., Lukacsovich, T., Pilz, P., Kmeczik, R., Csikos, G., Erdelyi, M., Sass, M., Kovacs, T., Vellai, T. and Billes, V. A. (2021). Condition-dependent functional shift of two Drosophila Mtmr lipid phosphatases in autophagy control. Autophagy: 1-19. PubMed ID: 33779490
Myotubularin (MTM) and myotubularin-related (MTMR) lipid phosphatases catalyze the removal of a phosphate group from certain phosphatidylinositol derivatives. Because some of these substrates are required for macroautophagy/autophagy, during which unwanted cytoplasmic constituents are delivered into lysosomes for degradation, MTM and MTMRs function as important regulators of the autophagic process. Despite its physiological and medical significance, the specific role of individual MTMR paralogs in autophagy control remains largely unexplored. This study examined two Drosophila MTMRs, EDTP and Mtmr6, the fly orthologs of mammalian MTMR14 and MTMR6 to MTMR8, respectively; these enzymes were found to affect the autophagic process in a complex, condition-dependent way. EDTP inhibited basal autophagy, but did not influence stress-induced autophagy. In contrast, Mtmr6 promoted the process under nutrient-rich settings, but effectively blocked its hyperactivation in response to stress. Thus, Mtmr6 is the first identified MTMR phosphatase with dual, antagonistic roles in the regulation of autophagy, and shows conditional antagonism/synergism with EDTP in modulating autophagic breakdown. These results provide a deeper insight into the adjustment of autophagy.
Zhao, H., Shi, L., Kong, R., Li, Z., Liu, F., Zhao, H. and Li, Z. (2021). Autophagy induction in tumor surrounding cells promotes tumor growth in adult Drosophila intestines. Dev Biol 476: 294-307. PubMed ID: 33940033
During tumorigenesis, tumor cells interact intimately with their surrounding cells (microenvironment) for their growth and progression. However, the roles of tumor microenvironment in tumor development and progression are not fully understood. Using an established benign tumor model in adult Drosophila intestines, this study found that non-cell autonomous autophagy (NAA) is induced in tumor surrounding neighbor cells. Tumor growth can be significantly suppressed by genetic ablation of autophagy induction in tumor neighboring cells, indicating that tumor neighboring cells act as tumor microenvironment to promote tumor growth. Autophagy in tumor neighboring cells is induced downstream of elevated ROS and activated JNK signaling in tumor cells. Interestingly, it was found that active transport of nutrients, such as amino acids, from tumor neighboring cells sustains tumor growth, and increasing nutrient availability could significantly restore tumor growth. Together, these data demonstrate that tumor cells take advantage of their surrounding normal neighbor cells as nutrient sources through NAA to meet their high metabolic demand for growth and progression. Thus this study provides insights into understanding of the mechanisms underlying the interaction between tumor cells and their microenvironment in tumor development.
Wang, W., Li, J., Tan, J., Wang, M., Yang, J., Zhang, Z. M., Li, C., Basnakian, A. G., Tang, H. W., Perrimon, N. and Zhou, Q. (2021). Endonuclease G promotes autophagy by suppressing mTOR signaling and activating the DNA damage response. Nat Commun 12(1): 476. PubMed ID: 33473107
Endonuclease G (ENDOG), a mitochondrial nuclease, is known to participate in many cellular processes, including apoptosis and paternal mitochondrial elimination, while its role in autophagy remains unclear. This study report that ENDOG released from mitochondria promotes autophagy during starvation, which this study finds to be evolutionally conserved across species by performing experiments in human cell lines, mice, Drosophila and C. elegans. Under starvation, Glycogen synthase kinase 3 beta-mediated phosphorylation of ENDOG at Thr-128 and Ser-288 enhances its interaction with 14-3-3γ, which leads to the release of Tuberin (TSC2) and Phosphatidylinositol 3-kinase catalytic subunit type 3 (Vps34) from 14-3-3γ, followed by mTOR pathway suppression and autophagy initiation. Alternatively, ENDOG activates DNA damage response and triggers autophagy through its endonuclease activity. These results demonstrate that ENDOG is a crucial regulator of autophagy, manifested by phosphorylation-mediated interaction with 14-3-3γ, and its endonuclease activity-mediated DNA damage response.
Nagai, H. and Yano, T. (2021). Selective autophagy tolerates symbiotic bacteria in the Drosophila intestine. Autophagy 17(4): 1057-1058. PubMed ID: 33734015
Intestinal epithelium functions as a barrier to protect the host from environmental microbes. Defects in macroautophagy/autophagy combined with intestinal microbes cause a disruption of homeostasis of the tissue, which is associated with the etiology of Crohn disease, an inflammatory bowel disease. However, the molecular mechanism of how autophagy interacts with microbes in the pathology are mostly unrevealed. Recent findings using Drosophila as a model system showed that autophagy in enterocytes suppresses a regenerative response triggered by reactive oxygen species (ROS) secreted by the host epithelia toward commensal bacteria in the intestine. Without this suppression, accumulation of a receptor protein of selective autophagy, ref(2)P, continuously acts as a signaling platform to cause excessive regeneration via cytokine production by yki (yorkie) activation. This chronic response leads to the acceleration of age-dependent barrier dysfunction, systemic inflammation, and shorter lifespan. These results uncover a novel regulatory network linking commensal bacteria, autophagy, and gut homeostasis, represented by ROS, ref(2)P, and the hippo pathway.
Joy, J., Barrio, L., Santos-Tapia, C., Romao, D., Giakoumakis, N. N., Clemente-Ruiz, M. and Milan, M. (2021). Proteostasis failure and mitochondrial dysfunction leads to aneuploidy-induced senescence. Dev Cell. PubMed ID: 34216545
Aneuploidy, an unbalanced number of chromosomes, is highly deleterious at the cellular level and leads to senescence, a stress-induced response characterized by permanent cell-cycle arrest and a well-defined associated secretory phenotype. This study used a Drosophila epithelial model to delineate the pathway that leads to the induction of senescence as a consequence of the acquisition of an aneuploid karyotype. Whereas aneuploidy induces, as a result of gene dosage imbalance, proteotoxic stress and activation of the major protein quality control mechanisms, near-saturation functioning of autophagy leads to compromised mitophagy, accumulation of dysfunctional mitochondria, and the production of radical oxygen species (ROS). This study uncovered a role of c-Jun N-terminal kinase (JNK) in driving senescence as a consequence of dysfunctional mitochondria and ROS. Activation of the major protein quality control mechanisms and mitophagy dampens the deleterious effects of aneuploidy, and this study has identified a role of senescence in proteostasis and compensatory proliferation for tissue repair.
Tarayrah-Ibraheim, L., Maurice, E. C., Hadary, G., Ben-Hur, S., Kolpakova, A., Braun, T., Peleg, Y., Yacobi-Sharon, K. and Arama, E. (2021). DNase II mediates a parthanatos-like developmental cell death pathway in Drosophila primordial germ cells. Nat Commun 12(1): 2285. PubMed ID: 33863891
During Drosophila embryonic development, cell death eliminates 30% of the primordial germ cells (PGCs). Inhibiting apoptosis does not prevent PGC death, suggesting a divergence from the conventional apoptotic program. This study demonstrates that PGCs normally activate an intrinsic alternative cell death (ACD) pathway mediated by DNase II release from lysosomes, leading to nuclear translocation and subsequent DNA double-strand breaks (DSBs). DSBs activate the DNA damage-sensing enzyme, Poly(ADP-ribose) (PAR) polymerase-1 (PARP-1) and the ATR/Chk1 branch of the DNA damage response. PARP-1 and DNase II engage in a positive feedback amplification loop mediated by the release of PAR polymers from the nucleus and the nuclear accumulation of DNase II in an AIF- and CypA-dependent manner, ultimately resulting in PGC death. Given the anatomical and molecular similarities with an ACD pathway called parthanatos, these findings reveal a parthanatos-like cell death pathway active during Drosophila development.

Thursday, July 22 - Adult neural development and function

Zatsepina, O. G., Nikitina, E. A., Shilova, V. Y., Chuvakova, L. N., Sorokina, S., Vorontsova, J. E., Tokmacheva, E. V., Funikov, S. Y., Rezvykh, A. P. and Evgen'ev, M. B. (2021). Hsp70 affects memory formation and behaviorally relevant gene expression in Drosophila melanogaster. Cell Stress Chaperones 26(3): 575-594. PubMed ID: 33829398
Heat shock proteins, in particular Hsp70, play a central role in proteostasis in eukaryotic cells. Due to its chaperone properties, Hsp70 is involved in various processes after stress and under normal physiological conditions. In contrast to mammals and many Diptera species, inducible members of the Hsp70 family in Drosophila are constitutively synthesized at a low level and undergo dramatic induction after temperature elevation or other forms of stress. In the courtship suppression paradigm used in this study, Drosophila males that have been repeatedly rejected by mated females during courtship are less likely than naive males to court other females. Although numerous genes with known function were identified to play important roles in long-term memory, there is no direct evidence implicating Hsp70 in this process. To elucidate a possible role of Hsp70 in memory formation, D. melanogaster strains containing different hsp70 copy numbers were used, including strains carrying a deletion of all six hsp70 genes. This investigations exploring the memory of courtship rejection paradigm demonstrated that a low constitutive level of Hsp70 is apparently required for learning and the formation of short and long-term memories in males. The performed transcriptomic studies demonstrate that males with different hsp70 copy numbers differ significantly in the expression of a few definite groups of genes involved in mating, reproduction, and immunity in response to rejection. Specifically, thus analysis reveals several major pathways that depend on the presence of hsp70 in the genome and participate in memory formation and consolidation, including the cAMP signaling cascade.
Turner, M. H., Mann, K. and Clandinin, T. R. (2021). The connectome predicts resting-state functional connectivity across the Drosophila brain. Curr Biol. PubMed ID: 33770490
Anatomical connectivity can constrain both a neural circuit's function and its underlying computation. This principle has been demonstrated for many small, defined neural circuits. For example, connectome reconstructions have informed models for direction selectivity in the vertebrate retina as well as the Drosophila visual system. In these cases, the circuit in question is relatively compact, well-defined, and has known functions. However, how the connectome constrains global properties of large-scale networks, across multiple brain regions or the entire brain, is incompletely understood. As the availability of partial or complete connectomes expands to more systems and species it becomes critical to understand how this detailed anatomical information can inform understanding of large-scale circuit function. This study used data from the Drosophila connectome in conjunction with whole-brain in vivo imaging to relate structural and functional connectivity in the central brain. A strong relationship was found between resting-state functional correlations and direct region-to-region structural connectivity. The relationship between structure and function varies across the brain, with some regions displaying a tight correspondence between structural and functional connectivity whereas others, including the mushroom body, are more strongly dependent on indirect connections. Throughout this work, features were observed of structural and functional networks in Drosophila that are strikingly similar to those seen in mammalian cortex, including in the human brain. Given the vast anatomical and functional differences between Drosophila and mammalian nervous systems, these observations suggest general principles that govern brain structure, function, and the relationship between the two.
Yin, J., Spillman, E., Cheng, E. S., Short, J., Chen, Y., Lei, J., Gibbs, M., Rosenthal, J. S., Sheng, C., Chen, Y. X., Veerasammy, K., Choetso, T., Abzalimov, R., Wang, B., Han, C., He, Y. and Yuan, Q. (2021). Brain-specific lipoprotein receptors interact with astrocyte derived apolipoprotein and mediate neuron-glia lipid shuttling. Nat Commun 12(1): 2408. PubMed ID: 33893307
Lipid shuttling between neurons and glia contributes to the development, function, and stress responses of the nervous system. To understand how a neuron acquires its lipid supply from specific lipoproteins and their receptors, A combined genetic, transcriptome, and biochemical analyses was performed in the developing Drosophila larval brain. This stud reports, the astrocyte-derived secreted lipocalin Glial Lazarillo (GLaz), a homolog of human Apolipoprotein D (APOD), and its neuronal receptor, the brain-specific short isoforms of Drosophila lipophorin receptor 1 (LpR1-short), cooperatively mediate neuron-glia lipid shuttling and support dendrite morphogenesis. The isoform specificity of LpR1 defines its distribution, binding partners, and ability to support proper dendrite growth and synaptic connectivity. By demonstrating physical and functional interactions between GLaz/APOD and LpR1, this study elucidated molecular pathways mediating lipid trafficking in the fly brain, and provide in vivo evidence indicating isoform-specific expression of lipoprotein receptors as a key mechanism for regulating cell-type specific lipid recruitment.
Zhao, C., Widmer, Y. F., Diegelmann, S., Petrovici, M. A., Sprecher, S. G. and Senn, W. (2021). Predictive olfactory learning in Drosophila. Sci Rep 11(1): 6795. PubMed ID: 33762640
Olfactory learning and conditioning in the fruit fly is typically modelled by correlation-based associative synaptic plasticity. It was shown that the conditioning of an odor-evoked response by a shock depends on the connections from Kenyon cells (KC) to mushroom body output neurons (MBONs). Although on the behavioral level conditioning is recognized to be predictive, it remains unclear how MBONs form predictions of aversive or appetitive values (valences) of odors on the circuit level. This study presents behavioral experiments that are not well explained by associative plasticity between conditioned and unconditioned stimuli, and two alternative models are suggested for how predictions can be formed. In error-driven predictive plasticity, dopaminergic neurons (DANs) represent the error between the predictive odor value and the shock strength. In target-driven predictive plasticity, the DANs represent the target for the predictive MBON activity. Predictive plasticity in KC-to-MBON synapses can also explain trace-conditioning, the valence-dependent sign switch in plasticity, and the observed novelty-familiarity representation. The model offers a framework to dissect MBON circuits and interpret DAN activity during olfactory learning.

Wednesday, July 22nd - Chromatin

Sabirov, M., Kyrchanova, O., Pokholkova, G. V., Bonchuk, A., Klimenko, N., Belova, E., Zhimulev, I. F., Maksimenko, O. and Georgiev, P. (2021). Mechanism and functional role of the interaction between CP190 and the architectural protein Pita in Drosophila melanogaster. Epigenetics Chromatin 14(1): 16. PubMed ID: 33752739
Pita is required for Drosophila development and binds specifically to a long motif in active promoters and insulators. Pita belongs to the Drosophila family of zinc-finger architectural proteins, which also includes Su(Hw) and the conserved among higher eukaryotes CTCF. The architectural proteins maintain the active state of regulatory elements and the long-distance interactions between them. In particular, Pita is involved in the formation of several boundaries between regulatory domains that controlled the expression of three hox genes in the Bithorax complex (BX-C). The CP190 protein is recruited to chromatin through interaction with the architectural proteins. Using in vitro pull-down analysis, two unstructured regions of Pita that interact with the BTB domain of CP190 were precisely mapped. Transgenic lines were constructed expressing the Pita protein of the wild-type and mutant variants lacking CP190-interacting regions. It was demonstrated that CP190-interacting region of the Pita can maintain nucleosome-free open chromatin and is critical for Pita-mediated enhancer blocking activity in BX-C. At the same time, interaction with CP190 is not required for the in vivo function of the mutant Pita protein, which binds to the same regions of the genome as the wild-type protein. Unexpectedly, it was found that CP190 was still associated with the most of genome regions bound by the mutant Pita protein, which suggested that other architectural proteins were continuing to recruit CP190 to these regions. The results directly demonstrate role of CP190 in insulation and support a model in which the regulatory elements are composed of combinations of binding sites that interact with several architectural proteins with similar functions.
Palmateer, C. M., Moseley, S. C., Ray, S., Brovero, S. G. and Arbeitman, M. N. (2021). Analysis of cell-type-specific chromatin modifications and gene expression in Drosophila neurons that direct reproductive behavior. PLoS Genet 17(4): e1009240. PubMed ID: 33901168
Examining the role of chromatin modifications and gene expression in neurons is critical for understanding how the potential for behaviors are established and maintained. This study investigate this question by examining Drosophila melanogaster fru P1 neurons that underlie reproductive behaviors in both sexes. A method was developed to purify cell-type-specific chromatin (Chromatag), using a tagged histone H2B variant that is expressed using the versatile Gal4/UAS gene expression system. This study used Chromatag to evaluate five chromatin modifications, at three life stages in both sexes. Substantial changes were found in chromatin modification profiles across development and fewer differences between males and females. Additionally, chromatin modifications were found that persist in different sets of genes from pupal to adult stages, which may point to genes important for cell fate determination in fru P1 neurons. Cell-type-specific RNA-seq data sets were generated, using translating ribosome affinity purification (TRAP). Actively translated genes were identified in fru P1 neurons, revealing novel stage- and sex-differences in gene expression. Chromatin modification enrichment patterns were also found that are associated with gene expression. Next, the chromatin modification data was used to identify cell-type-specific super-enhancer-containing genes. Genes with super-enhancers in fru P1 neurons differ across development and between the sexes. This study validated that a set of genes are expressed in fru P1 neurons, which were chosen based on having a super-enhancer and TRAP-enriched expression in fru P1 neurons.
Tauc, H. M., Rodriguez-Fernandez, I. A., Hackney, J. A., Pawlak, M., Ronnen Oron, T., Korzelius, J., Moussa, H. F., Chaudhuri, S., Modrusan, Z., Edgar, B. A. and Jasper, H. (2021). Age-related changes in polycomb gene regulation disrupt lineage fidelity in intestinal stem cells. Elife 10. PubMed ID: 33724181
Tissue homeostasis requires long-term lineage fidelity of somatic stem cells. Whether and how age-related changes in somatic stem cells impact the faithful execution of lineage decisions remains largely unknown. This study addressed this question using genome-wide chromatin accessibility and transcriptome analysis as well as single-cell RNA-seq to explore stem-cell-intrinsic changes in the aging Drosophila intestine. These studies indicate that in stem cells of old flies, promoters of Polycomb (Pc) target genes become differentially accessible, resulting in the increased expression of enteroendocrine (EE) cell specification genes. Consistently, age-related changes were found in the composition of the EE progenitor cell population in aging intestines, as well as a significant increase in the proportion of EE-specified intestinal stem cells (ISCs) and progenitors in aging flies. This study further confirmed that Pc-mediated chromatin regulation is a critical determinant of EE cell specification in the Drosophila intestine. Pc is required to maintain expression of stem cell genes while ensuring repression of differentiation and specification genes. These results identify Pc group proteins as central regulators of lineage identity in the intestinal epithelium and highlight the impact of age-related decline in chromatin regulation on tissue homeostasis.
Shindo, Y. and Amodeo, A. A. (2021). Excess histone H3 is a competitive Chk1 inhibitor that controls cell-cycle remodeling in the early Drosophila embryo. Curr Biol. PubMed ID: 33848457
The DNA damage checkpoint is crucial to protect genome integrity. However, the early embryos of many metazoans sacrifice this safeguard to allow for rapid cleavage divisions that are required for speedy development. At the mid-blastula transition (MBT), embryos switch from rapid cleavage divisions to slower, patterned divisions with the addition of gap phases and acquisition of DNA damage checkpoints. The timing of the MBT is dependent on the nuclear-to-cytoplasmic (N/C ratio) and the activation of the checkpoint kinase, Chk1. How Chk1 activity is coupled to the N/C ratio has remained poorly understood. This study shows that dynamic changes in histone H3 availability in response to the increasing N/C ratio control, Chk1 activity, and thus time the MBT in the Drosophila embryo. Excess H3 in the early cycles was shown to interfere with cell-cycle slowing independent of chromatin incorporation. The N-terminal tail of H3 acts as a competitive inhibitor of Chk1 in vitro and reduces Chk1 activity in vivo. Using a H3-tail mutant that has reduced Chk1 inhibitor activity, this study showed that the amount of available Chk1 sites in the H3 pool controls the dynamics of cell-cycle progression. Mathematical modeling quantitatively supports a mechanism where titration of H3 during early cleavage cycles regulates Chk1-dependent cell-cycle slowing. This study defines Chk1 regulation by H3 as a key mechanism that coordinates cell-cycle remodeling with developmental progression.
Shidlovskii, Y. V., Bylino, O. V., Shaposhnikov, A. V., Kachaev, Z. M., Lebedeva, L. A., Kolesnik, V. V., Amendola, D., De Simone, G., Formicola, N., Schedl, P., Digilio, F. A. and Giordano, E. (2021). Subunits of the PBAP Chromatin Remodeler Are Capable of Mediating Enhancer-Driven Transcription in Drosophila. Int J Mol Sci 22(6). PubMed ID: 33799739
The chromatin remodeler SWI/SNF is an important participant in gene activation, functioning predominantly by opening the chromatin structure on promoters and enhancers. This study describes its novel mode of action in which SWI/SNF factors mediate the targeted action of an enhancer. The functions of two signature subunits of PBAP subfamily, BAP170 and SAYP, were studied in Drosophila. These subunits were stably tethered to a transgene reporter carrying the hsp70 core promoter. The tethered subunits mediate transcription of the reporter in a pattern that is generated by enhancers close to the insertion site in multiple loci throughout the genome. Both tethered SAYP and BAP170 recruit the whole PBAP complex to the reporter promoter. However, it was found that BAP170-dependent transcription is more resistant to the depletion of other PBAP subunits, suggesting that BAP170 may play a more critical role in establishing enhancer-dependent transcription.
Yu, R., Cao, X., Sun, L., Zhu, J. Y., Wasko, B. M., Liu, W., Crutcher, E., Liu, H., Jo, M. C., Qin, L., Kaeberlein, M., Han, Z. and Dang, W. (2021). Inactivating histone deacetylase HDA promotes longevity by mobilizing trehalose metabolism. Nat Commun 12(1): 1981. PubMed ID: 33790287
Histone acetylations are important epigenetic markers for transcriptional activation in response to metabolic changes and various stresses. Using the high-throughput SEquencing-Based Yeast replicative Lifespan screen method and the yeast knockout collection, this study demonstrate that the HDA complex, a class-II histone deacetylase (HDAC), regulates aging through its target of acetylated H3K18 at storage carbohydrate genes. In addition to longer lifespan, disruption of HDA results in resistance to DNA damage and osmotic stresses. This study shows that these effects are due to increased promoter H3K18 acetylation and transcriptional activation in the trehalose metabolic pathway in the absence of HDA. Furthermore, it was determined that the longevity effect of HDA is independent of the Cyc8-Tup1 repressor complex known to interact with HDA and coordinate transcriptional repression. Silencing the HDA homologs in C. elegans and Drosophila increases their lifespan and delays aging-associated physical declines in adult flies. Hence, this study has demonstrated that this HDAC controls an evolutionarily conserved longevity pathway.

Tuesday, July 20th - Gonads

Yu, J., Zheng, Q., Li, Z., Wu, Y., Fu, Y., Wu, X., Lin, D., Shen, C., Zheng, B. and Sun, F. (2021). CG6015 controls spermatogonia transit-amplifying divisions by epidermal growth factor receptor signaling in Drosophila testes. Cell Death Dis 12(5): 491. PubMed ID: 33990549
Spermatogonia transit-amplifying (TA) divisions are crucial for the differentiation of germline stem cell daughters. However, the underlying mechanism is largely unknown. The present study demonstrated that CG6015 was essential for spermatogonia TA-divisions and elongated spermatozoon development in Drosophila melanogaster. Spermatogonia deficient in CG6015 inhibited germline differentiation leading to the accumulation of undifferentiated cell populations. Transcriptome profiling using RNA sequencing indicated that CG6015 was involved in spermatogenesis, spermatid differentiation, and metabolic processes. Gene Set Enrichment Analysis (GSEA) revealed the relationship between CG6015 and the epidermal growth factor receptor (EGFR) signaling pathway. Unexpectedly, it was discovered that phosphorylated extracellular regulated kinase (dpERK) signals were activated in germline stem cell (GSC)-like cells after reduction of CG6015 in spermatogonia. Moreover, Downstream of raf1 (Dsor1), a key downstream target of EGFR, mimicked the phenotype of CG6015, and germline dpERK signals were activated in spermatogonia of Dsor1 RNAi testes. Together, these findings revealed a potential regulatory mechanism of CG6015 via EGFR signaling during spermatogonia TA-divisions in Drosophila testes.
Wang, X., LaFever, K. S., Waghmare, I. and Page-McCaw, A. (2021). Extracellular spreading of Wingless is required for Drosophila oogenesis. PLoS Genet 17(4): e1009469. PubMed ID: 33798197
Recent studies have investigated whether the Wnt family of extracellular ligands can signal at long range, spreading from their source and acting as morphogens, or whether they signal only in a juxtacrine manner to neighboring cells. The original evidence for long-range Wnt signaling arose from studies of Wg, a Drosophila Wnt protein, which patterns the wing disc over several cell diameters from a central source of Wg ligand. However, the requirement of long-range Wg for patterning was called into question when it was reported that replacing the secreted protein Wg with a membrane-tethered version, NRT-Wg, results in flies with normally patterned wings. It has been reported that Wg spreads in the ovary about 50 μm or 5 cell diameters, from the cap cells to the follicle stem cells (FSCs) and that Wg stimulates FSC proliferation. The NRT-wg flies were used to analyze the consequence of tethering Wg to the cap cells. NRT-wg homozygous flies are sickly, but it was found that hemizygous NRT-wg/null flies, carrying only one copy of tethered Wingless, were significantly healthier. Despite their overall improved health, these hemizygous flies displayed dramatic reductions in fertility and in FSC proliferation. Further, FSC proliferation was nearly undetectable when the wg locus was converted to NRT-wg only in adults, and the resulting germarium phenotype was consistent with a previously reported wg loss-of-function phenotype. It is concluded that Wg protein spreads from its source cells in the germarium to promote FSC proliferation.
Yang, S. Y. (2021). Germline masculinization by Phf7 in D. melanogaster requires its evolutionarily novel C-terminus and the HP1-family protein HP1D3csd. Sci Rep 11(1): 6308. PubMed ID: 33737548
Germ cells in Drosophila melanogaster need intrinsic factors along with somatic signals to activate proper sexual programs. A key factor for male germline sex determination is PHD finger protein 7 (Phf7), a histone reader expressed in the male germline that can trigger sex reversal in female germ cells and is also important for efficient spermatogenesis. This study found that the evolutionarily novel C-terminus in Phf7 is necessary to turn on the complete male program in the early germline of D. melanogaster, suggesting that this domain may have been uniquely acquired to regulate sexual differentiation. Genes were sought that regulated by Phf7 related to sex determination in the embryonic germline by transcriptome profiling of FACS-purified embryonic gonads. One of the genes positively-regulated by Phf7 in the embryonic germline was an HP1family member, Heterochromatin Protein 1D3 chromoshadow domain (HP1D3csd). This gene is needed for Phf7 to induce male-like development in the female germline, indicating that HP1D3csd is an important factor acting downstream of Phf7 to regulate germline masculinization.
Vedelek, V., Kovacs, A. L., Juhasz, G., Alzyoud, E. and Sinka, R. (2021). The tumor suppressor archipelago E3 ligase is required for spermatid differentiation in Drosophila testis. Sci Rep 11(1): 8422. PubMed ID: 33875704
The human orthologue of the tumor suppressor protein FBW7 is encoded by the Drosophila archipelago (ago) gene. Ago is an F-box protein that gives substrate specificity to its SCF ubiquitin ligase complex. It has a central role in multiple biological processes in a tissue-specific manner such as cell proliferation, cellular differentiation, hypoxia-induced gene expression. This study presents a previously unknown tissue-specific role of Ago in spermatid differentiation. A classical mutant of ago was identified that is semi-lethal and male-sterile. During the characterization of ago function in testis, ago was found to play role in spermatid development, following meiosis. Spermatogenesis defects was confirmed by silencing ago by RNAi in testes. The v mutants show multiple abnormalities in elongating and elongated spermatids, including aberration of the cyst morphology, malformed mitochondrial structures, and individualization defects. Additionally, the subcellular localization of Ago protein was determined with mCherry-Ago transgene in spermatids. These findings highlight the potential roles of Ago in different cellular processes of spermatogenesis, like spermatid individualization, and regulation of mitochondrial morphology.
Zheng, Q., Chen, X., Qiao, C., Wang, M., Chen, W., Luan, X., Yan, Y., Shen, C., Fang, J., Hu, X., Zheng, B., Wu, Y. and Yu, J. (2021). Somatic CG6015 mediates cyst stem cell maintenance and germline stem cell differentiation via EGFR signaling in Drosophila testes. Cell Death Discov 7(1): 68. PubMed ID: 33824283
Stem cell niche is regulated by intrinsic and extrinsic factors. In the Drosophila testis, cyst stem cells (CySCs) support the differentiation of germline stem cells (GSCs). However, the underlying mechanisms remain unclear. This study found that somatic CG6015 is required for CySC maintenance and GSC differentiation in a Drosophila model. Knockdown of CG6015 in CySCs caused aberrant activation of dpERK in undifferentiated germ cells in the Drosophila testis, and disruption of key downstream targets of EGFR signaling (Dsor1 and rl) in CySCs results in a phenotype resembling that of CG6015 knockdown. CG6015, Dsor1, and rl are essential for the survival of Drosophila cell line Schneider 2 (S2) cells. The data showed that somatic CG6015 regulates CySC maintenance and GSC differentiation via EGFR signaling, and inhibits aberrant activation of germline dpERK signals. These findings indicate regulatory mechanisms of stem cell niche homeostasis in the Drosophila testis.
Wang, Q. Q., Zhao, P. A., Tastan O, Y. and Liu, J. L. (2021). Polarised maintenance of cytoophidia in Drosophila follicle epithelia. Exp Cell Res 402(2): 112564. PubMed ID: 33737069
The metabolic enzyme CTP synthase (CTPS) can form filamentous structures named cytoophidia in numerous types of cells, including follicle cells. However, the regulation of cytoophidium assembly remains elusive. The apicobasal polarity, a defining characteristic of Drosophila follicle epithelium, is established and regulated by a variety of membrane domains. This study shows that CTPS can form cytoophidia in Drosophila epithelial follicle cells. Cytoophidia localise to the basolateral side of follicle cells. If apical polarity regulators are knocked down, cytoophidia become unstable and distribute abnormally. Knockdown of basolateral polarity regulators has no significant effect on cytoophidia, even though the polarity is disturbed. These results indicate that cytoophidia are maintained via polarised distribution on the basolateral side of Drosophila follicle epithelia, which is primarily achieved through the apical polarity regulators.

Monday, July 19th - Signaling

Yang, M., Guo, Y., Wang, S., Chen, C., Chang, Y. H. and Ho, M. S. (2021). The F-Box Protein CG5003 Regulates Axon Pruning and the Integrity of the Drosophila Mushroom Body. Front Mol Neurosci 14: 634784. PubMed ID: 33716667
Protein homeostasis serves as an important step in regulating diverse cellular processes underlying the function and development of the nervous system. In particular, the ubiquitination proteasome system (UPS), a universal pathway mediating protein degradation, contributes to the development of numerous synaptic structures, including the Drosophila olfactory-associative learning center mushroom body (MB), thereby affecting associated function. This study describes the function of a newly characterized Drosophila F-box protein CG5003, an adaptor for the RING-domain type E3 ligase (SCF complex), in MB development. Lacking CG5003 ubiquitously causes MB γ axon pruning defects and selective CG5003 expression in pan-neurons leads to both γ axon and α/β lobe abnormalities. Interestingly, change in CG5003 expression in MB neurons does not cause any abnormalities in axons, suggesting that CG5003 functions in cells extrinsic to MB to regulate its development. Mass spectrum analysis indicates that silencing CG5003 expression in all neurons affects expression levels of proteins in the cell and structural morphogenesis, transcription regulator activity, and catalytic activity. These findings reinforce the importance of UPS and identify a new factor in regulating neuronal development as exemplified by the synaptic structure of the MB.
Yin, Y., Wu, Y., Zhang, X., Zhu, Y., Sun, Y., Yu, J., Gong, Y., Sun, P., Lin, H. and Han, X. (2021). PPA1 Regulates Systemic Insulin Sensitivity by Maintaining Adipocyte Mitochondria Function as a Novel PPARgamma Target Gene. Diabetes. PubMed ID: 33722839
Downregulation of mitochondrial function in adipose tissue is considered as one important driver for the development of obesity-associated metabolic disorders. Inorganic Pyrophosphatase 1 (PPA1) is an enzyme catalyzes the hydrolysis of PPi to Pi, and is required for anabolism to take place in cells. Although alternation of PPA1 has been related to some diseases, the importance of PPA1 in metabolic syndromes has never been discussed before. This study found that global PPA1 knockout mice (PPA1(+/-)) showed impaired glucose tolerance and severe insulin resistance under HFD feeding. In addition, impaired adipose tissue development and ectopic lipid accumulation were also observed. Conversely, overexpression of PPA1 in adipose tissue by AAV injection can partly reverse the metabolic disorders in PPA1(+/-) mice, suggesting that impaired adipose tissue function is responsible for the metabolic disorders observed in PPA1(+/-) mice. Mechanistic studies revealed that PPA1 acted as a PPARγ (E75 and E78) target gene to maintain mitochondrial function in adipocytes. Furthermore, specific knockdown of PPA1 in fat body of Drosophila led to impaired mitochondria morphology, decreased lipid storage, and made Drosophila more sensitive to starvation. In conclusion, these finding findings demonstrated the importance of PPA1 in maintaining adipose tissue function and whole body metabolic homeostasis.
Yamamoto, R., Palmer, M., Koski, H., Curtis-Joseph, N. and Tatar, M. (2021). Aging modulated by the Drosophila insulin receptor through distinct structure-defined mechanisms. Genetics 217(2). PubMed ID: 33724413
Mutations of the Drosophila melanogaster insulin/IGF signaling system slow aging, while also affecting growth and reproduction. To understand this pleiotropy, an allelic series of single codon substitutions was produced in the Drosophila insulin receptor, InR. Substitutions were generated using homologous recombination, and each was related to emerging models of receptor tyrosine kinase structure and function. Three mutations when combined as trans-heterozygotes extended lifespan while retarding growth and fecundity. These genotypes reduced insulin-stimulated Akt phosphorylation, suggesting they impede kinase catalytic domain function. Among these genotypes, longevity was negatively correlated with egg production, consistent with life-history trade-off theory. In contrast, one mutation (InR353) was located in the kinase insert domain, a poorly characterized element found in all receptor tyrosine kinases. Remarkably, wild-type heterozygotes with InR353 robustly extended lifespan without affecting growth or reproduction and retained capacity to fully phosphorylate Akt. The Drosophila insulin receptor kinase insert domain contains a previously unrecognized SH2 binding motif. It is proposed the kinase insert domain interacts with SH2-associated adapter proteins to affect aging through mechanisms that retain insulin sensitivity and are independent of reproduction.
Wang, X., Liang, H., Xu, W. and Ma, X. (2021). Wallenda-Nmo Axis Regulates Growth via Hippo Signaling. Front Cell Dev Biol 9: 658288. PubMed ID: 33937258
Both Hippo signaling pathways and cell polarity regulation are critical for cell proliferation and the maintenance of tissue homeostasis, despite the well-established connections between cell polarity disruption and Hippo inactivation, the molecular mechanism by which aberrant cell polarity induces Hippo-mediated overgrowth remains underexplored. This study used Drosophila wing discs as a model and identify the Wnd-Nmo axis as an important molecular link that bridges loss-of-cell polarity-triggered Hippo inactivation and overgrowth. Wallenda (Wnd), a MAPKKK (mitogen-activated protein kinase kinase kinase) family member, is shown to be a novel regulator of Hippo pathways in Drosophila; overexpression of Wnd promotes growth via Nemo (Nmo)- mediated Hippo pathway inactivation. It was further demonstrated that both Wnd and Nmo are required for loss-of-cell polarity-induced overgrowth and Hippo inactivation. In summary, these findings provide a novel insight on how cell polarity loss contributes to overgrowth and uncover the Wnd-Nmo axis as an essential additional branch that regulates Hippo pathways in Drosophila.

Friday, July 16th - Disease Models

Yap, Z. Y., Park, Y. H., Wortmann, S. B., Gunning, A. C., Ezer, S., Lee, S., Duraine, L., Wilichowski, E., Wilson, K., Mayr, J. A., Wagner, M., Li, H., Kini, U., Black, E. D., Monaghan, K. G., Lupski, J. R., Ellard, S., Westphal, D. S., Harel, T. and Yoon, W. H. (2021). Functional interpretation of ATAD3A variants in neuro-mitochondrial phenotypes. Genome Med 13(1): 55. PubMed ID: 33845882
ATPase family AAA-domain containing protein 3A (ATAD3A) is a nuclear-encoded mitochondrial membrane-anchored protein involved in diverse processes including mitochondrial dynamics, mitochondrial DNA organization, and cholesterol metabolism. Biallelic deletions (null), recessive missense variants (hypomorph), and heterozygous missense variants or duplications (antimorph) in ATAD3A lead to neurological syndromes in humans. To expand the mutational spectrum of ATAD3A variants and to provide functional interpretation of missense alleles in trans to deletion alleles, exome sequencing was performed for identification of single nucleotide variants (SNVs) and copy number variants (CNVs) in ATAD3A in individuals with neurological and mitochondrial phenotypes. A Drosophila Atad3a Gal4 knockin-null allele was generated using CRISPR-Cas9 genome editing technology to aid the interpretation of variants. This study reports 13 individuals from 8 unrelated families with biallelic ATAD3A variants. The variants included four missense variants inherited in trans to loss-of-function alleles (p.(Leu77Val), p.(Phe50Leu), p.(Arg170Trp), p.(Gly236Val)), a homozygous missense variant p.(Arg327Pro), and a heterozygous non-frameshift indel p.(Lys568del). Affected individuals exhibited findings previously associated with ATAD3A pathogenic variation, including developmental delay, hypotonia, congenital cataracts, hypertrophic cardiomyopathy, and cerebellar atrophy. Drosophila studies indicated that Phe50Leu, Gly236Val, Arg327Pro, and Lys568del are severe loss-of-function alleles leading to early developmental lethality. Further, Phe50Leu, Gly236Val, and Arg327Pro were shown to cause neurogenesis defects. On the contrary, Leu77Val and Arg170Trp are partial loss-of-function alleles that cause progressive locomotion defects and whose expression leads to an increase in autophagy and mitophagy in adult muscles. These findings expand the allelic spectrum of ATAD3A variants and exemplify the use of a functional assay in Drosophila to aid variant interpretation.
Wang, J., Qiao, J. D., Liu, X. R., Liu, D. T., Chen, Y. H., Wu, Y., Sun, Y., Yu, J., Ren, R. N., Mei, Z., Liu, Y. X., Shi, Y. W., Jiang, M., Lin, S. M., He, N., Li, B., Bian, W. J., Li, B. M., Yi, Y. H., Su, T., Liu, H. K., Gu, W. Y. and Liao, W. P. (2021). UNC13B variants associated with partial epilepsy with favourable outcome. Brain. PubMed ID: 33876820
The unc-13 homolog B (UNC13B) gene encodes a presynaptic protein, mammalian uncoordinated 13-2 (Munc13-2), that is highly expressed in the brain-predominantly in the cerebral cortex-and plays an essential role in synaptic vesicle priming and fusion, potentially affecting neuronal excitability. However, the functional significance of UNC13B mutation in human disease is not known. This study screened for novel genetic variants in a cohort of 446 unrelated cases (families) with partial epilepsy without acquired causes by trio-based whole-exome sequencing. UNC13B variants were identified in 12 individuals affected by partial epilepsy and/or febrile seizures from eight unrelated families. The eight probands all had focal seizures and focal discharges in EEG recordings, including two patients who experienced frequent daily seizures and one who showed abnormalities in the hippocampus by brain MRI; however, all of the patients showed favorable outcome without intellectual or developmental abnormalities. The identified UNC13B variants included one nonsense variant, two variants at or around a splice site, one compound heterozygous missense variant, and four missense variants that cosegregated in the families. The frequency of UNC13B variants identified in the present study was significantly higher than that in a control cohort of Han Chinese and controls of the East Asian and all populations in the Genome Aggregation Database. Computational modeling, including hydrogen bond and docking analyses, suggested that the variants lead to functional impairment. In Drosophila, seizure rate and duration were increased by Unc13b knockdown compared to wild-type flies, but these effects were less pronounced than in sodium voltage-gated channel alpha subunit 1 (Scn1a; Paralytic) knockdown Drosophila. Electrophysiologic recordings showed that excitatory neurons in Unc13b-deficient flies exhibited increased excitability. These results suggest that UNC13B is potentially associated with epilepsy. The frequent daily seizures and hippocampal abnormalities but ultimately favorable outcome under antiepileptic therapy in patients indicate that partial epilepsy caused by UNC13B variant is a clinically manageable condition.
Wang, Y., Burra, S. and Galko, M. J. (2021). Drosophila larval epidermal cells only exhibit epidermal aging when they persist to the adult stage. J Exp Biol. PubMed ID: 33795421
Holometabolous insects undergo a complete transformation of the body plan from the larval to the adult stage. In Drosophila, this transformation includes replacement of larval epidermal cells (LECs) by adult epidermal cells (AECs). AECs in Drosophila undergo a rapid and stereotyped aging program where they lose both cell membranes and nuclei. Whether LECs are capable of undergoing aging in a manner similar to AECs remains unknown. This question was addressed in two ways. First, hallmarks were sought of epidermal aging in larvae that have a greatly extended third instar and/or carry mutations that would cause premature epidermal aging at the adult stage. Such larvae, irrespective of genotype, did not show any of the signs of epidermal aging observed in the adult. Second, a procedure was developed to effect a heterochronic persistence of LECs into the adult epidermal sheet. Lineage tracing verified that presumptive LECs in the adult epidermis are not derived from imaginal epidermal histoblasts. LECs embedded within the adult epidermal sheet undergo clear signs of epidermal aging; they form multinucleate cells with each other and with the surrounding AECs. The incidence of adult cells with mixed AEC nuclei (small) and persistent LEC nuclei (large) increased with age. These data reveals that epidermal aging in holometabolous Drosophila is a stage-specific phenomenon and that the capacity of LECs to respond to aging signals does exist.
Tatar, M. (2021). Aging Regulated Through a Stability Model of Insulin/Insulin Growth Factor Receptor Function. Front Endocrinol (Lausanne) 12: 649880. PubMed ID: 33776941
Mutations of the insulin-like receptor in Drosophila extend lifespan. New research suggests this receptor operates in two modes. The first extends lifespan while slowing reproduction and reducing growth. The second strongly extends lifespan without impairing growth or reproduction; it confers longevity assurance. The mutation that confers longevity assurance resides in the kinase insert domain, which contains a potential SH2 binding site for substrate proteins. A recent model for the function of receptor tyrosine kinases was applied to propose how insulin receptor structure can modulate aging. This concept hypothesizes that strong insulin-like ligands promote phosphorylation of high threshold substrate binding sites to robustly induce reproduction, which impairs survival as a consequence of trade-offs. Lower levels of receptor stimulation provide less kinase dimer stability, which reduces reproduction and extends lifespan by avoiding reproductive costs. Environmental conditions that favor diapause alter the expression of insulin ligands to further repress the stability of the interacting kinase domains, block phosphorylation of low threshold substrates and thus induce a unique molecular program that confers longevity assurance. Mutations of the insulin receptor that block low-phosphorylation site interactions, such as within the kinase insert domain, can extend lifespan while maintaining overall dimer stability. These flies are long-lived while maintaining reproduction and growth. The kinase insert domain of Drosophila provides a novel avenue from which to seek signaling of the insulin/insulin-like growth factor system of humans that modulate aging without impacting reproduction and growth, or incurring insulin resistance pathology.
Xie, J., Han, Y. and Wang, T. (2021). RACK1 modulates polyglutamine-induced neurodegeneration by promoting ERK degradation in Drosophila. PLoS Genet 17(5): e1009558. PubMed ID: 33983927
Polyglutamine diseases are neurodegenerative diseases caused by the expansion of polyglutamine (polyQ) tracts within different proteins. Although multiple pathways have been found to modulate aggregation of the expanded polyQ proteins, the mechanisms by which polyQ tracts induced neuronal cell death remain unknown. A genome-wide genetic screen was conducted to identify genes that suppress polyQ-induced neurodegeneration when mutated. Loss of the scaffold protein RACK1 alleviated cell death associated with the expression of polyQ tracts alone, as well as in models of Machado-Joseph disease (MJD) and Huntington's disease (HD), without affecting proteostasis of polyQ proteins. A genome-wide RNAi screen for modifiers of this rack1 suppression phenotype revealed that knockdown of the E3 ubiquitin ligase, POE (Purity of essence), further suppressed polyQ-induced cell death, resulting in nearly wild-type looking eyes. Biochemical analyses demonstrated that RACK1 interacts with POE and ERK to promote ERK degradation. These results suggest that RACK1 plays a key role in polyQ pathogenesis by promoting POE-dependent degradation of ERK, and implicate RACK1/POE/ERK as potent drug targets for treatment of polyQ diseases.
Westin, I. M., Jonsson, F., Osterman, L., Holmberg, M., Burstedt, M. and Golovleva, I. (2021). EYS mutations and implementation of minigene assay for variant classification in EYS-associated retinitis pigmentosa in northern Sweden. Sci Rep 11(1): 7696. PubMed ID: 33833316
Retinitis pigmentosa (RP) is a clinically and genetically heterogeneous group of inherited retinal degenerations. The ortholog of Drosophila eyes shut/spacemaker, EYS on chromosome 6q12 is a major genetic cause of recessive RP worldwide, with prevalence of 5 to 30%. In this study, by using targeted NGS, MLPA and Sanger sequencing, the EYS gene was identifed as one of the most common genetic cause of autosomal recessive RP in northern Sweden accounting for at least 16%. The most frequent pathogenic variant was c.8648_8655del that in some patients was identified in cis with c.1155T>A, indicating Finnish ancestry. Two novel EYS variants, c.2992_2992+6delinsTG and c.3877+1G>A caused exon skipping in human embryonic kidney cells, HEK293T and in retinal pigment epithelium cells, ARPE-19 demonstrating that in vitro minigene assay is a straightforward tool for the analysis of intronic variants. It is concluded, that whenever it is possible, functional testing is of great value for classification of intronic EYS variants and the following molecular testing of family members, their genetic counselling, and inclusion of RP patients to future treatment studies.

Thursday, July 15th - Adult Physiology

Wang, Y., Zhang, H., Zhang, Z., Sun, B., Tang, C., Zhang, L., Jiang, Z., Ding, B., Liao, Y. and Cai, P. (2021). Simulated mobile communication frequencies (3.5 GHz) emitted by a signal generator affects the sleep of Drosophila melanogaster. Environ Pollut 283: 117087. PubMed ID: 33894629
With the rapid development of science and technology, 5G technology will be widely used, and biosafety concerns about the effects of 5G radiofrequency radiation on health have been raised. Drosophila melanogaster was selected as the model organism for this study, in which a 3.5 GHz radiofrequency radiation (RF-EMR) environment was simulated at intensities of 0.1 W/m(2), 1 W/m(2), and 10 W/m(2). The activity of parent male and offspring (F1) male flies was measured using a Drosophila activity monitoring system under short-term and long-term 3.5 GHz RF-EMR exposure. Core genes associated with heat stress, the circadian clock and neurotransmitters were detected by QRT-PCR technology, and the contents of GABA and glutamate were detected by UPLC-MS. The results show that short-term RF-EMR exposure increased the activity level and reduced the sleep duration while long-term RF-EMR exposure reduced the activity level and increased the sleep duration of F1 male flies. Under long-term RF-EMR, the expression of heat stress response-related hsp22, hsp26 and hsp70 genes was increased, the expression of circadian clock-related per, cyc, clk, cry, and tim genes was altered, the content of GABA and glutamate was reduced, and the expression levels of synthesis, transport and receptor genes were altered. In conclusion, long-term RF-EMR exposure enhances the heat stress response of offspring flies and then affects the expression of circadian clock and neurotransmitter genes, which leads to decreased activity, prolonged sleep duration, and improved sleep quality.
Wong, K. C., Sankaran, S., Jayapalan, J. J., Subramanian, P. and Abdul-Rahman, P. S. (2021). Melatonin improves cognitive behavior, oxidative stress, and metabolism in tumor-prone lethal giant larvae mutant of Drosophila melanogaster. Arch Insect Biochem Physiol 107(1): e21785. PubMed ID: 33818826
Mutant lethal giant larvae (lgl) flies (Drosophila melanogaster) are known to develop epithelial tumors with invasive characteristics. The present study has been conducted to investigate the influence of melatonin (0.025 mM) on behavioral responses of lgl mutant flies as well as on biochemical indices (redox homeostasis, carbohydrate and lipid metabolism, transaminases, and minerals) in hemolymph, and head and intestinal tissues. Behavioral abnormalities were quantitatively observed in lgl flies but were found normalized among melatonin-treated lgl flies. Significantly decreased levels of lipid peroxidation products and antioxidants involved in redox homeostasis were observed in hemolymph and tissues of lgl flies, but had restored close to normalcy in melatonin-treated flies. Carbohydrates including glucose, trehalose, and glycogen were decreased and increased in the hemolymph and tissues of lgl and melatonin-treated lgl flies, respectively. Key enzymes of carbohydrate metabolism showed a significant increment in their levels in lgl mutants but had restored close to wild-type baseline levels in melatonin-treated flies. Variables of lipid metabolism showed significantly inverse levels in hemolymph and tissues of lgl flies, while normalization of most of these variables was observed in melatonin-treated mutants. Lipase, chitinase, transaminases, and alkaline phosphatase showed an increment in their activities and minerals exhibited decrement in lgl flies; reversal of changes was observed under melatonin treatment. The impairment of cognition, disturbance of redox homeostasis and metabolic reprogramming in lgl flies, and restoration of normalcy in all these cellular and behavioral processes indicate that melatonin could act as oncostatic and cytoprotective agents in Drosophila.
Wang, L., Wen, P., van de Leemput, J., Zhao, Z. and Han, Z. (2021). Slit diaphragm maintenance requires dynamic clathrin-mediated endocytosis facilitated by AP-2, Lap, Aux and Hsc70-4 in nephrocytes. Cell Biosci 11(1): 83. PubMed ID: 33975644
The Slit diaphragm (SD) is the key filtration structure in human glomerular kidney that is affected in many types of renal diseases. SD proteins are known to undergo endocytosis and recycling to maintain the integrity of the filtration structure. However, the key components of this pathway remain unclear. Using the Drosophila nephrocyte as a genetic screen platform, most genes involved in endocytosis and cell trafficking were screened, and the key components were identified of the cell trafficking pathway required for SD protein endocytosis and recycling. The SD protein endocytosis and recycling pathway was found to contain clathrin, dynamin, AP-2 complex, like-AP180 (Lap), auxilin and Hsc70-4 (the endocytosis part) followed by Rab11 and the exocyst complex (the recycling part). Disrupting any component in this pathway led to disrupted SD on the cell surface and intracellular accumulation of mislocalized SD proteins. This study provides the first in vivo evidence of trapped SD proteins in clathrin-coated pits at the plasma membrane when this pathway is disrupted. All genes in this SD protein endocytosis and recycling pathway, as well as SD proteins themselves, are highly conserved from flies to humans. Thus, these results suggest that the SD proteins in human kidney undergo the same endocytosis and recycling pathway to maintain the filtration structure, and mutations in any genes in this pathway could lead to abnormal SD and renal diseases.
Wang, T., Wiater, E., Zhang, X., Thomas, J. B. and Montminy, M. (2021). Crtc modulates fasting programs associated with 1-C metabolism and inhibition of insulin signaling. Proc Natl Acad Sci U S A 118(12). PubMed ID: 33723074
Fasting in mammals promotes increases in circulating glucagon and decreases in circulating insulin that stimulate catabolic programs and facilitate a transition from glucose to lipid burning. The second messenger cAMP mediates effects of glucagon on fasting metabolism, in part by promoting the phosphorylation of CREB and the dephosphorylation of the cAMP-regulated transcriptional coactivators (CRTCs) in hepatocytes. In Drosophila, fasting also triggers activation of the single Crtc homolog in neurons, via the PKA-mediated phosphorylation and inhibition of salt-inducible kinases. Crtc mutant flies are more sensitive to starvation and oxidative stress, although the underlying mechanism remains unclear. This study used RNA sequencing to identify Crtc target genes that are up-regulated in response to starvation. Crtc was found to stimulate a subset of fasting-inducible genes that have conserved CREB binding sites. In keeping with its role in the starvation response, Crtc was found to induce the expression of genes that inhibit insulin secretion (Lst) and insulin signaling (Impl2). In parallel, Crtc also promoted the expression of genes involved in one-carbon (1-C) metabolism. Within the 1-C pathway, Crtc stimulated the expression of enzymes that encode modulators of S-adenosyl-methionine metabolism (Gnmt and Sardh) and purine synthesis (ade2 and AdSl) Collectively, these results point to an important role for the CREB/CRTC pathway in promoting energy balance in the context of nutrient stress.
van Heerwaarden, B. and Sgro, C. M. (2021). Male fertility thermal limits predict vulnerability to climate warming. Nat Commun 12(1): 2214. PubMed ID: 33850157
Forecasting which species/ecosystems are most vulnerable to climate warming is essential to guide conservation strategies to minimize extinction. Tropical/mid-latitude species are predicted to be most at risk as they live close to their upper critical thermal limits (CTLs). However, these assessments assume that upper CTL estimates, such as CTmax, are accurate predictors of vulnerability and ignore the potential for evolution to ameliorate temperature increases. This study used experimental evolution to assess extinction risk and adaptation in tropical and widespread Drosophila species. Tropical species were found to succumb to extinction before widespread species. Male fertility thermal limits, which are much lower than CTmax, are better predictors of species' current distributions and extinction in the laboratory. Little evidence was found of adaptive responses to warming in any species. These results suggest that species are living closer to their upper thermal limits than currently presumed and evolution/plasticity are unlikely to rescue populations from extinction.
Tzou, F. Y., Su, T. Y., Lin, W. S., Kuo, H. C., Yu, Y. L., Yeh, Y. H., Liu, C. C., Kuo, C. H., Huang, S. Y. and Chan, C. C. (2021). Dihydroceramide desaturase regulates the compartmentalization of Rac1 for neuronal oxidative stress. Cell Rep 35(2): 108972. PubMed ID: 33852856
Disruption of sphingolipid homeostasis is known to cause neurological disorders, but the mechanisms by which specific sphingolipid species modulate pathogenesis remain unclear. The last step of de novo sphingolipid synthesis is the conversion of dihydroceramide to ceramide by dihydroceramide desaturase (human DEGS1; Drosophila Ifc). Loss of ifc leads to dihydroceramide accumulation, oxidative stress, and photoreceptor degeneration, whereas human DEGS1 variants are associated with leukodystrophy and neuropathy. This work demonstrates that DEGS1/ifc regulates Rac1 compartmentalization in neuronal cells and that dihydroceramide alters the association of active Rac1 with organelle-mimicking membranes. The Rac1-NADPH oxidase (NOX) complex was identified as the major cause of reactive oxygen species (ROS) accumulation in ifc-knockout (ifc-KO) photoreceptors and in SH-SY5Y cells with the leukodystrophy-associated DEGS1(H132R) variant. Suppression of Rac1-NOX activity rescues degeneration of ifc-KO photoreceptors and ameliorates oxidative stress in DEGS1(H132R)-carrying cells. Therefore, it is concluded that DEGS1/ifc deficiency causes dihydroceramide accumulation, resulting in Rac1 mislocalization and NOX-dependent neurodegeneration.

Wednesday, July 14th - Signaling

Wong, C. O., Karagas, N. E., Jung, J., Wang, Q., Rousseau, M. A., Chao, Y., Insolera, R., Soppina, P., Collins, C. A., Zhou, Y., Hancock, J. F., Zhu, M. X. and Venkatachalam, K. (2021). Regulation of longevity by depolarization-induced activation of PLC-beta-IP(3)R signaling in neurons. Proc Natl Acad Sci U S A 118(16). PubMed ID: 33859040
Mitochondrial ATP production is a well-known regulator of neuronal excitability. The reciprocal influence of plasma-membrane potential on ATP production, however, remains poorly understood. This study describes a mechanism by which depolarized neurons elevate the somatic ATP/ADP ratio in Drosophila glutamatergic neurons. Depolarization increased phospholipase-Cβ (PLC-β) activity by promoting the association of the enzyme with its phosphoinositide substrate. Augmented PLC-β activity led to greater release of endoplasmic reticulum Ca(2+) via the inositol trisphosphate receptor (IP(3)R), increased mitochondrial Ca(2+) uptake, and promoted ATP synthesis. Perturbations that decoupled membrane potential from this mode of ATP synthesis led to untrammeled PLC-β-IP(3)R activation and a dramatic shortening of Drosophila lifespan. Upon investigating the underlying mechanisms, this study found that increased sequestration of Ca(2+) into endolysosomes was an intermediary in the regulation of lifespan by IP(3)Rs. Manipulations that either lowered PLC-β/IP(3)R abundance or attenuated endolysosomal Ca(2+) overload restored animal longevity. Collectively, these findings demonstrate that depolarization-dependent regulation of PLC-β-IP(3)R signaling is required for modulation of the ATP/ADP ratio in healthy glutamatergic neurons, whereas hyperactivation of this axis in chronically depolarized glutamatergic neurons shortens animal lifespan by promoting endolysosomal Ca(2+) overload.
Wolf, D. B., Maier, D. and Nagel, A. C. (2021). Nucleo-cytoplasmic shuttling of murine RBPJ by Hairless protein matches that of Su(H) protein in the model system Drosophila melanogaster. Hereditas 158(1): 11. PubMed ID: 33775255
CSL transcription factors are central to signal transduction in the highly conserved Notch signaling pathway. CSL acts as a molecular switch: depending on the cofactors recruited, CSL induces either activation or repression of Notch target genes. Unexpectedly, CSL depends on its cofactors for nuclear entry, despite its role as gene regulator. In Drosophila, the CSL homologue Suppressor of Hairless (Su(H)), recruits Hairless (H) for repressor complex assembly, and eventually for nuclear import. It was recently found that Su(H) is subjected to a dynamic nucleo-cytoplasmic shuttling, thereby strictly following H subcellular distribution. Hence, regulation of nuclear availability of Su(H) by H may represent a new layer of control of Notch signaling activity. This study extended this work on the murine CSL homologue RBPJ. Using a 'murinized' fly model bearing RBPJ(wt) in place of Su(H) at the endogenous locus this study demonstrated that RBPJ protein likewise follows H subcellular distribution. For example, overexpression of a H(*NLS3) protein variant defective of nuclear import resulted in a cytosolic localization of RBPJ protein, whereas the overexpression of a H(*NES) protein variant defective in the nuclear export signal caused the accumulation of RBPJ protein in the nucleus. Evidently, RBPJ is exported from the nucleus as well. Overall these data demonstrate that in the fly model, RBPJ is subjected to H-mediated nucleo-cytoplasmic shuttling as is Su(H). These data raise the possibility that nuclear availability of mammalian CSL proteins is likewise restricted by cofactors, and may hence present a more general mode of regulating Notch signaling activity.
Wang, L. I., DeFosse, T., Jang, J. K., Battaglia, R. A., Wagner, V. F. and McKim, K. S. (2021). Borealin directs recruitment of the CPC to oocyte chromosomes and movement to the microtubules. J Cell Biol 220(6). PubMed ID: 33836043
The chromosomes in the oocytes of many animals appear to promote bipolar spindle assembly. In Drosophila oocytes, spindle assembly requires the chromosome passenger complex (CPC), which consists of INCENP, Borealin, Survivin, and Aurora B. To determine what recruits the CPC to the chromosomes and its role in spindle assembly, a strategy was developed to manipulate the function and localization of INCENP, which is critical for recruiting the Aurora B kinase. An interaction between Borealin and the chromatin was found to be crucial for the recruitment of the CPC to the chromosomes and is sufficient to build kinetochores and recruit spindle microtubules. HP1 colocalizes with the CPC on the chromosomes and together they move to the spindle microtubules. It is proposed that the Borealin interaction with HP1 promotes the movement of the CPC from the chromosomes to the microtubules. In addition, within the central spindle, rather than at the centromeres, the CPC and HP1 are required for homologous chromosome bi-orientation.
Wang, M., Han, X., Liu, C., Takayama, R., Yasugi, T., Ei, S. I., Nagayama, M., Tanaka, Y. and Sato, M. (2021). Intracellular trafficking of Notch orchestrates temporal dynamics of Notch activity in the fly brain. Nat Commun 12(1): 2083. PubMed ID: 33828096
While Delta non-autonomously activates Notch in neighboring cells, it autonomously inactivates Notch through cis-inhibition, the molecular mechanism and biological roles of which remain elusive. The wave of differentiation in the Drosophila brain, the 'proneural wave', is an excellent model for studying Notch signaling in vivo. This study shows that strong nonlinearity in cis-inhibition reproduces the second peak of Notch activity behind the proneural wave in silico. Based on this, Delta expression was demonstrated to induce a quick degradation of Notch in late endosomes and the formation of the twin peaks of Notch activity in vivo. Indeed, the amount of Notch is upregulated and the twin peaks are fused forming a single peak when the function of Delta or late endosomes is compromised. Additionally, this study showed that the second Notch peak behind the wavefront controls neurogenesis. Thus, intracellular trafficking of Notch orchestrates the temporal dynamics of Notch activity and the temporal patterning of neurogenesis.
Shweta, K., Basargekar, A. and Ratnaparkhi, A. (2021). FGFR/Heartless and Smog interact synergistically to negatively regulate Fog mediated G-protein coupled receptor signaling in the Drosophila nervous system. G3 (Bethesda) 11(3). PubMed ID: 33729500
Folded gastrulation (Fog) is a secreted ligand that signals through the G-protein-coupled receptors Mist and Smog and the G-protein Concertina to activate downstream effectors to elicit cell-shape change during gastrulation. In the embryonic central nervous system (CNS), Fog has roles in axon guidance and glial morphogenesis. However, the elements of the pathway as well as mechanisms required for transducing the signal in this context have not been determined. This study finds that while Concertina is essential for Fog signaling, Mist is dispensable and Smog, surprisingly, functions as a negative regulator of the pathway in the CNS. Interestingly Heartless, a fibroblast growth factor receptor, also functions as a negative regulator. Furthermore, both Heartless and Smog interact in a synergistic manner to regulate Fog signaling. These results thus identify Heartless and Smog as part of a common regulatory pathway that functions to restrict Fog signaling in the embryonic CNS and highlights the context-specific role for Fog receptors during development.
Tamada, M., Shi, J., Bourdot, K. S., Supriyatno, S., Palmquist, K. H., Gutierrez-Ruiz, O. L. and Zallen, J. A. (2021). Toll receptors remodel epithelia by directing planar-polarized Src and PI3K activity. Dev Cell. PubMed ID: 33932332
Toll-like receptors are essential for animal development and survival, with conserved roles in innate immunity, tissue patterning, and cell behavior. The mechanisms by which Toll receptors signal to the nucleus are well characterized, but how Toll receptors generate rapid, localized signals at the cell membrane to produce acute changes in cell polarity and behavior is not known. This study shows that Drosophila Toll receptors direct epithelial convergent extension by inducing planar-polarized patterns of Src and PI3-kinase (PI3K) activity. Toll receptors target Src activity to specific sites at the membrane, and Src recruits PI3K to the Toll-2 complex through tyrosine phosphorylation of the Toll-2 cytoplasmic domain. Reducing Src or PI3K activity disrupts planar-polarized myosin assembly, cell intercalation, and convergent extension, whereas constitutive Src activity promotes ectopic PI3K and myosin cortical localization. These results demonstrate that Toll receptors direct cell polarity and behavior by locally mobilizing Src and PI3K activity.

Tuesday, July 13th - Adult neural development and function

Spierer, A. N., Mossman, J. A., Smith, S. P., Crawford, L., Ramachandran, S. and Rand, D. M. (2021). Natural variation in the regulation of neurodevelopmental genes modifies flight performance in Drosophila. PLoS Genet 17(3): e1008887. PubMed ID: 33735180
The winged insects of the order Diptera are colloquially named for their most recognizable phenotype: flight. These insects rely on flight for a number of important life history traits, such as dispersal, foraging, and courtship. Despite the importance of flight, relatively little is known about the genetic architecture of flight performance. Accordingly, this study sought to uncover the genetic modifiers of flight using a measure of flies' reaction and response to an abrupt drop in a vertical flight column. A genome wide association study (GWAS) was conducted using 197 of the Drosophila Genetic Reference Panel (DGRP) lines, and a combination was identified of additive and marginal variants, epistatic interactions, whole genes, and enrichment across interaction networks. Egfr, a highly pleiotropic developmental gene, was among the most significant additive variants identified. 13 of the additive candidate genes (Adgf-A/Adgf-A2/CG32181, bru1, CadN, flapper (CG11073), CG15236, flippy (CG9766), CREG, Dscam4, form3, fry, Lasp/CG9692, Pde6, Snoo) were functionally validated, and a novel approach was introduced to whole gene significance screens: PEGASUS_flies. Additionally, ppk23, an Acid Sensing Ion Channel (ASIC) homolog, was identified as an important hub for epistatic interactions. A model is proposed that suggests genetic modifiers of wing and muscle morphology, nervous system development and function, BMP signaling, sexually dimorphic neural wiring, and gene regulation are all important for the observed differences flight performance in a natural population. Additionally, these results represent a snapshot of the genetic modifiers affecting drop-response flight performance in Drosophila, with implications for other insects.
Santiago, I. J., Zhang, D., Saras, A., Pontillo, N., Xu, C., Chen, X., Weirauch, M. T., Mistry, M., Ginty, D. D., Pecot, M. Y. and Peng, J. (2021). Drosophila Fezf functions as a transcriptional repressor to direct layer-specific synaptic connectivity in the fly visual system. Proc Natl Acad Sci U S A 118(13). PubMed ID: 33766917
The layered compartmentalization of synaptic connections, a common feature of nervous systems, underlies proper connectivity between neurons and enables parallel processing of neural information. However, the stepwise development of layered neuronal connections is not well understood. The medulla neuropil of the Drosophila visual system, which comprises 10 discrete layers (M1 to M10), where neural computations underlying distinct visual features are processed, serves as a model system for understanding layered synaptic connectivity. The first step in establishing layer-specific connectivity in the outer medulla (M1 to M6) is the innervation by lamina (L) neurons of one of two broad, primordial domains that will subsequently expand and transform into discrete layers. Previous work found that the transcription factor dFezf (Earmuff) cell-autonomously directs L3 lamina neurons to their proper primordial broad domain before they form synapses within the developing M3 layer. This study shows that dFezf controls L3 broad domain selection through temporally precise transcriptional repression of the transcription factor slp1 (sloppy paired 1). In wild-type L3 neurons, slp1 is transiently expressed at a low level during broad domain selection. When dFezf is deleted, slp1 expression is up-regulated, and ablation of slp1 fully rescues the defect of broad domain selection in dFezf-null L3 neurons. Although the early, transient expression of slp1 is expendable for broad domain selection, it is surprisingly necessary for the subsequent L3 innervation of the M3 layer. DFezf thus functions as a transcriptional repressor to coordinate the temporal dynamics of a transcriptional cascade that orchestrates sequential steps of layer-specific synapse formation.
Ryabova, E. V., Melentev, P. A., Komissarov, A. E., Surina, N. V., Ivanova, E. A., Matiytsiv, N., Shcherbata, H. R. and Sarantseva, S. V. (2021). Morpho-Functional Consequences of Swiss Cheese Knockdown in Glia of Drosophila melanogaster. Cells 10(3). PubMed ID: 33801404
Glia are crucial for the normal development and functioning of the nervous system in many animals. Insects are widely used for studies of glia genetics and physiology. Drosophila melanogaster surface glia (perineurial and subperineurial) form a blood-brain barrier in the central nervous system and blood-nerve barrier in the peripheral nervous system. Under the subperineurial glia layer, in the cortical region of the central nervous system, cortex glia encapsulate neuronal cell bodies, whilst in the peripheral nervous system, wrapping glia ensheath axons of peripheral nerves. This study shows that the expression of the evolutionarily conserved swiss cheese gene is important in several types of glia. swiss cheese knockdown in subperineurial glia leads to morphological abnormalities of these cells.The number of subperineurial glia nuclei is reduced under swiss cheese knockdown, possibly due to apoptosis. In addition, the downregulation of swiss cheese in wrapping glia causes a loss of its integrity. Transcriptome changes were revealed under swiss cheese knockdown in subperineurial glia and in cortex + wrapping glia; the downregulation of swiss cheese in these types of glia provokes reactive oxygen species acceleration. These results are accompanied by a decline in animal mobility measured by the negative geotaxis performance assay.
Thoener, J., Konig, C., Weiglein, A., Toshima, N., Mancini, N., Amin, F. and Schleyer, M. (2021). Associative learning in larval and adult Drosophila is impaired by the dopamine-synthesis inhibitor 3-Iodo-L-tyrosine. Biol Open. PubMed ID: 33978715
Across the animal kingdom, dopamine plays a crucial role in conferring reinforcement signals that teach animals about the causal structure of the world. In the fruit fly Drosophila melanogaster, dopaminergic reinforcement has largely been studied using genetics, whereas pharmacological approaches have received less attention. This study applied the dopamine-synthesis inhibitor 3-Iodo-L-tyrosine (3IY), which causes acute systemic inhibition of dopamine signaling, and investigated its effects on Pavlovian conditioning. 3IY feeding impairs sugar reward learning in larvae while leaving task-relevant behavioral faculties intact, and additional feeding of a precursor of dopamine (L-3,4-dihydroxyphenylalanine, L-DOPA), rescues this impairment. Concerning a different developmental stage and for the aversive valence domain, it was furthermore demonstrated that punishment learning by activating the dopaminergic neuron PPL1-γ1pedc in adult flies is also impaired by 3IY feeding and can likewise be rescued by L-DOPA. These findings exemplify the advantages of using a pharmacological approach in combination with the genetic techniques available in D. melanogaster to manipulate neuronal and behavioral function.
Saikia, C., Dym, O., Altman-Gueta, H., Gordon, D., Reuveny, E. and Karbat, I. (2021). A Molecular Lid Mechanism of K(+) Channel Blocker Action Revealed by a Cone Peptide. J Mol Biol: 166957. PubMed ID: 33771569
Many venomous organisms carry in their arsenal short polypeptides that block K(+) channels in a highly selective manner. These toxins may compete with the permeating ions directly via a "plug" mechanism or indirectly via a "pore-collapse" mechanism. An alternative "lid" mechanism was proposed but remained poorly defined. The Drosophila Shaker channel block by Conkunitzin-S1 and Conkunitzin-C3, two highly similar toxins derived from cone venom, was examined in this study. Despite their similarity, the two peptides exhibited differences in their binding poses and biophysical assays, implying discrete action modes. While Conkunitzin-S1 binds tightly to the channel turret and acts via a "pore-collapse" mechanism, Conkunitzin-C3 does not contact this region. Instead, Conk-C3 uses a non-conserved Arg to divert the permeant ions and trap them in off-axis cryptic sites above the SF, a mechanism termed a "molecular-lid". This study provides an atomic description of the "lid" K(+) blocking mode and offers valuable insights for the design of therapeutics based on venom peptides.
Song, B. J., Sharp, S. J. and Rogulja, D. (2021). Daily rewiring of a neural circuit generates a predictive model of environmental light. Sci Adv 7(13). PubMed ID: 33762336
Behavioral responsiveness to external stimulation is shaped by context. This study examined how sensory information can be contextualized, by examining light-evoked locomotor responsiveness of Drosophila relative to time of day. Light elicits an acute increase in locomotion (startle) that is modulated in a time-of-day-dependent manner: Startle is potentiated during the nighttime, when light is unexpected, but is suppressed during the daytime. The internal daytime-nighttime context is generated by two interconnected and functionally opposing populations of circadian neurons-LNvs generating the daytime state and DN1as generating the nighttime state. Switching between the two states requires daily remodeling of LNv and DN1a axons such that the maximum presynaptic area in one population coincides with the minimum in the other. It is proposed that a dynamic model of environmental light resides in the shifting connectivities of the LNv-DN1a circuit, which helps animals evaluate ongoing conditions and choose a behavioral response.

Monday, July 12th - Immune Response

Tardy, O. R., Armitage, E. L., Prince, L. R. and Evans, I. R. (2021). The Epidermal Growth Factor Ligand Spitz Modulates Macrophage Efferocytosis, Wound Responses and Migration Dynamics During Drosophila Embryogenesis. Front Cell Dev Biol 9: 636024. PubMed ID: 33898424
How multifunctional cells such as macrophages interpret the different cues within their environment and undertake an appropriate response is a key question in developmental biology. Understanding how cues are prioritized is critical to answering this - both the clearance of apoptotic cells (efferocytosis) and the migration toward damaged tissue is dependent on macrophages being able to interpret and prioritize multiple chemoattractants, polarize, and then undertake an appropriate migratory response. This study investigate the role of Spitz, the cardinal Drosophila epidermal growth factor (EGF) ligand, in regulation of macrophage behavior in the developing fly embryo, using activated variants with differential diffusion properties. The results show that misexpression of activated Spitz can impact macrophage polarity and lead to clustering of cells in a variant-specific manner, when expressed either in macrophages or the developing fly heart. Spitz can also alter macrophage distribution and perturb apoptotic cell clearance undertaken by these phagocytic cells without affecting the overall levels of apoptosis within the embryo. Expression of active Spitz, but not a membrane-bound variant, can also increase macrophage migration speeds and impair their inflammatory responses to injury. The fact that the presence of Spitz specifically undermines the recruitment of more distal cells to wound sites suggests that Spitz desensitizes macrophages to wounds or is able to compete for their attention where wound signals are weaker. Taken together these results suggest this molecule regulates macrophage migration and their ability to dispose of apoptotic cells. This work identifies a novel regulator of Drosophila macrophage function and provides insights into signal prioritization and integration in vivo. Given the importance of apoptotic cell clearance and inflammation in human disease, this work may help in understanding the role EGF ligands play in immune cell recruitment during development and at sites of disease pathology.
Huang, C., Xu, R., Liegeois, S., Chen, D., Li, Z. and Ferrandon, D. (2020). Differential Requirements for Mediator Complex Subunits in Drosophila melanogaster Host Defense Against Fungal and Bacterial Pathogens. Front Immunol 11: 478958. PubMed ID: 33746938
The humoral immune response to bacterial or fungal infections in Drosophila relies largely on a transcriptional response mediated by the Toll and Immune deficiency NF-κB pathways. Antimicrobial peptides are potent effectors of these pathways and allow the organism to attack invading pathogens. Dorsal-related Immune Factor (DIF), a transcription factor regulated by the Toll pathway, is required in the host defense against fungal and some Gram-positive bacterial infections. The Mediator complex is involved in the initiation of transcription of most RNA polymerase B (PolB)-dependent genes by forming a functional bridge between transcription factors bound to enhancer regions and the gene promoter region and then recruiting the PolB pre-initiation complex. Mediator is formed by several modules that each comprises several subunits. The Med17 subunit of the head module of Mediator has been shown to be required for the expression of Drosomycin, which encodes a potent antifungal peptide, by binding to DIF. Thus, Mediator is expected to mediate the host defense against pathogens controlled by the Toll pathway-dependent innate immune response. This study first focused on the Med31 subunit of the middle module of Mediator and find that it is required in host defense against Aspergillus fumigatus, Enterococcus faecalis, and injected but not topically-applied Metarhizium robertsii. Thus, host defense against M. robertsii requires Dif but not necessarily Med31 in the two distinct infection models. The induction of some Toll-pathway-dependent genes is decreased after a challenge of Med31 RNAi-silenced flies with either A. fumigatus or E. faecalis, while these flies exhibit normal phagocytosis and melanization. This study further tested most Mediator subunits using RNAi by monitoring their survival after challenges to several other microbial infections known to be fought off through DIF. The host defense against specific pathogens involves a distinct set of Mediator subunits with only one subunit for C. glabrata or Erwinia carotovora carotovora, at least one for M. robertsii or a somewhat extended repertoire for A. fumigatus (at least eight subunits) and E. faecalis (eight subunits), with two subunits, Med6 and Med11 being required only against A. fumigatus. Med31 but not Med17 is required in fighting off injected M. robertsii conidia. Thus, the involvement of Mediator in Drosophila innate immunity is more complex than expected.
Ramirez-Corona, B. A., Fruth, S. M., Ofoegbu, O. and Wunderlich, Z. (2021). The mode of expression divergence in Drosophila fat body is infection-specific. Genome Res. PubMed ID: 33858842
Transcription is controlled by interactions of cis-acting DNA elements with diffusible trans-acting factors. Changes in cis or trans factors can drive expression divergence within and between species, and their relative prevalence can reveal the evolutionary history and pressures that drive expression variation. Previous work delineating the mode of expression divergence in animals has largely used whole body expression measurements in one condition. Since cis-acting elements often drive expression in a subset of cell types or conditions, these measurements may not capture the complete contribution of cis-acting changes. This study quantified the mode of expression divergence in the Drosophila fat body, the primary immune organ, in several conditions, using two geographically distinct lines of D. melanogaster and their F1 hybrids. Expression was measured in the absence of infection and in infections with Gram-negative S. marcescens or Gram-positive E. faecalis bacteria, which trigger the two primary signaling pathways in the Drosophila innate immune response. The mode of expression divergence strongly depends on the condition, with trans-acting effects dominating in response to Gram-positive infection and cis-acting effects dominating in Gram-negative and pre-infection conditions. Expression divergence in several receptor proteins may underlie the infection-specific trans effects. Before infection, when the fat body has a metabolic role, there are many compensatory effects, changes in cis and trans that counteract each other to maintain expression levels. This work demonstrates that within a single tissue, the mode of expression divergence varies between conditions and suggests that these differences reflect the diverse evolutionary histories of host-pathogen interactions.
Schlamp, F., Delbare, S. Y. N., Early, A. M., Wells, M. T., Basu, S. and Clark, A. G. (2021). Dense time-course gene expression profiling of the Drosophila melanogaster innate immune response. BMC Genomics 22(1): 304. PubMed ID: 33902461
Immune responses need to be initiated rapidly, and maintained as needed, to prevent establishment and growth of infections. At the same time, resources need to be balanced with other physiological processes. On the level of transcription, studies have shown that this balancing act is reflected in tight control of the initiation kinetics and shutdown dynamics of specific immune genes. To investigate genome-wide expression dynamics and trade-offs after infection at a high temporal resolution, an RNA-seq time course was performed on D. melanogaster with 20 time points post Imd stimulation. A combination of methods, including spline fitting, cluster analysis, and Granger causality inference, allowed detailed dissection of expression profiles, lead-lag interactions, and functional annotation of genes through guilt-by-association. Imd-responsive genes and co-expressed, less well characterized genes, with an immediate-early response and sustained up-regulation up to 5 days after stimulation were identified. In contrast, stress response and Toll-responsive genes, among which were Bomanins, demonstrated early and transient responses. This study further observed a strong trade-off with metabolic genes, which strikingly recovered to pre-infection levels before the immune response was fully resolved. This high-dimensional dataset enabled the comprehensive study of immune response dynamics through the parallel application of multiple temporal data analysis methods. The well annotated data set should also serve as a useful resource for further investigation of the D. melanogaster innate immune response, and for the development of methods for analysis of a post-stress transcriptional response time-series at whole-genome scale.
Chen, D., Roychowdhury-Sinha, A., Prakash, P., Lan, X., Fan, W., Goto, A. and Hoffmann, J. A. (2021). A time course transcriptomic analysis of host and injected oncogenic cells reveals new aspects of Drosophila immune defenses. Proc Natl Acad Sci U S A 118(12). PubMed ID: 33737397
Oncogenic RasV12 cells injected into adult males proliferated massively after a lag period of several days, and led to the demise of the flies after 2 to 3 wk. The injection induced an early massive transcriptomic response that, unexpectedly, included more than 100 genes encoding chemoreceptors of various families. The kinetics of induction and the identities of the induced genes differed markedly from the responses generated by injections of microbes. Subsequently, hundreds of genes were up-regulated, attesting to intense catabolic activities in the flies, active tracheogenesis, and cuticulogenesis, as well as stress and inflammation-type responses. At 11 d after the injections, GFP-positive oncogenic cells isolated from the host flies exhibited a markedly different transcriptomic profile from that of the host and distinct from that at the time of their injection, including in particular up-regulated expression of genes typical for cells engaged in the classical antimicrobial response of Drosophila.
Singh, R. K., Jonely, M., Leslie, E., Rejali, N. A., Noriega, R. and Bass, B. L. (2021). Transient kinetic studies of the antiviral Drosophila Dicer-2 reveal roles of ATP in self-nonself discrimination. Elife 10. PubMed ID: 33787495
Some RIG-I-like receptors (RLRs) discriminate viral and cellular dsRNA by their termini, and Drosophila melanogaster Dicer-2 (dmDcr-2) differentially processes dsRNA with blunt or 2 nucleotide 3'-overhanging termini. This study investigated the transient kinetic mechanism of the dmDcr-2 reaction using a rapid reaction stopped-flow technique and time-resolved fluorescence spectroscopy. Indeed, it was found that ATP binding to dmDcr-2's helicase domain impacts association and dissociation kinetics of dsRNA in a termini-dependent manner, revealing termini-dependent discrimination of dsRNA on a biologically relevant time scale (seconds). ATP hydrolysis promotes transient unwinding of dsRNA termini followed by slow rewinding, and directional translocation of the enzyme to the cleavage site. Time-resolved fluorescence anisotropy reveals a nucleotide-dependent modulation in conformational fluctuations (nanoseconds) of the helicase and Platform-PAZ domains that is correlated with termini-dependent dsRNA cleavage. This study offers a kinetic framework for comparison to other Dicers, as well as all members of the RLRs involved in innate immunity.

Friday, July 9th - Behavior

Leitch, K. J., Ponce, F. V., Dickson, W. B., van Breugel, F. and Dickinson, M. H. (2021). The long-distance flight behavior of Drosophila supports an agent-based model for wind-assisted dispersal in insects. Proc Natl Acad Sci U S A 118(17). PubMed ID: 33879607
Despite the ecological importance of long-distance dispersal in insects, its mechanistic basis is poorly understood in genetic model species, in which advanced molecular tools are readily available. One critical question is how insects interact with the wind to detect attractive odor plumes and increase their travel distance as they disperse. To gain insight into dispersal, release-and-recapture experiments were conducted in the Mojave Desert using the fruit fly, Drosophila melanogaster. Chemically baited traps were deployed in a 1 km radius ring around the release site, equipped with cameras that captured the arrival times of flies as they landed. In each experiment, between 30,000 and 200,000 flies were released. By repeating the experiments under a variety of conditions, it was possible to quantify the influence of wind on flies' dispersal behavior. The results confirm that even tiny fruit flies could disperse ∼12 km in a single flight in still air and might travel many times that distance in a moderate wind. The dispersal behavior of the flies is well explained by an agent-based model in which animals maintain a fixed body orientation relative to celestial cues, actively regulate groundspeed along their body axis, and allow the wind to advect them sideways. The model accounts for the observation that flies actively fan out in all directions in still air but are increasingly advected downwind as winds intensify. The results suggest that dispersing insects may strike a balance between the need to cover large distances while still maintaining the chance of intercepting odor plumes from upwind sources.
Ruf, F., Mitesser, O., Mungwa, S. T., Horn, M., Rieger, D., Hovestadt, T. and Wegener, C. (2021). Natural Zeitgebers Under Temperate Conditions Cannot Compensate for the Loss of a Functional Circadian Clock in Timing of a Vital Behavior in Drosophila. J Biol Rhythms 36(3): 271-285. PubMed ID: 33745356
The adaptive significance of adjusting behavioral activities to the right time of the day seems obvious. Laboratory studies implicated an important role of circadian clocks in behavioral timing and rhythmicity. Yet, recent studies on clock-mutant animals questioned this importance under more naturalistic settings, as various clock mutants showed nearly normal diel activity rhythms under seminatural zeitgeber conditions. This study reports evidence that proper timing of eclosion, a vital behavior of the fruit fly Drosophila melanogaster, requires a functional molecular clock under quasi-natural conditions. In contrast to wild-type flies, period(01) mutants with a defective molecular clock showed impaired rhythmicity and gating in a temperate environment even in the presence of a full complement of abiotic zeitgebers. Although period(01) mutants still eclosed during a certain time window during the day, this time window was much broader and loosely defined, and rhythmicity was lower or lost as classified by various statistical measures. Moreover, peak eclosion time became more susceptible to variable day-to-day changes of light. In contrast, flies with impaired peptidergic interclock signaling (Pdf(01) and han(5304) PDF receptor mutants) eclosed mostly rhythmically with normal gate sizes, similar to wild-type controls. The results suggest that the presence of natural zeitgebers is not sufficient, and a functional molecular clock is required to induce stable temporal eclosion patterns in flies under temperate conditions with considerable day-to-day variation in light intensity and temperature. Temperate zeitgebers are, however, sufficient to functionally rescue a loss of PDF-mediated clock-internal and -output signaling.
Tanizawa, F. and Takemoto, H. (2021). Sleep contributes to preference for novel food odours in Drosophila melanogaster. Sci Rep 11(1): 9395. PubMed ID: 33931708
The importance of sleep in maintaining cognitive functions such as learning and memory has been reported in both vertebrates and invertebrates. Previous studies demonstrated that sleep deprivation impaired the olfactory memory retention of fruit flies as described in the classical conditioning paradigm. This study shows that sleep deprivation leads to a preference for the odours of the rearing environment in Drosophila melanogaster. Flies whose sleep had been disturbed with periodic rotation stimuli during night-time preferred apple cider vinegar (ACV) to broth, while this preference was lower in flies without sleep deprivation and those rotated during daytime. Experiments using single odours showed an increase in responses to ACV due to sleep deprivation. These results suggest that sleep functions in food odour preference. Flies grown on medium supplemented with ACV showed greater preference for ACV, and those grown with broth supplementation showed a greater preference for broth under sleep-deprived conditions. These results suggest that flies with night-time sleep deprivation become attached to the environment on which they have developed, and that sleep contributes to preference for novel food odours. This study offers an approach to investigating the interaction between sleep and neural disorders concerning cognitive deficits towards novel stimuli.
Ryvkin, J., Bentzur, A., Shmueli, A., Tannenbaum, M., Shallom, O., Dokarker, S., Benichou, J. I. C., Levi, M. and Shohat-Ophir, G. (2021). Transcriptome Analysis of NPFR Neurons Reveals a Connection Between Proteome Diversity and Social Behavior. Front Behav Neurosci 15: 628662. PubMed ID: 33867948
Social behaviors are mediated by the activity of highly complex neuronal networks, the function of which is shaped by their transcriptomic and proteomic content. Contemporary advances in neurogenetics, genomics, and tools for automated behavior analysis make it possible to functionally connect the transcriptome profile of candidate neurons to their role in regulating behavior. This study used Drosophila melanogaster to explore the molecular signature of neurons expressing receptor for neuropeptide F (NPF), the fly homolog of neuropeptide Y (NPY). By comparing the transcription profile of NPFR neurons to those of nine other populations of neurons, this study discovered that NPFR neurons exhibit a unique transcriptome, enriched with receptors for various neuropeptides and neuromodulators, as well as with genes known to regulate behavioral processes, such as learning and memory. By manipulating RNA editing and protein ubiquitination programs specifically in NPFR neurons, this study demonstrated that the proper expression of their unique transcriptome and proteome is required to suppress male courtship and certain features of social group interaction. The results highlight the importance of transcriptome and proteome diversity in the regulation of complex behaviors and pave the path for future dissection of the spatiotemporal regulation of genes within highly complex tissues, such as the brain.
Krams, I. A., Krama, T., Krams, R., Trakimas, G., Popovs, S., Joers, P., Munkevics, M., Elferts, D., Rantala, M. J., Makņa, J. and de Bivort, B. L. (2021). Serotoninergic Modulation of Phototactic Variability Underpins a Bet-Hedging Strategy in Drosophila melanogaster. Front Behav Neurosci 15: 659331. PubMed ID: 33935664
When organisms' environmental conditions vary unpredictably in time, it can be advantageous for individuals to hedge their phenotypic bets. It has been shown that a bet-hedging strategy possibly underlies the high inter-individual diversity of phototactic choice in Drosophila melanogaster. This study shows that fruit flies from a population living in a boreal and relatively unpredictable climate have more variable variable phototactic biases than fruit flies from a more stable tropical climate, consistent with bet-hedging theory. This study experimentally showed that phototactic variability of D. melanogaster is regulated by the neurotransmitter serotonin (5-HT), which acts as a suppressor of the variability of phototactic choices. When fed 5-HT precursor, boreal flies exhibited lower variability, and they were insensitive to 5-HT inhibitor. The opposite pattern was seen in the tropical flies. Thus, the reduction of 5-HT in fruit flies' brains may be the mechanistic basis of an adaptive bet-hedging strategy in a less predictable boreal climate.
Mahishi, D., Triphan, T., Hesse, R. and Huetteroth, W. (2021). The Panopticon-Assessing the Effect of Starvation on Prolonged Fly Activity and Place Preference. Front Behav Neurosci 15: 640146. PubMed ID: 33841109
Animal behaviours are demonstrably governed by sensory stimulation, previous experience and internal states like hunger. With increasing hunger, priorities shift towards foraging and feeding. During foraging, flies are known to employ efficient path integration strategies. However, general long-term activity patterns for both hungry and satiated flies in conditions of foraging remain to be better understood. Similarly, little is known about how permanent contact chemosensory stimulation affects locomotion. To address these questions, a novel, simplistic fly activity tracking setup, the Panopticon, was developed. Using a 3D-printed Petri dish inset, this assay allows recording of walking behaviour, of several flies in parallel, with all arena surfaces covered by a uniform substrate layer. Two constellations of providing food were tested: (i) in single patches and (ii) omnipresent within the substrate layer. Fly tracking is done with FIJI, further assessment, analysis and presentation is done with a custom-built MATLAB analysis framework. This study found that starvation history leads to a long-lasting reduction in locomotion, as well as a delayed place preference for food patches which seems to be not driven by immediate hunger motivation.

Thursday, July 8th - Adult Physiology

Santiago, J. C., Boylan, J. M., Lemieux, F. A., Gruppuso, P. A., Sanders, J. A. and Rand, D. M. (2021). Mitochondrial genotype alters the impact of rapamycin on the transcriptional response to nutrients in Drosophila. BMC Genomics 22(1): 213. PubMed ID: 33761878
In addition to their well characterized role in cellular energy production, new evidence has revealed the involvement of mitochondria in diverse signaling pathways that regulate a broad array of cellular functions. The mitochondrial genome (mtDNA) encodes essential components of the oxidative phosphorylation (OXPHOS) pathway whose expression must be coordinated with the components transcribed from the nuclear genome. Mitochondrial dysfunction is associated with disorders including cancer and neurodegenerative diseases, yet the role of the complex interactions between the mitochondrial and nuclear genomes are poorly understood. Using a Drosophila model in which alternative mtDNAs are present on a common nuclear background, the effects of this altered mitonuclear communication on the transcriptomic response to altered nutrient status were studied. Adult flies with the 'native' and 'disrupted' genotypes were re-fed following brief starvation, with or without exposure to rapamycin, the cognate inhibitor of the nutrient-sensing target of rapamycin (TOR). RNAseq showed that alternative mtDNA genotypes affect the temporal transcriptional response to nutrients in a rapamycin-dependent manner. Pathways most greatly affected were OXPHOS, protein metabolism and fatty acid metabolism. A distinct set of testis-specific genes was also differentially regulated in the experiment. It is concluded that any of the differentially expressed genes between alternative mitonuclear genotypes have no direct interaction with mtDNA gene products, suggesting that the mtDNA genotype contributes to retrograde signaling from mitochondria to the nucleus. The interaction of mitochondrial genotype (mtDNA) with rapamycin treatment identifies new links between mitochondria and the nutrient-sensing mTORC1 (mechanistic target of rapamycin complex 1) signaling pathway.
Shin, G. J., Pero, M. E., Hammond, L. A., Burgos, A., Kumar, A., Galindo, S. E., Lucas, T., Bartolini, F. and Grueber, W. B. (2021). Integrins protect sensory neurons in models of paclitaxel-induced peripheral sensory neuropathy. Proc Natl Acad Sci U S A 118(15). PubMed ID: 33876743
Chemotherapy-induced peripheral neuropathy (CIPN) is a major side effect from cancer treatment with no known method for prevention or cure in clinics. CIPN often affects unmyelinated nociceptive sensory terminals. Despite the high prevalence, molecular and cellular mechanisms that lead to CIPN are still poorly understood. In this study a genetically tractable Drosophila model and primary sensory neurons isolated from adult mouse to examine the mechanisms underlying CIPN and identify protective pathways. Chronic treatment of Drosophila larvae with paclitaxel caused degeneration and altered the branching pattern of nociceptive neurons, and reduced thermal nociceptive responses. It was further found that nociceptive neuron-specific overexpression of integrins (see Drosophila Myospheroid), which are known to support neuronal maintenance in several systems, conferred protection from paclitaxel-induced cellular and behavioral phenotypes. Live imaging and superresolution approaches provide evidence that paclitaxel treatment causes cellular changes that are consistent with alterations in endosome-mediated trafficking of integrins. Paclitaxel-induced changes in recycling endosomes precede morphological degeneration of nociceptive neuron arbors, which could be prevented by integrin overexpression. Primary dorsal root ganglia (DRG) neuron cultures to test conservation of integrin-mediated protection. Transduction of a human integrin β-subunit 1 also prevented degeneration following paclitaxel treatment. Furthermore, endogenous levels of surface integrins were decreased in paclitaxel-treated mouse DRG neurons, suggesting that paclitaxel disrupts recycling in vertebrate sensory neurons. Altogether, this study supports conserved mechanisms of paclitaxel-induced perturbation of integrin trafficking and a therapeutic potential of restoring neuronal interactions with the extracellular environment to antagonize paclitaxel-induced toxicity in sensory neurons.
Semaniuk, U., Gospodaryov, D., Mishchanyn, K., Storey, K. and Lushchak, O. (2021). Drosophila insulin-like peptides regulate concentration-dependent changes of appetite to different carbohydrates. Zoology (Jena) 146: 125927. PubMed ID: 33894679
The volumes of sugar solutions ingested and amounts of different carbohydrates eaten were measured in fruit fly lines with mutated genes for Drosophila insulin-like peptides (DILPs). The wild type w(1118) flies consumed 20-40 μg of fructose or glucose per day regardless of carbohydrate concentration. This relatively constant amount of consumed carbohydrate was regulated due to satiety-driven decreases in the ingested volume of sugar solution, a so-called "compensatory feeding" strategy. This decrease was not observed for flies fed sucrose solutions. The dilp3 mutant and quadruple mutant dilp1-4 showed no "compensatory feeding" when fed glucose but these two mutants consumed larger amounts of sucrose than the wild type from solutions with carbohydrate concentrations equal to or higher than 4%. Flies with mutations of dilp2, dilp3, dilp4, dilp5, and dilp6 genes consumed larger amounts of carbohydrate from 4-10% sucrose solutions as compared to the wild type. Mutations of DILPs affected appetite mainly for sucrose and glucose, but the least for fructose. The presented data confirm the hypothesis that DILPs are involved in the regulation of fly appetite in response to type and concentration of carbohydrate.
Shah, E. J., Huttemann, M., Sanderson, T. H., Gurdziel, K. and Ruden, D. M. (2021). Inhibiting Mitochondrial Cytochrome c Oxidase Downregulates Gene Transcription After Traumatic Brain Injury in Drosophila. Front Physiol 12: 628777. PubMed ID: 33790803
Traumatic brain injuries (TBIs) caused by a sudden impact to the head alter behavior and impair physical and cognitive function. Besides the severity, type and area of the brain affected, the outcome of TBI is also influenced by the patient's biological sex. Previous studies reporting mitochondrial dysfunction mainly focused on exponential reactive oxygen species (ROS) generation, increased mitochondrial membrane potential, and altered mitochondrial dynamics as a key player in the outcome to brain injury. This study evaluated the effect of a near-infrared (NIR) light exposure on gene expression in a Drosophila TBI model. NIR interacts with cytochrome c oxidase (COX) of the electron transport chain to reduce mitochondrial membrane potential hyperpolarization, attenuate ROS generation, and apoptosis. W (1118) male and female flies were sugjected to TBI using a high-impact trauma (HIT) device and subsequently exposed the isolated fly brains to a COX-inhibitory wavelength of 750 nm for 2 hours (hr). Genome-wide 3'-mRNA-sequencing of fly brains revealed that injured w (1118) females exhibit greater changes in transcription compared to males at 1, 2, and 4 hours (hr) after TBI. Inhibiting COX by exposure to NIR downregulates gene expression in injured females but has minimal effect in injured males. These results suggest that mitochondrial COX modulation with NIR alters gene expression in Drosophila following TBI and the response to injury and NIR exposure varies by biological sex.
Simoes, J. M., Levy, J. I., Zaharieva, E. E., Vinson, L. T., Zhao, P., Alpert, M. H., Kath, W. L., Para, A. and Gallio, M. (2021). Robustness and plasticity in Drosophila heat avoidance. Nat Commun 12(1): 2044. PubMed ID: 33824330
Simple innate behavior is often described as hard-wired and largely inflexible. This study shows that the avoidance of hot temperature, a simple innate behavior, contains unexpected plasticity in Drosophila. First, it was demonstrate that hot receptor neurons of the antenna and their molecular heat sensor, Gr28B.d, are essential for flies to produce escape turns away from heat. High-resolution fly tracking combined with a 3D simulation of the thermal environment shows that, in steep thermal gradients, the direction of escape turns is determined by minute temperature differences between the antennae (0.1°-1 °C). In parallel, live calcium imaging confirms that such small stimuli reliably activate both peripheral thermosensory neurons and central circuits. Next, based on these measurements, a fly/vehicle model with two symmetrical sensors and motors (a "Braitenberg vehicle") was evolved which closely approximates basic fly thermotaxis. Critical differences between real flies and the hard-wired vehicle reveal that fly heat avoidance involves decision-making, relies on rapid learning, and is robust to new conditions, features generally associated with more complex behavior.
Tamamouna, V., Rahman, M. M., Petersson, M., Charalambous, I., Kux, K., Mainor, H., Bolender, V., Isbilir, B., Edgar, B. A. and Pitsouli, C. (2021). Remodelling of oxygen-transporting tracheoles drives intestinal regeneration and tumorigenesis in Drosophila. Nat Cell Biol 23(5): 497-510. PubMed ID: 33972730
The Drosophila trachea, as the functional equivalent of mammalian blood vessels, senses hypoxia and oxygenates the body. This study shows that the adult intestinal tracheae are dynamic and respond to enteric infection, oxidative agents and tumours with increased terminal branching. Increased tracheation is necessary for efficient damage-induced intestinal stem cell (ISC)-mediated regeneration and is sufficient to drive ISC proliferation in undamaged intestines. Gut damage or tumours induce HIF-1α (Sima in Drosophila), which stimulates tracheole branching via the FGF (Branchless (Bnl))-FGFR (Breathless (Btl)) signalling cascade. Bnl-Btl signalling is required in the intestinal epithelium and the trachea for efficient damage-induced tracheal remodelling and ISC proliferation. Chemical or Pseudomonas-generated reactive oxygen species directly affect the trachea and are necessary for branching and intestinal regeneration. Similarly, tracheole branching and the resulting increase in oxygenation are essential for intestinal tumour growth. This study has identified a mechanism of tracheal-intestinal tissue communication, whereby damage and tumours induce neo-tracheogenesis in Drosophila, a process reminiscent of cancer-induced neoangiogenesis in mammals.

Wednesday, July 7th - Adult Physiology

Zhou, H., Whitworth, C., Pozmanter, C., Neville, M. C. and Van Doren, M. (2021). Doublesex regulates fruitless expression to promote sexual dimorphism of the gonad stem cell niche. PLoS Genet 17(3): e1009468. PubMed ID: 33788836
Doublesex (Dsx) and Fruitless (Fru) are the two downstream transcription factors that actuate Drosophila sex determination. While Dsx assists Fru to regulate sex-specific behavior, whether Fru collaborates with Dsx in regulating other aspects of sexual dimorphism remains unknown. One important aspect of sexual dimorphism is found in the gonad stem cell (GSC) niches, where male and female GSCs are regulated to create large numbers of sperm and eggs. This study reports that Fru is expressed male-specifically in the GSC niche and plays important roles in the development and maintenance of these cells. Unlike previously-studied aspects of sex-specific Fru expression, which are regulated by Transformer (Tra)-mediated alternative splicing, this study shows that male-specific expression of fru in the gonad is regulated downstream of dsx, and is independent of tra. fru genetically interacts with dsx to support maintenance of the niche throughout development. Ectopic expression of fru inhibited female niche formation and partially masculinized the ovary. fru is also required autonomously for cyst stem cell maintenance and cyst cell survival. Finally, this study identified a conserved Dsx binding site upstream of fru promoter P4 that regulates fru expression in the niche, indicating that fru is likely a direct target for transcriptional regulation by Dsx. These findings demonstrate that fru acts outside the nervous system to influence sexual dimorphism and reveal a new mechanism for regulating sex-specific expression of fru that is regulated at the transcriptional level by Dsx, rather than by alternative splicing by Tra.
Sadanandappa, M. K., Sathyanarayana, S. H., Kondo, S. and Bosco, G. (2021). Neuropeptide F signaling regulates parasitoid-specific germline development and egg-laying in Drosophila. PLoS Genet 17(3): e1009456. PubMed ID: 33770070
Drosophila larvae and pupae are at high risk of parasitoid infection in nature. To circumvent parasitic stress, fruit flies have developed various survival strategies, including cellular and behavioral defenses. This study shows that adult Drosophila females exposed to the parasitic wasps, Leptopilina boulardi, decrease their total egg-lay by deploying at least two strategies: Retention of fully developed follicles reduces the number of eggs laid, while induction of caspase-mediated apoptosis eliminates the vitellogenic follicles. These reproductive defense strategies require both visual and olfactory cues, but not the MB247-positive mushroom body neuronal function, suggesting a novel mode of sensory integration mediates reduced egg-laying in the presence of a parasitoid. It was further shown that neuropeptide F (NPF) signaling is necessary for both retaining matured follicles and activating apoptosis in vitellogenic follicles. Whereas previous studies have found that gut-derived NPF controls germ stem cell proliferation, this study shows that sensory-induced changes in germ cell development specifically require brain-derived NPF signaling, which recruits a subset of NPFR-expressing cell-types that control follicle development and retention. Importantly, it was found that reduced egg-lay behavior is specific to parasitic wasps that infect the developing Drosophila larvae, but not the pupae. These findings demonstrate that female fruit flies use multimodal sensory integration and neuroendocrine signaling via NPF to engage in parasite-specific cellular and behavioral survival strategies.
Stark, K., Crowe, O. and Lewellyn, L. (2021). Precise levels of the Drosophila adaptor protein Dreadlocks maintain the size and stability of germline ring canals. J Cell Sci 134(8). PubMed ID: 33912915
Intercellular bridges are essential for fertility in many organisms. The developing fruit fly egg has become the premier model system to study intercellular bridges. During oogenesis, the oocyte is connected to supporting nurse cells by relatively large intercellular bridges, or ring canals. Once formed, the ring canals undergo a 20-fold increase in diameter to support the movement of materials from the nurse cells to the oocyte. This study demonstrates a novel role for the conserved SH2/SH3 adaptor protein Dreadlocks (Dock) in regulating ring canal size and structural stability in the germline. Dock localizes at germline ring canals throughout oogenesis. Loss of Dock leads to a significant reduction in ring canal diameter, and overexpression of Dock causes dramatic defects in ring canal structure and nurse cell multinucleation. The SH2 domain of Dock is required for ring canal localization downstream of Src64 (also known as Src64B), and the function of one or more of the SH3 domains is necessary for the strong overexpression phenotype. Genetic interaction and localization studies suggest that Dock promotes WASp-mediated Arp2/3 activation in order to determine ring canal size and regulate growth.
McDonough-Goldstein, C. E., Borziak, K., Pitnick, S. and Dorus, S. (2021). Drosophila female reproductive tract gene expression reveals coordinated mating responses and rapidly evolving tissue-specific genes. G3 (Bethesda) 11(3). PubMed ID: 33890615
Sexual reproduction in internally fertilizing species requires complex coordination between female and male reproductive systems and among the diverse tissues of the female reproductive tract (FRT). This study reports a comprehensive, tissue-specific investigation of Drosophila melanogaster FRT gene expression before and after mating. Expression profiles were identified that distinguished each tissue, including major differences between tissues with glandular or primarily nonglandular epithelium. All tissues were enriched for distinct sets of genes possessing secretion signals that exhibited accelerated evolution, as might be expected for genes participating in molecular interactions between the sexes within the FRT extracellular environment. Despite robust transcriptional differences between tissues, postmating responses were dominated by coordinated transient changes indicative of an integrated systems-level functional response. This comprehensive characterization of gene expression throughout the FRT identifies putative female contributions to postcopulatory events critical to reproduction and potentially reproductive isolation, as well as the putative targets of sexual selection and conflict.
Jianbo, W., Xin, L., Zhiyang, G., Lin, P., Xian, L., Xiahe, H., Yingchun, W. and Zhaohui, W. (2021). RNA Kinase CLP1/Cbc Regulates Meiosis Initiation in Spermatogenesis. Hum Mol Genet. PubMed ID: 33864361
CLP1, TSEN complex, and VCP are evolutionarily conserved proteins whose mutations are associated with neurodegenerative diseases. This study found that they are also involved in germline differentiation. To optimize both quantity and quality in gametes production, germ cells expand themselves through limited mitotic cycles prior to meiosis. Stemming from previous findings on the correlation between mRNA 3'-processing and meiosis entry, this study identified that the RNA kinase Cbc, the Drosophila member of the highly conserved CLP1 family, is a component of the program regulating the transition from mitosis to meiosis. Using genetic manipulations in Drosophila testis, this study demonstrates that nuclear Cbc is required to promote meiosis entry. Combining biochemical and genetic methods, it is revealed that Cbc physically and/or genetically intersects with Tsen54 and TER94 (VCP ortholog) in this process. The C-terminal half of Tsen54 is both necessary and sufficient for its binding with Cbc. Further, this study illustrates the functional conservation between Cbc and mammalian CLP1 in the assays of subcellular localization and Drosophila fertility. As CLP1, TSEN complex, and VCP have also been identified in neurodegenerations of animal models, a mechanism involving these factors seems to be shared in gametogenesis and neurogenesis.
Li, Y. R., Lai, H. W., Huang, H. H., Chen, H. C., Fugmann, S. D. and Yang, S. Y. (2021). Trajectory mapping of the early Drosophila germline reveals controls of zygotic activation and sex differentiation. Genome Res. PubMed ID: 33858841
Germ cells in Drosophila melanogaster are specified maternally shortly after fertilization and are transcriptionally quiescent until their zygotic genome is activated to sustain further development. To understand the molecular basis of this process, this study analyzed the progressing transcriptomes of early male and female germ cells at the single-cell level between germline specification and coalescence with somatic gonadal cells. The data comprehensively covered zygotic activation in the germline genome, and analyses on genes that exhibit germline-restricted expression revealed that polymerase pausing and differential RNA stability are important mechanisms that establish gene expression differences between the germline and soma. In addition, an immediate bifurcation was observed between the male and female germ cells as zygotic transcription begins. The main difference between the two sexes is an elevation in X Chromosome expression in females relative to males signifying incomplete dosage compensation with a few select genes exhibiting even higher expression increases. These indicate that the male program is the default mode in the germline that is driven to female development with a second X Chromosome.
Row, S., Huang, Y. C. and Deng, W. M. (2021). Developmental regulation of oocyte lipid intake through 'patent' follicular epithelium in Drosophila melanogaster. iScience 24(4): 102275. PubMed ID: 33817579
Epithelia form protective permeability barriers that selectively allow the exchange of material while maintaining tissue integrity under extreme mechanical, chemical, and bacterial loads. This study in the Drosophila follicular epithelium reports a developmentally regulated and evolutionarily conserved process "patency", wherein a breach is created in the epithelium at tricellular contacts during mid-vitellogenesis. In Drosophila, patency exhibits a strict temporal range potentially delimited by the transcription factor Tramtrack69 and a spatial pattern influenced by the dorsal-anterior signals of the follicular epithelium. Crucial for growth and lipid uptake by the oocyte, patency is also exploited by endosymbionts such as Spiroplasma pulsonii. These findings reveal an evolutionarily conserved and developmentally regulated non-typical epithelial function in a classic model system.
Tanaka, T., Tani, N. and Nakamura, A. (2021). Receptor-mediated yolk uptake is required for oskar mRNA localization and cortical anchorage of germ plasm components in the Drosophila oocyte. PLoS Biol 19(4): e3001183. PubMed ID: 33891588
The Drosophila germ plasm is responsible for germ cell formation. Its assembly begins with localization of oskar mRNA to the posterior pole of the oocyte. The oskar translation produces 2 isoforms with distinct functions: short Oskar recruits germ plasm components, whereas long Oskar remodels actin to anchor the components to the cortex. The mechanism by which long Oskar anchors them remains elusive. This study reports that Yolkless, which facilitates uptake of nutrient yolk proteins into the oocyte, is a key cofactor for long Oskar. Loss of Yolkless or depletion of yolk proteins disrupts the microtubule alignment and oskar mRNA localization at the posterior pole of the oocyte, whereas microtubule-dependent localization of bicoid mRNA to the anterior and gurken mRNA to the anterior-dorsal corner remains intact. Furthermore, these mutant oocytes do not properly respond to long Oskar, causing defects in the actin remodeling and germ plasm anchoring. Thus, the yolk uptake is not merely the process for nutrient incorporation, but also crucial for oskar mRNA localization and cortical anchorage of germ plasm components in the oocyte.

Tuesday, July 6th - Disease Models

Nisha and Sarkar, S. (2021). Downregulation of glob1 suppresses pathogenesis of human neuronal tauopathies in Drosophila by regulating tau phosphorylation and ROS generation. Neurochem Int 146: 105040. PubMed ID: 33865914
Human tauopathies represent a group of neurodegenerative disorders, characterized by abnormal hyperphosphorylation and aggregation of tau protein, which ultimately cause neurodegeneration. The aberrant tau hyperphosphorylation is mostly attributed to the kinases/phosphatases imbalance, which is majorly contributed by the generation of reactive oxygen species (ROS). Globin(s) represent a well-conserved group of proteins which are involved in O(2) management, regulation of cellular ROS in different cell types. Similarly, Drosophila globin1 (a homologue of human globin) with its known roles in oxygen management and development of nervous system exhibits striking similarities with the mammalian neuroglobin. Several recent evidences support the hypothesis that neuroglobins are associated with Alzheimer's disease pathogenesis. It is noted that targeted expression of human-tau induces the cellular level of Glob1 protein in Drosophila tauopathy models. Subsequently, RNAi mediated restored level of Glob1 restricts the pathogenic effect of human-tau by minimizing its hyperphosphorylation via GSK-3β/p-Akt and p-JNK pathways. In addition, it also activates the Nrf2-keap1-ARE cascade to stabilize the tau-mediated increased level of ROS. These two parallel cellular events provide a significant rescue against human tau-mediated neurotoxicity in the fly models. This study reports a direct involvement of an oxygen sensing globin gene in tau etiology. In view of the fact that human genome encodes for the multiple Globin proteins including a nervous system specific neuroglobin; and therefore, these findings may pave the way to investigate if the conserved oxygen sensing globin gene(s) can be exploited in devising novel therapeutic strategies against tauopathies.
Niccoli, T., Kerr, F., Snoeren, I., Fabian, D., Aleyakpo, B., Ivanov, D., Sofola-Adesakin, O., Cryar, A., Adcott, J., Thornton, J. and Partridge, L. (2021). Activating transcription factor 4-dependent lactate dehydrogenase activation as a protective response to amyloid beta toxicity. Brain Commun 3(2): fcab053. PubMed ID: 33977265
Accumulation of amyloid beta peptides is thought to initiate the pathogenesis of Alzheimer's disease. Microarray analyses have shown that, in Drosophila models of amyloid beta 42 toxicity, genes involved in the unfolded protein response and metabolic processes are upregulated in brain. Comparison with the brain transcriptome of early-stage Alzheimer's patients revealed a common transcriptional signature, but with generally opposing directions of gene expression changes between flies and humans. Among these differentially regulated genes, lactate dehydrogenase (Ldh) was up-regulated by the greatest degree in amyloid beta 42 flies and the human orthologues (LDHA and LDHB) were down-regulated in patients. Functional analyses revealed that either over-expression or inhibition of Ldh by RNA interference (RNAi) slightly exacerbated climbing defects in both healthy and amyloid beta 42-induced Drosophila. This suggests that metabolic responses to lactate dehydrogenase must be finely-tuned, and that its observed upregulation following amyloid beta 42 production could potentially represent a compensatory protection to maintain pathway homeostasis in this model, with further manipulation leading to detrimental effects. The increased Ldh expression in amyloid beta 42 flies was regulated partially by unfolded protein response signalling, as ATF4 RNAi diminished the transcriptional response and enhanced amyloid beta 42-induced climbing phenotypes. This study thus reveals dysregulation of lactate dehydrogenase signalling in Drosophila models and patients with Alzheimer's disease, which may lead to a detrimental loss of metabolic homeostasis. Importantly, it was observed that down-regulation of ATF4-dependent endoplasmic reticulum-stress signalling in this context appears to prevent Ldh compensation and to exacerbate amyloid beta 42-dependent neuronal toxicity.
Singh, A. and Agrawal, N. (2021). Deciphering the key mechanisms leading to alteration of lipid metabolism in Drosophila model of Huntington's disease. Biochim Biophys Acta Mol Basis Dis 1867(7): 166127. PubMed ID: 33722743
Huntington's disease (HD) is an inherited, progressively debilitating disorder marked by prominent degeneration in striatal and cortical brain regions. HD is caused by (CAG)(n) repeat expansion in huntingtin (HTT) gene that translates into a mutant form of the ubiquitously present Huntingtin (HTT) protein. Extensive metabolic dysfunction coexisting with overt neuropathies has been evidenced in clinical and experimental settings of HD. Body weight loss despite normal to high caloric intake remains a critical determinant of the disease progression and a challenge for therapeutic interventions. This study, monitored the cellular and molecular perturbations in Drosophila, caused by pan-neuronal expression of mHTT (mutant Huntingtin) protein. Aberrant transcription profile of key lipolytic and lipogenic genes was found in whole-body of the fly with disease progression. Interestingly, fat body undergoes extensive alteration of vital cellular processes and eventually surrenders to increased apoptotic cell death in terminal stage of the disease. Extensive mitochondrial dysfunction from early disease stage along with calcium derangement at terminal stage were observed in fat body, which contribute to its deteriorating integrity. All the mechanisms were monitored progressively, at different disease stages, and many alterations were documented in the early stage itself. This study hence provides insight into the mechanisms through which neuronal expression of mHTT might be inflicting the profound systemic effects, specifically on lipid metabolism, and may open new therapeutic avenues for alleviation of the multidimensional disease.
Pallos, J., Jeng, S., McWeeney, S. and Martin, I. (2021). Dopamine neuron-specific LRRK2 G2019S effects on gene expression revealed by translatome profiling. Neurobiol Dis: 105390. PubMed ID: 33984508
Leucine-rich repeat kinase 2 (LRRK2) mutations are the most common genetic cause of late-onset autosomal dominant Parkinson's disease. The pathogenic G2019S mutation enhances LRRK2 kinase activity and induces neurodegeneration in C. elegans, Drosophila and rodent models through unclear mechanisms. Gene expression profiling has the potential to provide detailed insight into the biological pathways modulated by LRRK2 kinase activity in vivo. Prior studies have surveyed the effects of LRRK2 G2019S on genome-wide mRNA expression in complex brain tissues with high cellular heterogeneity, limiting their power to detect more restricted gene expression changes occurring in a cell type-specific manner. This study used translating ribosome affinity purification (TRAP) coupled to RNA-seq to profile dopamine neuron-specific gene expression changes caused by LRRK2 G2019S in the Drosophila CNS. A modest number of genes were differentially expressed in the presence of mutant LRRK2 that represent a broad range of molecular functions including DNA repair (RfC3), mRNA metabolism and translation (Ddx1 and lin-28), calcium homeostasis (MCU), and other categories (Ugt37c1, disp, l(1)G0196, CG6602, CG1126 and CG11068). Further analysis on a subset of these genes revealed that LRRK2 G2019S did not alter their expression across the whole brain, consistent with dopamine neuron-specific effects uncovered by the TRAP approach that may offer insight into the neurodegenerative process. This is the first study to profile the effects of LRRK2 G2019S on DA neuron gene expression in vivo. Beyond providing a set of differentially expressed gene candidates relevant to LRRK2, this study demonstrates the effective use of TRAP to perform high-resolution assessment of dopamine neuron gene expression for the study of PD.
Onur, T. S., Laitman, A., Zhao, H., Keyho, R., Kim, H., Wang, J., Mair, M., Wang, H., Li, L., Perez, A., de Haro, M., Wan, Y. W., Allen, G., Lu, B., Al-Ramahi, I., Liu, Z. and Botas, J. (2021). Downregulation of glial genes involved in synaptic function mitigates Huntington's Disease pathogenesis. Elife 10. PubMed ID: 33871358
Most research on neurodegenerative diseases has focused on neurons, yet glia help form and maintain the synapses whose loss is so prominent in these conditions. To investigate the contributions of glia to Huntington's disease (HD), this study profiled the gene expression alterations of Drosophila expressing human mutant Huntingtin (mHTT) in either glia or neurons and compared these changes to what is observed in HD human and HD mice striata. A large portion of conserved genes are concordantly dysregulated across the three species; these genes were tested in a high-throughput behavioral assay, and it was found that downregulation of genes involved in synapse assembly mitigated pathogenesis and behavioral deficits. Surprisingly, reducing dNRXN3 (also known as nrx-1) function in glia was sufficient to improve the phenotype of flies expressing mHTT in neurons, suggesting that mHTT's toxic effects in glia ramify throughout the brain. This supports a model in which dampening synaptic function is protective because it attenuates the excitotoxicity that characterizes HD.
Nangia, V., O'Connell, J., Chopra, K., Qing, Y., Reppert, C., Chai, C. M., Bhasiin, K. and Colodner, K. J. (2021). Genetic reduction of tyramine beta hydroxylase suppresses Tau toxicity in a Drosophila model of tauopathy. Neurosci Lett 755: 135937. PubMed ID: 33910059
Tauopathies are a class of neurodegenerative diseases characterized by the abnormal phosphorylation and accumulation of the microtubule-associated protein, Tau. These diseases are associated with degeneration and dysfunction of the noradrenergic system, a critical regulator of memory, locomotion, and the fight or flight response. Though Tau pathology accumulates early in noradrenergic neurons, the relationship between noradrenaline signaling and tauopathy pathogenesis remains unclear. The fruit fly, Drosophila melanogaster, is a valuable model organism commonly used to investigate factors that promote Tau-mediated degeneration. Moreover, Drosophila contain the biogenic amine, octopamine, which is the functional homolog to noradrenaline. Using a Drosophila model of tauopathy, a candidate modifier screen was conducted targeting tyramine β hydroxylase (tβh), the enzyme that controls the production of octopamine in the fly, to determine if levels of this enzyme modulate Tau-induced degeneration in the fly eye. Genetic reduction of tβh was found to suppress Tau toxicity, independent of Tau phosphorylation. These findings show that reduction of tβh, a critical enzyme in the octopaminergic pathway, suppresses Tau pathogenicity and establishes an interaction that can be further utilized to determine the relationship between noradrenergic-like signaling and Tau toxicity in Drosophila.
Suart, C. E., Perez, A. M., Al-Ramahi, I., Maiuri, T., Botas, J. and Truant, R. (2021). Spinocerebellar Ataxia Type 1 protein Ataxin-1 is signalled to DNA damage by Ataxia Telangiectasia Mutated kinase. Hum Mol Genet. PubMed ID: 33772540
Spinocerebellar Ataxia Type 1 (SCA1) is an autosomal dominant neurodegenerative disorder caused by a polyglutamine expansion in the ataxin-1 protein. Recent genetic correlational studies have implicated DNA damage repair pathways in modifying the age at onset of disease symptoms in SCA1 and Huntington's Disease, another polyglutamine expansion disease. This study demonstrates that both endogenous and transfected ataxin-1 localizes to sites of DNA damage, which is impaired by polyglutamine expansion. This response is dependent on ataxia telangiectasia mutated (ATM) kinase activity. Further, an ATM phosphorylation motif within ataxin-1 at serine 188 was characterized. Reduction of the Drosophila ATM homolog levels in a ATXN1[82Q] Drosophila model through shRNA or genetic cross ameliorates motor symptoms. These findings offer a possible explanation as to why DNA repair was implicated in SCA1 pathogenesis by past studies. The similarities between the ataxin-1 and the huntingtin responses to DNA damage provide further support for a shared pathogenic mechanism for polyglutamine expansion diseases.
Tain, L. S., Sehlke, R., Meilenbrock, R. L., Leech, T., Paulitz, J., Chokkalingam, M., Nagaraj, N., Gronke, S., Frohlich, J., Atanassov, I., Mann, M., Beyer, A. and Partridge, L. (2021). Tissue-specific modulation of gene expression in response to lowered insulin signalling in Drosophila. Elife 10. PubMed ID: 33879316
Reduced activity of the insulin/IGF signalling network increases health during ageing in multiple species. Diverse and tissue-specific mechanisms drive the health improvement. Tissue-specific transcriptional and proteomic profiling were performed of long-lived Drosophila dilp2-3,5 mutants, and tissue-specific regulation was identified of &gy;3600 transcripts and >3700 proteins. Most expression changes were regulated post-transcriptionally in the fat body, and only in mutants infected with the endosymbiotic bacteria, Wolbachia pipientis, which increases their lifespan. Bioinformatic analysis identified reduced co-translational ER targeting of secreted and membrane-associated proteins and increased DNA damage/repair response proteins. Accordingly, age-related DNA damage and genome instability were lower in fat body of the mutant, and overexpression of a minichromosome maintenance protein subunit extended lifespan. Proteins involved in carbohydrate metabolism showed altered expression in the mutant intestine, and gut-specific overexpression of a lysosomal mannosidase increased autophagy, gut homeostasis, and lifespan. These processes are candidates for combatting ageing-related decline in other organisms.

Friday, July 2nd - Adult Physiology

Manenti, T., Juul Sten, L. and Loeschcke, V. (2021). Daily increasing or decreasing photoperiod affects stress resistance and life history traits in four Drosophila species. J Insect Physiol: 104251. PubMed ID: 33971199
Photoperiod is one of the most reliable seasonal cues that organisms can use to prepare for upcoming environmental changes. Evidence suggests that exposure to different photoperiod can activate plastic responses in stress resistance traits, while there is limited evidence on the plastic response induced by daily progressive cumulative changes in photoperiod. This study assayed the effect of within generation daily uni-directional and cumulative changes in photoperiod on stress resistance and life history traits in four Drosophila species. It was predicted that daily increasing photoperiod, mimicking upcoming summer conditions, should lead to an increase in heat resistance and establish trade-offs with other fitness related traits. On the other hand, it was predicted that daily decreasing photoperiod should reflect upcoming winter conditions leading to an increase in cold resistance. It was found that within genreation changes in photoperiod had a significant effect on life history and stress resistance traits in the four Drosophila species. The observed response was different across species, with D. melanogaster showing five out of six studied traits affected, while in D. mercatorum only one trait was significantly affected. The exposure to changing photoperiod led to an increased upper thermal resistance in D. melanogaster and D. mercatorum and a decreased lower thermal resistance in D. melanogaster and D. simulans, as well as a decreased starvation and desiccation resistance in D. virilis. The developmental time was shorter when flies were exposed to the two photoperiod regimes compared to constant day length control in D. melanogaster and D. simulans. A limited effect was observed on egg-to-adult-viability and desiccation resistance. The results of this study show that daily change in photoperiod induced a plastic response in different traits of drosophilids, suggesting that this environmental parameter needs to be carefully considered in evolutionary studies.
Poetini, M. R., Musachio, E. A. S., et al. (2021). Iron overload during the embryonic period develops hyperactive like behavior and dysregulation of biogenic amines in Drosophila melanogaster. Dev Biol 475: 80-90. PubMed ID: 33741348
Iron (Fe) is used in various cellular functions, and a constant balance between its uptake, transport, storage, and use is necessary to maintain its homeostasis in the body. Changes in Fe metabolism with a consequent overload of this metal are related to neurological changes and cover a broad spectrum of diseases, mainly when these changes occur during the embryonic period. This work aimed to evaluate the effect of exposure to Fe overload during the embryonic period of Drosophila melanogaster. Progenitor flies (male and female) were exposed to ferrous sulfate (FeSO(4)) for ten days in concentrations of 0.5, 1, and 5 ​mM. After mating and oviposition, the progenitors were removed and the treatment bottles preserved, and the number of daily hatches and cumulative hatching of the first filial generation (F1) were counted. Subsequently, F1 flies (separated by sex) were subjected to behavioral tests such as negative geotaxis test, open field test, grooming, and aggression test. They have evaluated the levels of dopamine (DA), serotonin (5-HT), octopamine (OA), tryptophan and tyrosine hydroxylase (TH), acetylcholinesterase, reactive species, and the levels of Fe in the progenitor flies and F1. The Fe levels of F1 flies are directly proportional to what is incorporated during the period of embryonic development; a delay in hatching and a reduction in the number of the hatch of F1 flies exposed during the embryonic period to the 5mM Fe diet were observed, a fact that may be related to the reduction of the cell viability of the ovarian tissue of progenitor flies. The flies exposed to Fe (1 and 5 ​mM) showed an increase in locomotor activity (hyperactivity) and a significantly higher number of repetitive movements. In addition to a high number of aggressive encounters when compared to control flies. An increase was observed in the levels of biogenic amines DA and 5-HT and an increase in TH activity in flies exposed to Fe (1 and 5 ​mM) compared to the control group. It is concluded that the hyperactive-like behavior demonstrated in both sexes by F1 flies exposed to Fe may be associated with a dysregulation in the levels of DA and 5-HT since Fe is a cofactor of TH, which had its activity increased in this study. Therefore, more attention is needed during the embryonic development period for exposure to Fe overload.
Privalova, V., Szlachcic, E., Sobczyk, Ł., Szabla, N. and Czarnoleski, M. (2021). Oxygen Dependence of Flight Performance in Ageing Drosophila melanogaster. Biology (Basel) 10(4). PubMed ID: 33919761
Similar to humans, insects lose their physical and physiological capacities with age, which makes them a convenient study system for human ageing. Although insects have an efficient oxygen-transport system, little is known about how their flight capacity changes with age and environmental oxygen conditions. Two types of locomotor performance in ageing Drosophila melanogaster flies: the frequency of wing beats and the capacity to climb vertical surfaces. Flight performance was measured under normoxia and hypoxia. As anticipated, ageing flies showed systematic deterioration of climbing performance, and low oxygen impeded flight performance. Against predictions, flight performance did not deteriorate with age, and younger and older flies showed similar levels of tolerance to low oxygen during flight. It is suggested that among different insect locomotory activities, flight performance deteriorates slowly with age, which is surprising, given that insect flight is one of the most energy-demanding activities in animals. Apparently, the superior capacity of insects to rapidly deliver oxygen to flight muscles remains little altered by ageing, but this study showed that insects can become oxygen limited in habitats with a poor oxygen supply (e.g., those at high elevations) during highly oxygen-demanding activities such as flight.
Rai, M., Coleman, Z., Curley, M., Nityanandam, A., Platt, A., Robles-Murguia, M., Jiao, J., Finkelstein, D., Wang, Y. D., Xu, B., Fan, Y. and Demontis, F. (2021). Proteasome stress in skeletal muscle mounts a long-range protective response that delays retinal and brain aging. Cell Metab. PubMed ID: 33773104
Neurodegeneration in the central nervous system (CNS) is a defining feature of organismal aging that is influenced by peripheral tissues. Clinical observations indicate that skeletal muscle influences CNS aging, but the underlying muscle-to-brain signaling remains unexplored. In Drosophila, this study found that moderate perturbation of the proteasome in skeletal muscle induces compensatory preservation of CNS proteostasis during aging. Such long-range stress signaling depends on muscle-secreted Amyrel amylase. Mimicking stress-induced Amyrel upregulation in muscle reduces age-related accumulation of poly-ubiquitinated proteins in the brain and retina via chaperones. Preservation of proteostasis stems from the disaccharide maltose, which is produced via Amyrel amylase activity. Correspondingly, RNAi for SLC45 maltose transporters reduces expression of Amyrel-induced chaperones and worsens brain proteostasis during aging. Moreover, maltose preserves proteostasis and neuronal activity in human brain organoids challenged by thermal stress. Thus, proteasome stress in skeletal muscle hinders retinal and brain aging by mounting an adaptive response via amylase/maltose.
Perochon, J., Yu, Y., Aughey, G. N., Medina, A. B., Southall, T. D. and Cordero, J. B. (2021). Dynamic adult tracheal plasticity drives stem cell adaptation to changes in intestinal homeostasis in Drosophila. Nat Cell Biol 23(5): 485-496. PubMed ID: 33972729
Coordination of stem cell function by local and niche-derived signals is essential to preserve adult tissue homeostasis and organismal health. The vasculature is a prominent component of multiple stem cell niches. However, its role in adult intestinal homeostasis remains largely understudied. This study has uncover a previously unrecognised crosstalk between adult intestinal stem cells in Drosophila and the vasculature-like tracheal system, which is essential for intestinal regeneration. Following damage to the intestinal epithelium, gut-derived reactive oxygen species activate tracheal HIF-1α and bidirectional FGF/FGFR signalling, leading to reversible remodelling of gut-associated terminal tracheal cells and intestinal stem cell proliferation following damage. Unexpectedly, reactive oxygen species-induced adult tracheal plasticity involves downregulation of the tracheal specification factor trachealess (trh) and upregulation of IGF2 messenger RNA-binding protein (IGF2BP2/Imp). These results reveal an intestine-vasculature inter-organ communication programme that is essential to adapt the stem cell response to the proliferative demands of the intestinal epithelium.
Schroeder, S., Hofer, S. J., et al. (2021). Dietary spermidine improves cognitive function. Cell Rep 35(2): 108985. PubMed ID: 33852843
Decreased cognitive performance is a hallmark of brain aging, but the underlying mechanisms and potential therapeutic avenues remain poorly understood. Recent studies have revealed health-protective and lifespan-extending effects of dietary spermidine, a natural autophagy-promoting polyamine. This study shows that dietary spermidine passes the blood-brain barrier in mice and increases hippocampal eIF5A hypusination and mitochondrial function. Spermidine feeding in aged mice affects behavior in homecage environment tasks, improves spatial learning, and increases hippocampal respiratory competence. In a Drosophila aging model, spermidine boosts mitochondrial respiratory capacity, an effect that requires the autophagy regulator Atg7 and the mitophagy mediators Parkin and Pink1. Neuron-specific Pink1 knockdown abolishes spermidine-induced improvement of olfactory associative learning. This suggests that the maintenance of mitochondrial and autophagic function is essential for enhanced cognition by spermidine feeding. Finally, this study showed large-scale prospective data linking higher dietary spermidine intake with a reduced risk for cognitive impairment in humans.

Thursday, July 1st - Signaling

Tandon, S. and Sarkar, S. (2021). The S6k/4E-BP mediated growth promoting sub-pathway of insulin signalling cascade is essential to restrict pathogenesis of poly(Q) disorders in Drosophila. Life Sci 275: 119358. PubMed ID: 33744321
Human neurodegenerative polyglutamine [poly(Q)] disorders, such as Huntington's disease (HD) and spinocerebellar ataxias (SCA), are characterised by an abnormal expansion of CAG repeats in the affected gene. The mutated proteins misfold and aggregate to form inclusion bodies that sequester important factors involved in cellular transcription, growth, stress and autophagic response and other essential functions. The insulin signalling pathway has been demonstrated as a major modifier and a potential drug target to ameliorate the poly(Q) mediated neurotoxicity in various model systems. Insulin signalling cascade harbours several downstream sub-pathways, which are synergistically involved in discharging indispensable biological functions such as growth and proliferation, metabolism, autophagy, regulation of cell death pathways etc. Hence, it is difficult to conclude whether the mitigation of poly(Q) neurotoxicity is an accumulative outcome of the insulin cascade, or the result of a specific sub-pathway. This study reports that the ligand binding domain of insulin receptor mediated downstream growth promoting sub-pathway plays the pivotal role in operating the rescue event. The growth promoting activity of insulin cascade is essential to minimize the abundance of inclusion bodies, to restrict neurodegeneration, and to restore the cellular transcriptional balance. Subsequently, it was noted the involvement of the mTOR/S6k/4E-BP candidates in mitigating poly(Q) mediated neurotoxicity. Due to the conserved cellular functioning of the insulin cascade across species, and availability of several growth promoting molecules, these results in Drosophila poly(Q) models indicate towards a possibility of designing novel therapeutic strategies to restrict the pathogenesis of devastating human poly(Q) disorders.
McDonald, J. M. C., Nabili, P., Thorsen, L., Jeon, S. and Shingleton, A. W. (2021). Sex-specific plasticity and the nutritional geometry of insulin-signaling gene expression in Drosophila melanogaster. Evodevo 12(1): 6. PubMed ID: 33990225
Sexual-size dimorphism (SSD) is replete among animals, but while the selective pressures that drive the evolution of SSD have been well studied, the developmental mechanisms upon which these pressures act are poorly understood. SSD in D. melanogaster reflects elevated levels of nutritional plasticity in females versus males, such that SSD increases with dietary intake and body size, a phenomenon called sex-specific plasticity (SSP). Additional data indicate that while body size in both sexes responds to variation in protein level, only female body size is sensitive to variation in carbohydrate level. This study explored whether these difference in sensitivity at the morphological level are reflected by differences in how the insulin/IGF-signaling (IIS) and TOR-signaling pathways respond to changes in carbohydrates and proteins in females versus males, using a nutritional geometry approach.The IIS-regulated transcripts of 4E-BP and InR most strongly correlated with body size in females and males, respectively, but neither responded to carbohydrate level and so could not explain the sex-specific response to body size to dietary carbohydrate. Transcripts regulated by TOR-signaling did, however, respond to dietary carbohydrate in a sex-specific manner. In females, expression of dILP5 positively correlated with body size, while expression of dILP2,3 and 8, was elevated on diets with a low concentration of both carbohydrate and protein. In contrast, lower levels of dILP2 and 5 protein were observed in the brains of females fed on low concentration diets. No effect of diet on dILP expression in males was detectec. Although females and males show sex-specific transcriptional responses to changes in protein and carbohydrate, the patterns of expression do not support a simple model of the regulation of body-size SSP by either insulin- or TOR-signaling. The data also indicate a complex relationship between carbohydrate and protein level, dILP expression and dILP peptide levels in the brain. In general, diet quality and sex both affect the transcriptional response to changes in diet quantity, and so should be considered in future studies that explore the effect of nutrition on body size.
Millington, J. W., Brownrigg, G. P., Basner-Collins, P. J., Sun, Z. and Rideout, E. J. (2021). Genetic manipulation of insulin/insulin-like growth factor signaling pathway activity has sex-biased effects on Drosophila body size. G3 (Bethesda) 11(3). PubMed ID: 33793746
In Drosophila raised in nutrient-rich conditions, female body size is approximately 30% larger than male body size due to an increased rate of growth and differential weight loss during the larval period. While the mechanisms that control this sex difference in body size remain incompletely understood, recent studies suggest that the insulin/insulin-like growth factor signaling pathway (IIS) plays a role in the sex-specific regulation of processes that influence body size during development. In larvae, IIS activity differs between the sexes, and there is evidence of sex-specific regulation of IIS ligands. Yet, knowledge is lacking of how changes to IIS activity impact body size in each sex, as the majority of studies on IIS and body size use single- or mixed-sex groups of larvae and/or adult flies. The goal of the current study was to clarify the body size requirement for IIS activity in each sex. To achieve this goal, established genetic approaches were used to enhance, or inhibit, IIS activity, and pupal size was quantified in males and females. Overall, genotypes that inhibited IIS activity caused a female-biased decrease in body size, whereas genotypes that augmented IIS activity caused a male-specific increase in body size. These data extend the current understanding of body size regulation by showing that most changes to IIS pathway activity have sex-biased effects, and highlights the importance of analyzing body size data according to sex.
Sardi, J., Bener, M. B., Simao, T., Descoteaux, A. E., Slepchenko, B. M. and Inaba, M. (2021). Mad dephosphorylation at the nuclear pore is essential for asymmetric stem cell division. Proc Natl Acad Sci U S A 118(13). PubMed ID: 33753475
Stem cells divide asymmetrically to generate a stem cell and a differentiating daughter cell. Yet, it remains poorly understood how a stem cell and a differentiating daughter cell can receive distinct levels of niche signal and thus acquire different cell fates (self-renewal versus differentiation), despite being adjacent to each other and thus seemingly exposed to similar levels of niche signaling. In the Drosophila ovary, germline stem cells (GSCs) are maintained by short range bone morphogenetic protein (BMP) signaling; the BMP ligands activate a receptor that phosphorylates the downstream molecule mothers against decapentaplegic (Mad). Phosphorylated Mad (pMad) accumulates in the GSC nucleus and activates the stem cell transcription program. This study demonstrates that pMad is highly concentrated in the nucleus of the GSC, while it quickly decreases in the nucleus of the differentiating daughter cell, the precystoblast (preCB), before the completion of cytokinesis. A known Mad phosphatase, Dullard (Dd), is required for the asymmetric partitioning of pMad. Mathematical modeling recapitulates the high sensitivity of the ratio of pMad levels to the Mad phosphatase activity and explains how the asymmetry arises in a shared cytoplasm. Together, these studies reveal a mechanism for breaking the symmetry of daughter cells during asymmetric stem cell division.
Palmerini, V., Monzani, S., Laurichesse, Q., Loudhaief, R., Mari, S., Cecatiello, V., Olieric, V., Pasqualato, S., Colombani, J., Andersen, D. S. and Mapelli, M. (2021). Drosophila TNFRs Grindelwald and Wengen bind Eiger with different affinities and promote distinct cellular functions. Nat Commun 12(1): 2070. PubMed ID: 33824334
The Drosophila tumour necrosis factor (TNF) ligand-receptor system consists of a unique ligand, Eiger (Egr), and two receptors, Grindelwald (Grnd) and Wengen (Wgn), and therefore provides a simple system for exploring the interplay between ligand and receptors, and the requirement for Grnd and Wgn in TNF/Egr-mediated processes. This study reports the crystallographic structure of the extracellular domain (ECD) of Grnd in complex with Egr, a high-affinity hetero-hexameric assembly reminiscent of human TNF:TNFR complexes. WEctopic expression of Egr results in internalisation of Egr:Grnd complexes in vesicles, a step preceding and strictly required for Egr-induced apoptosis. It was further demonstrated that Wgn binds Egr with much reduced affinity and is localised in intracellular vesicles that are distinct from those containing Egr:Grnd complexes. Altogether, these data provide insight into ligand-mediated activation of Grnd and suggest that distinct affinities of TNF ligands for their receptors promote different and non-redundant cellular functions.
Nishida, H., Okada, M., Yang, L., Takano, T., Tabata, S., Soga, T., Ho, D. M., Chung, J., Minami, Y. and Yoo, S. K. (2021). Methionine restriction breaks obligatory coupling of cell proliferation and death by an oncogene Src in Drosophila. Elife 10. PubMed ID: 33902813
Oncogenes often promote cell death as well as proliferation. How oncogenes drive these diametrically opposed phenomena remains to be solved. A key question is whether cell death occurs as a response to aberrant proliferation signals or through a proliferation-independent mechanism. This study revealed that Src, the first identified oncogene, simultaneously drives cell proliferation and death in an obligatorily coupled manner through parallel MAPK pathways. The two MAPK pathways diverge from a lynchpin protein Slpr. A MAPK p38 drives proliferation whereas another MAPK JNK drives apoptosis independently of proliferation signals. Src-p38-induced proliferation is regulated by methionine-mediated Tor signaling. Reduction of dietary methionine uncouples the obligatory coupling of cell proliferation and death, suppressing tumorigenesis and tumor-induced lethality. These findings provide an insight into how cells evolved to have a fail-safe mechanism that thwarts tumorigenesis by the oncogene Src. This study also exemplifies a diet-based approach to circumvent oncogenesis by exploiting the fail-safe mechanism.

Wednesday, May 30th - Adult Neural Development and Function

Ruiz, D., Bajwa, S. T., Vanani, N., Bajwa, T. A. and Cavanaugh, D. J. (2021). Slowpoke functions in circadian output cells to regulate rest:activity rhythms. PLoS One 16(3): e0249215. PubMed ID: 33765072
The circadian system produces ~24-hr oscillations in behavioral and physiological processes to ensure that they occur at optimal times of day and in the correct temporal order. At its core, the circadian system is composed of dedicated central clock neurons that keep time through a cell-autonomous molecular clock. To produce rhythmic behaviors, time-of-day information generated by clock neurons must be transmitted across output pathways to regulate the downstream neuronal populations that control the relevant behaviors. An understanding of the manner through which the circadian system enacts behavioral rhythms therefore requires the identification of the cells and molecules that make up the output pathways. To that end, the Drosophila pars intercerebralis (PI) was recently characterized as a major circadian output center that lies downstream of central clock neurons in a circuit controlling rest:activity rhythms. Single-cell RNA sequencing (scRNAseq) was performed to identify potential circadian output genes expressed by PI cells, and cell-specific RNA interference (RNAi) was used to knock down expression of ~40 of these candidate genes selectively within subsets of PI cells. Knockdown of the slowpoke (slo) potassium channel in PI cells reliably decreases circadian rest:activity rhythm strength. Interestingly, slo mutants have previously been shown to have aberrant rest:activity rhythms, in part due to a necessary function of slo within central clock cells. However, rescue of slo in all clock cells does not fully reestablish behavioral rhythms, indicating that expression in non-clock neurons is also necessary. These results demonstrate that slo exerts its effects in multiple components of the circadian circuit, including PI output cells in addition to clock neurons, and it is hypothesized that it does so by contributing to the generation of daily neuronal activity rhythms that allow for the propagation of circadian information throughout output circuits.
Pizzo, L., Lasser, M., Yusuff, T., Jensen, M., Ingraham, P., Huber, E., Singh, M. D., Monahan, C., Iyer, J., Desai, I., Karthikeyan, S., Gould, D. J., Yennawar, S., Weiner, A. T., Pounraja, V. K., Krishnan, A., Rolls, M. M., Lowery, L. A. and Girirajan, S. (2021). Functional assessment of the "two-hit" model for neurodevelopmental defects in Drosophila and X. laevis. PLoS Genet 17(4): e1009112. PubMed ID: 33819264
Previous work identified a deletion on chromosome 16p12.1 that is mostly inherited and associated with multiple neurodevelopmental outcomes, where severely affected probands carried an excess of rare pathogenic variants compared to mildly affected carrier parents. It was hypothesized that the 16p12.1 deletion sensitizes the genome for disease, while "second-hits" in the genetic background modulate the phenotypic trajectory. To test this model, this study examined how neurodevelopmental defects conferred by knockdown of individual 16p12.1 homologs are modulated by simultaneous knockdown of homologs of "second-hit" genes in Drosophila melanogaster and Xenopus laevis. Knockdown of 16p12.1 homologs affected multiple phenotypic domains, leading to delayed developmental timing, seizure susceptibility, brain alterations, abnormal dendrite and axonal morphology, and cellular proliferation defects. Compared to genes within the 16p11.2 deletion, which has higher de novo occurrence, 16p12.1 homologs were less likely to interact with each other in Drosophila models or a human brain-specific interaction network, suggesting that interactions with "second-hit" genes may confer higher impact towards neurodevelopmental phenotypes. Assessment of 212 pairwise interactions in Drosophila between 16p12.1 homologs and 76 homologs of patient-specific "second-hit" genes (such as ARID1B and CACNA1A), genes within neurodevelopmental pathways (such as PTEN and UBE3A), and transcriptomic targets (such as DSCAM and TRRAP) identified genetic interactions in 63% of the tested pairs. In 11 out of 15 families, patient-specific "second-hits" enhanced or suppressed the phenotypic effects of one or many 16p12.1 homologs in 32/96 pairwise combinations tested. In fact, homologs of SETD5 synergistically interacted with homologs of MOSMO in both Drosophila and X. laevis, leading to modified cellular and brain phenotypes, as well as axon outgrowth defects that were not observed with knockdown of either individual homolog. These results suggest that several 16p12.1 genes sensitize the genome towards neurodevelopmental defects, and complex interactions with "second-hit" genes determine the ultimate phenotypic manifestation.
Poudel, S., Guo, H. and Smith, D. P. (2021). PKC98E Regulates Odorant Responses in Drosophila melanogaster. J Neurosci 41(18): 3948-3957. PubMed ID: 33789918
Drosophila odorant receptors (Ors) are ligand gated ion channels composed of a common receptor subunit Or co-receptor (ORCO) and one of 62 "tuning" receptor subunits that confer odorant specificity to olfactory neuron responses. Like other sensory systems studied to date, exposing Drosophila olfactory neurons to activating ligands results in reduced responses to subsequent exposures through a process called desensitization. It was recently shown that phosphorylation of serine 289 on the common Or subunit ORCO is required for normal peak olfactory neuron responses. Dephosphorylation of this residue occurs on prolonged odorant exposure, and underlies the slow modulation of olfactory neuron responses termed "slow desensitization." Slow desensitization results in the reduction of peak olfactory neuron responses and flattening of dose-response curves, implicating changes in ORCO(S289) phosphorylation state as an important modulator of olfactory neuron responses. This study reports the identification of the primary kinase responsible for ORCO(S289) phosphorylation, PKC98E. Antiserum localizes the kinase to the dendrites of the olfactory neurons. Deletion of the kinase from olfactory neurons in the naive state (the absence of prolonged odor exposure) reduces ORCO(S289) phosphorylation and reduces peak odorant responses without altering receptor localization or expression levels. Genetic rescue with a PKC98E predicted to be constitutively active restores ORCO S289 phosphorylation and olfactory neuron sensitivity to the PKC98E mutants in the naive state. However, the dominant kinase is defective for slow desensitization. Together, these findings reveal that PKC98E is an important regulator of ORCO receptors and olfactory neuron function.
Raji, J. I. and Potter, C. J. (2021). The number of neurons in Drosophila and mosquito brains. PLoS One 16(5): e0250381. PubMed ID: 33989293
Various insect species serve as valuable model systems for investigating the cellular and molecular mechanisms by which a brain controls sophisticated behaviors. In particular, the nervous system of Drosophila melanogaster has been extensively studied, yet experiments aimed at determining the number of neurons in the Drosophila brain are surprisingly lacking. Using isotropic fractionator coupled with immunohistochemistry, this study counted the total number of neuronal and non-neuronal cells in the whole brain, central brain, and optic lobe of Drosophila melanogaster. For comparison, neuronal populations were counted in three divergent mosquito species: Aedes aegypti, Anopheles coluzzii and Culex quinquefasciatus. The average number of neurons in a whole adult brain was determined to be 199,380 ±3,400 cells in D. melanogaster, 217,910 ±6,180 cells in Ae. aegypti, 223,020 ± 4,650 cells in An. coluzzii and 225,911±7,220 cells in C. quinquefasciatus. The mean neuronal cell count in the central brain vs. optic lobes for D. melanogaster (101,140 ±3,650 vs. 107,270 ± 2,720), Ae. aegypti (109,140 ± 3,550 vs. 112,000 ± 4,280), An. coluzzii (105,130 ± 3,670 vs. 107,140 ± 3,090), and C. quinquefasciatus (108,530 ±7,990 vs. 110,670 ± 3,950) was also estimated. Each insect brain was comprised of 89% ± 2% neurons out of its total cell population. Isotropic fractionation analyses did not identify obvious sexual dimorphism in the neuronal and non-neuronal cell population of these insects. This study provides experimental evidence for the total number of neurons in Drosophila and mosquito brains.
Lu, Y. X., Regan, J. C., Esser, J., Drews, L. F., Weinseis, T., Stinn, J., Hahn, O., Miller, R. A., Gronke, S. and Partridge, L. (2021). A TORC1-histone axis regulates chromatin organisation and non-canonical induction of autophagy to ameliorate ageing. Elife 10. PubMed ID: 33988501
Age-related changes to histone levels are seen in many species. However, it is unclear whether changes to histone expression could be exploited to ameliorate the effects of ageing in multicellular organisms. This study shows that inhibition of mTORC1 by the lifespan-extending drug rapamycin increases expression of histones H3 and H4 post-transcriptionally, through eIF3-mediated translation. Elevated expression of H3/H4 in intestinal enterocytes in Drosophila alters chromatin organization, induces intestinal autophagy through transcriptional regulation, prevents age-related decline in the intestine. Importantly, it also mediates rapamycin-induced longevity and intestinal health. Histones H3/H4 regulate expression of an autophagy cargo adaptor Blue Cheese (Bchs (WDFY3 in mammals), increased expression of which in enterocytes mediates increased H3/H4-dependent healthy longevity. In mice, rapamycin treatment increases expression of histone proteins and Wdfy3 transcription, and alters chromatin organisation in the small intestine, suggesting the mTORC1-histone axis is at least partially conserved in mammals and may offer new targets for anti-ageing interventions.
Pidathala, S., Mallela, A. K., Joseph, D. and Penmatsa, A. (2021). Structural basis of norepinephrine recognition and transport inhibition in neurotransmitter transporters. Nat Commun 12(1): 2199. PubMed ID: 33850134
Norepinephrine is a biogenic amine neurotransmitter that has widespread effects on alertness, arousal and pain sensation. Consequently, blockers of norepinephrine uptake have served as vital tools to treat depression and chronic pain. This study employed the Drosophila melanogaster dopamine transporter as a surrogate for the norepinephrine transporter and determine X-ray structures of the transporter in its substrate-free and norepinephrine-bound forms. This study also reports structures of the transporter in complex with inhibitors of chronic pain including duloxetine, milnacipran and a synthetic opioid, tramadol. When compared to dopamine, it was observed that norepinephrine binds in a different pose, in the vicinity of subsite C within the primary binding site. These experiments reveal that this region is the binding site for chronic pain inhibitors and a determinant for norepinephrine-specific reuptake inhibition, thereby providing a paradigm for the design of specific inhibitors for catecholamine neurotransmitter transporters.
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