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Current papers in developmental biology and gene function


Wednesday, March 31st, 2021 - Stem Cells

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Magadi, S. S., Voutyraki, C., Anagnostopoulos, G., Zacharioudaki, E., Poutakidou, I. K., Efraimoglou, C., Stapountzi, M., Theodorou, V., Nikolaou, C., Koumbanakis, K. A., Fullard, J. F. and Delidakis, C. (2020). Dissecting Hes-centred transcriptional networks in neural stem cell maintenance and tumorigenesis in Drosophila. Development 147(22). PubMed ID: 33229432
Neural stem cells divide during embryogenesis and juvenile life to generate the entire complement of neurons and glia in the nervous system of vertebrates and invertebrates. Studies of the mechanisms controlling the fine balance between neural stem cells and more differentiated progenitors have shown that, in every asymmetric cell division, progenitors send a Delta-Notch signal to their sibling stem cells. This study shows that excessive activation of Notch or overexpression of its direct targets of the Hes family causes stem-cell hyperplasias in the Drosophila larval central nervous system, which can progress to malignant tumours after allografting to adult hosts. Transcriptomic data from these hyperplasias were combined with chromatin occupancy data for Dpn, a Hes transcription factor, to identify genes regulated by Hes factors in this process. The Notch/Hes axis represses a cohort of transcription factor genes. These are excluded from the stem cells and promote early differentiation steps, most likely by preventing the reversion of immature progenitors to a stem-cell fate. The impact is described of two of these 'anti-stemness' factors, Zfh1 and Gcm, on Notch/Hes-triggered tumorigenesis.
Giannios, P. and Casanova, J. (2021). Systemic and local effect of the Drosophila headcase gene and its role in stress protection of Adult Progenitor Cells. PLoS Genet 17(2): e1009362. PubMed ID: 33556132
During the development of a holometabolous insect such as Drosophila, specific group of cells in the larva survive during metamorphosis, unlike the other larval cells, and finally give rise to the differentiated adult structures. These cells, also known as Adult Progenitor Cells (APCs), maintain their multipotent capacity, differentially respond to hormonal and nutritional signals, survive the intrinsic and environmental stress and respond to the final differentiation cues. However, not much is known about the specific molecular mechanisms that account for their unique characteristics. This study shows that a specific Drosophila APC gene, headcase (hdc), has a dual role in the normal development of these cells. It acts at a systemic level by controlling the hormone ecdysone in the prothoracic gland and at the same time it acts locally as a tissue growth suppressor in the APC clusters, where it modulates the activity of the TOR pathway and promotes their survival by contributing in the regulation of the Unfolded Protein Response. This study also showed that hdc provides protection against stress in the APCs and that its ectopic expression in cells that do not usually express hdc can confer these cells with an additional stress protection. Hdc is the founding member of a group of homolog proteins identified from C. elegans to humans, where has been found associated with cancer progression. The finding that the Drosophilahdc is specifically expressed in progenitor cells and that it provides protection against stress opens up a new hypothesis to be explored regarding the role of the human Heca and its contribution to carcinogenesis.
Al Hayek, S., Alsawadi, A., Kambris, Z., Boquete, J. P., Bohere, J., Immarigeon, C., Ronsin, B., Plaza, S., Lemaitre, B., Payre, F. and Osman, D. (2020). Steroid-dependent switch of OvoL/Shavenbaby controls self-renewal versus differentiation of intestinal stem cells. Embo j: e104347. PubMed ID: 33372708
Adult stem cells must continuously fine-tune their behavior to regenerate damaged organs and avoid tumors. While several signaling pathways are well known to regulate somatic stem cells, the underlying mechanisms remain largely unexplored. This study demonstrates a cell-intrinsic role for the OvoL family transcription factor, Shavenbaby (Svb), in balancing self-renewal and differentiation of Drosophila intestinal stem cells. svb is a downstream target of Wnt and EGFR pathways, mediating their activity for stem cell survival and proliferation. This requires post-translational processing of Svb into a transcriptional activator, whose upregulation induces tumor-like stem cell hyperproliferation. In contrast, the unprocessed form of Svb acts as a repressor that imposes differentiation into enterocytes, and suppresses tumors induced by altered signaling. The switch between Svb repressor and activator is triggered in response to systemic steroid hormone, which is produced by ovaries. Therefore, the Svb axis allows intrinsic integration of local signaling cues and inter-organ communication to adjust stem cell proliferation versus differentiation, suggesting a broad role of OvoL/Svb in adult and cancer stem cells.
Ferguson, M., Petkau, K., Shin, M., Galenza, A., Fast, D. and Foley, E. (2021). Differential effects of commensal bacteria on progenitor cell adhesion, division symmetry and tumorigenesis in the Drosophila intestine. Development 148(5). PubMed ID: 33593820
Microbial factors influence homeostatic and oncogenic growth in the intestinal epithelium. However, little is known about immediate effects of commensal bacteria on stem cell division programs. This study examined the effects of commensal Lactobacillus species on homeostatic and tumorigenic stem cell proliferation in the female Drosophila intestine. Lactobacillus brevis was identified as a potent stimulator of stem cell divisions. In a wild-type midgut, L. brevis activates growth regulatory pathways that drive stem cell divisions. In a Notch-deficient background, L. brevis-mediated proliferation causes rapid expansion of mutant progenitors, leading to accumulation of large, multi-layered tumors throughout the midgut. Mechanistically, this study showed that L. brevis disrupts expression and subcellular distribution of progenitor cell integrins, supporting symmetric divisions that expand intestinal stem cell populations. Collectively, these data emphasize the impact of commensal microbes on division and maintenance of the intestinal progenitor compartment.
Harrison, N. J., Connolly, E., Gascon Gubieda, A., Yang, Z., Altenhein, B., Losada Perez, M., Moreira, M., Sun, J. and Hidalgo, A. (2021). Regenerative neurogenic response from glia requires insulin-driven neuron-glia communication. Elife 10. PubMed ID: 33527895
Understanding how injury to the central nervous system induces de novo neurogenesis in animals would help promote regeneration in humans. Regenerative neurogenesis could originate from glia and glial neuron-glia antigen-2 (NG2) may sense injury-induced neuronal signals, but these are unknown. This used Drosophila to search for genes functionally related to the NG2 homologue kon-tiki (kon), and identified Islet Antigen-2 (Ia-2), required in neurons for insulin secretion. Both loss and over-expression of ia-2 induced neural stem cell gene expression, injury increased ia-2 expression and induced ectopic neural stem cells. Using genetic analysis and lineage tracing, this study demonstrated that Ia-2 and Kon regulate Drosophila insulin-like peptide 6 (Dilp-6) to induce glial proliferation and neural stem cells from glia. Ectopic neural stem cells can divide, and limited de novo neurogenesis could be traced back to glial cells. Altogether, Ia-2 and Dilp-6 drive a neuron-glia relay that restores glia and reprogrammes glia into neural stem cells for regeneration.
Amcheslavsky, A., Lindblad, J. L. and Bergmann, A. (2020). Transiently "Undead" Enterocytes Mediate Homeostatic Tissue Turnover in the Adult Drosophila Midgut. Cell Rep 33(8): 108408. PubMed ID: 33238125
This study reveals surprising similarities between homeostatic cell turnover in adult Drosophila midguts and "undead" apoptosis-induced compensatory proliferation (AiP) in imaginal discs. During undead AiP, immortalized cells signal for AiP, allowing its analysis. Critical for undead AiP is the Myo1D-dependent localization of the initiator caspase Dronc to the plasma membrane. This study shows that Myo1D functions in mature enterocytes (ECs) to control mitotic activity of intestinal stem cells (ISCs). In Myo1D mutant midguts, many signaling events involved in AiP (ROS generation, hemocyte recruitment, and JNK signaling) are affected. Importantly, similar to AiP, Myo1D is required for membrane localization of Dronc in ECs. It is proposed that ECs destined to die transiently enter an undead-like state through Myo1D-dependent membrane localization of Dronc, which enables them to generate signals for ISC activity and their replacement. The concept of transiently "undead" cells may be relevant for other stem cell models in flies and mammals.

Tuesday, March 30th - Synapse and Vesicles

Birnbaum, A., Sodders, M., Bouska, M., Chang, K., Kang, P., McNeill, E. and Bai, H. (2020). FOXO Regulates Neuromuscular Junction Homeostasis During Drosophila Aging. Front Aging Neurosci 12: 567861. PubMed ID: 33584240
The transcription factor Foxo is a known regulator of lifespan extension and tissue homeostasis. It has been linked to the maintenance of neuronal processes across many species and has been shown to promote youthful characteristics by regulating cytoskeletal flexibility and synaptic plasticity at the neuromuscular junction (NMJ). However, the role of foxo in aging neuromuscular junction function has yet to be determined. This study profiled adult Drosophila foxo- null mutant abdominal ventral longitudinal muscles and found that young mutants exhibited morphological profiles similar to those of aged wild-type flies, such as larger bouton areas and shorter terminal branches. Changes to the axonal cytoskeleton and an accumulation of late endosomes were also observed in foxo null mutants and motor neuron-specific foxo knockdown flies, similar to those of aged wild-types. Motor neuron-specific overexpression of foxo can delay age-dependent changes to NMJ morphology, suggesting foxo is responsible for maintaining NMJ integrity during aging. Through genetic screening, several downstream factors mediated through foxo-regulated NMJ homeostasis were identified, including genes involved in the MAPK pathway. Interestingly, the phosphorylation of p38 was increased in the motor neuron-specific foxo knockdown flies, suggesting foxo acts as a suppressor of p38/MAPK activation. This work reveals that foxo is a key regulator for NMJ homeostasis, and it may maintain NMJ integrity by repressing MAPK signaling.
Brady, J., Vasin, A. and Bykhovskaia, M. (2021). The Accessory Helix of Complexin Stabilizes a Partially Unzippered State of the SNARE Complex and Mediates the Complexin Clamping Function in Vivo. eNeuro. PubMed ID: 33692090
Spontaneous synaptic transmission is regulated by the protein Complexin (Cpx). Cpx binds the SNARE complex, a coil-coiled four-helical bundle that mediates the attachment of a synaptic vesicle (SV) to the presynaptic membrane (PM). Cpx is thought to clamp spontaneous fusion events by stabilizing a partially unraveled state of the SNARE bundle; however, the molecular detail of this mechanism is still debated. This study combined electrophysiology, molecular modeling, and site-directed mutagenesis in Drosophila to develop and validate the atomic model of the Cpx-mediated clamped state of the SNARE complex. Advantage was taken of botulinum neurotoxins (BoNT) B and G, which cleave the SNARE protein synaptobrevin (Syb) at different sites. Monitoring synaptic depression upon BoNT loading revealed that the clamped state of the SNARE complex has two or three unraveled helical turns of Syb. Site-directed mutagenesis showed that the Cpx clamping function is predominantly maintained by its accessory helix (AH), while molecular modeling suggested that the Cpx AH interacts with the unraveled C-terminus of Syb and the SV lipid bilayer. The developed molecular model was employed to design new Cpx poor-clamp and super-clamp mutations and to tested the predictions in silico employing molecular dynamics simulations. Subsequently, Drosophila lines were generated harboring these mutations, and the poor-clamp and super-clamp phenotypes were confirmed in vivo. Altogether, these results validate the atomic model of the Cpx-mediated fusion clamp, wherein the Cpx AH inserts between the SNARE bundle and the SV lipid bilayer, simultaneously binding the unraveled C-terminus of Syb and preventing full SNARE assembly.
Du, G., Liu, Z., Yu, Z., Zhuo, Z., Zhu, Y., Zhou, J., Li, Y. and Chen, H. (2021). Taurine represses age-associated gut hyperplasia in Drosophila via counteracting endoplasmic reticulum stress. Aging Cell: e13319. PubMed ID: 33559276
As they age, adult stem cells become more prone to functional decline, which is responsible for aging-associated tissue degeneration and diseases. One goal of aging research is to identify drugs that can repair age-associated tissue degeneration. Multiple organ development-related signaling pathways have recently been demonstrated to have functions in tissue homeostasis and aging process. Therefore, in this study, several chemicals that are essential for organ development were tested to assess their ability to delay intestinal stem cell (ISC) aging and promote gut function in adult Drosophila. Taurine, a free amino acid that supports neurological development and tissue metabolism in humans, was found to repress ISC hyperproliferation and restrain the intestinal functional decline seen in aged animals. Taurine was found to repress age-associated ISC hyperproliferation through a mechanism that eliminated endoplasmic reticulum (ER) stress by upregulation of the target genes of unfolded protein response in the ER (UPR(ER)) and inhibiting the c-Jun N-terminal kinase (JNK) signaling. These findings show that taurine plays a critical role in delaying the aging process in stem cells and suggest that it may be used as a natural compound for the treatment of age-associated, or damage-induced intestinal dysfunction in humans.
Fujii, T., Sakurai, A., Littleton, J. T. and Yoshihara, M. (2021). Synaptotagmin 7 switches short-term synaptic plasticity from depression to facilitation by suppressing synaptic transmission. Sci Rep 11(1): 4059. PubMed ID: 33603074
Short-term synaptic plasticity is a fast and robust modification in neuronal presynaptic output that can enhance release strength to drive facilitation or diminish it to promote depression. The mechanisms that determine whether neurons display short-term facilitation or depression are still unclear. This study shows that the Ca(2+)-binding protein Synaptotagmin 7 (Syt7) determines the sign of short-term synaptic plasticity by controlling the initial probability of synaptic vesicle (SV) fusion. Electrophysiological analysis of Syt7 null mutants at Drosophila embryonic neuromuscular junctions demonstrate loss of the protein converts the normally observed synaptic facilitation response during repetitive stimulation into synaptic depression. In contrast, overexpression of Syt7 dramatically enhanced the magnitude of short-term facilitation. These changes in short-term plasticity were mirrored by corresponding alterations in the initial evoked response, with SV release probability enhanced in Syt7 mutants and suppressed following Syt7 overexpression. Indeed, Syt7 mutants were able to display facilitation in lower [Ca(2+)] where release was reduced. These data suggest Syt7 does not act by directly sensing residual Ca(2+) and argues for the existence of a distinct Ca(2+) sensor beyond Syt7 that mediates facilitation. Instead, Syt7 normally suppresses synaptic transmission to maintain an output range where facilitation is available to the neuron.
Pannen, H., Rapp, T. and Klein, T. (2020). The ESCRT Machinery regulates Retromer dependent Transcytosis of Septate Junction Components in Drosophila. Elife 9. PubMed ID: 33377869
Loss of ESCRT function in Drosophila imaginal discs is known to cause neoplastic overgrowth fuelled by mis-regulation of signalling pathways. Its impact on junctional integrity, however, remains obscure. To dissect the events leading to neoplasia, transmission electron microscopy (TEM)was used on wing imaginal discs temporally depleted of the ESCRT-III core component Shrub. A specific requirement for Shrub was found in maintaining Septate Junction (SJ) integrity by transporting the Claudin Megatrachea (Mega) to the SJ. In absence of Shrub function, Mega is lost from the SJ and becomes trapped on endosomes coated with the endosomal retrieval machinery Retromer. ESCRT function is required for apical localization and mobility of Retromer positive carrier vesicles, which mediate the biosynthetic delivery of Mega to the SJ. Accordingly, loss of Retromer function impairs the anterograde transport of several SJ core components, revealing a novel physiological role for this ancient endosomal agent.
Belalcazar, H. M., Hendricks, E. L., Zamurrad, S., Liebl, F. L. W. and Secombe, J. (2021). The histone demethylase KDM5 is required for synaptic structure and function at the Drosophila neuromuscular junction. Cell Rep 34(7): 108753. PubMed ID: 33596422
Mutations in the genes encoding the lysine demethylase 5 (KDM5) family of histone demethylases are observed in individuals with intellectual disability (ID). Despite clear evidence linking KDM5 function to neurodevelopmental pathways, how this family of proteins impacts transcriptional programs to mediate synaptic structure and activity remains unclear. Using the Drosophila larval neuromuscular junction (NMJ), this study shows that KDM5 is required presynaptically for neuroanatomical development and synaptic function. The Jumonji C (JmjC) domain-encoded histone demethylase activity of KDM5, which is expected to be diminished by many ID-associated alleles, is required for appropriate synaptic morphology and neurotransmission. The activity of the C5HC2 zinc finger is also required, as an ID-associated mutation in this motif reduces NMJ bouton number, increases bouton size, and alters microtubule dynamics. KDM5 therefore uses demethylase-dependent and independent mechanisms to regulate NMJ structure and activity, highlighting the complex nature by which this chromatin modifier carries out its neuronal gene-regulatory programs.

Monday, April 29th - Signaling

DaCrema, D., Bhandari, R., Karanja, F., Yano, R. and Halme, A. (2021). Ecdysone regulates the Drosophila imaginal disc epithelial barrier, determining the length of regeneration checkpoint delay. Development. PubMed ID: 33658221
Regeneration of Drosophila imaginal discs, larval precursors to adult tissues, activates a regeneration checkpoint that coordinates regenerative growth with developmental progression. This regeneration checkpoint results from the release of the relaxin-family peptide Dilp8 from regenerating imaginal tissues. Secreted Dilp8 protein is detected within the imaginal disc lumen, where it is separated from its receptor target Lgr3, expressed in the brain and prothoracic gland, by the disc epithelial barrier. This study demonstrates that following damage the imaginal disc epithelial barrier limits Dilp8 signaling and the duration of regeneration checkpoint delay. It was also found that the barrier becomes increasingly impermeable to the transepithelial diffusion of labeled dextran during the second half of the third instar. This change in barrier permeability is driven by the steroid hormone ecdysone and correlates with changes in localization of Coracle, a component of the septate junctions that is required for the late-larval, impermeable epithelial barrier. Based on these observations, it is proposed that the imaginal disc epithelial barrier regulates the duration of the regenerative checkpoint, providing a mechanism by which tissue function can signal the completion of regeneration.
Diegmiller, R., Zhang, L., Gameiro, M., Barr, J., Imran Alsous, J., Schedl, P., Shvartsman, S. Y. and Mischaikow, K. (2021). Mapping parameter spaces of biological switches. PLoS Comput Biol 17(2): e1008711. PubMed ID: 33556054
Since the seminal 1961 paper of Monod and Jacob, mathematical models of biomolecular circuits have guided understanding of cell regulation. Model-based exploration of the functional capabilities of any given circuit requires systematic mapping of multidimensional spaces of model parameters. Despite significant advances in computational dynamical systems approaches, this analysis remains a nontrivial task. This study used a nonlinear system of ordinary differential equations to model oocyte selection in Drosophila, a robust symmetry-breaking event that relies on autoregulatory localization of oocyte-specification factors. By applying an algorithmic approach that implements symbolic computation and topological methods, all phase portraits were enumerated of stable steady states in the limit when nonlinear regulatory interactions become discrete switches. Leveraging this initial exact partitioning and further using numerical exploration, parameter regions were located that are dense in purely asymmetric steady states when the nonlinearities are not infinitely sharp, enabling systematic identification of parameter regions that correspond to robust oocyte selection. This framework can be generalized to map the full parameter spaces in a broad class of models involving biological switches.
Emmons-Bell, M. and Hariharan, I. K. (2021). Membrane potential regulates Hedgehog signalling in the Drosophila wing imaginal disc. EMBO Rep: e51861. PubMed ID: 33629503
While the membrane potential of cells has been shown to be patterned in some tissues, specific roles for membrane potential in regulating signalling pathways that function during development are still being established. In the Drosophila wing imaginal disc, Hedgehog (Hh) from posterior cells activates a signalling pathway in anterior cells near the boundary which is necessary for boundary maintenance. This study shows that membrane potential is patterned in the wing disc. Anterior cells near the boundary, where Hh signalling is most active, are more depolarized than posterior cells across the boundary. Elevated expression of the ENaC channel Ripped Pocket (Rpk), observed in these anterior cells, requires Hh. Antagonizing Rpk reduces depolarization and Hh signal transduction. Using genetic and optogenetic manipulations, in both the wing disc and the salivary gland, it was shown that membrane depolarization promotes membrane localization of Smoothened and augments Hh signalling, independently of Patched. Thus, membrane depolarization and Hh-dependent signalling mutually reinforce each other in cells immediately anterior to the compartment boundary.
Fic, W., Bastock, R., Raimondi, F., Los, E., Inoue, Y., Gallop, J. L., Russell, R. B. and St Johnston, D. (2021). RhoGAP19D inhibits Cdc42 laterally to control epithelial cell shape and prevent invasion. J Cell Biol 220(4). PubMed ID: 33646271
Cdc42-GTP is required for apical domain formation in epithelial cells, where it recruits and activates the Par-6-aPKC polarity complex, but how the activity of Cdc42 itself is restricted apically is unclear. This study used sequence analysis and 3D structural modeling to determine which Drosophila GTPase-activating proteins (GAPs) are likely to interact with Cdc42 and identified RhoGAP19D as the only high-probability Cdc42GAP required for polarity in the follicular epithelium. RhoGAP19D is recruited by α-catenin to lateral E-cadherin adhesion complexes, resulting in exclusion of active Cdc42 from the lateral domain. rhogap19d mutants therefore lead to lateral Cdc42 activity, which expands the apical domain through increased Par-6/aPKC activity and stimulates lateral contractility through the myosin light chain kinase, Genghis khan (MRCK). This causes buckling of the epithelium and invasion into the adjacent tissue, a phenotype resembling that of precancerous breast lesions. Thus, RhoGAP19D couples lateral cadherin adhesion to the apical localization of active Cdc42, thereby suppressing epithelial invasion.
Erez, N., Israitel, L., Bitman-Lotan, E., Wong, W. H., Raz, G., Cornelio-Parra, D. V., Danial, S., Flint Brodsly, N., Belova, E., Maksimenko, O., Georgiev, P., Druley, T., Mohan, R. D. and Orian, A. (2021). A Non-stop identity complex (NIC) supervises enterocyte identity and protects from premature aging. Elife 10. PubMed ID: 33629655
A hallmark of aging is loss of differentiated cell identity. Aged Drosophila midgut differentiated enterocytes (ECs) lose their identity, impairing tissue homeostasis. To discover identity regulators, an RNAi screen targeting ubiquitin-related genes was performed in ECs. Seventeen genes were identified, including the deubiquitinase Non-stop (CG4166). Lineage tracing established that acute loss of Non-stop in young ECs phenocopies aged ECs at cellular and tissue levels. Proteomic analysis unveiled that Non-stop maintains identity as part of a Non-stop identity complex (NIC) containing E(y)2, Sgf11, Cp190, (Mod) mdg4, and Nup98. Non-stop ensured chromatin accessibility, maintaining the EC-gene signature, and protected NIC subunit stability. Upon aging, the levels of Non-stop and NIC subunits declined, distorting the unique organization of the EC nucleus. Maintaining youthful levels of Non-stop in wildtype aged ECs safeguards NIC subunits, nuclear organization, and suppressed aging phenotypes. Thus, Non-stop and NIC, supervise EC identity and protects from premature aging.
Fatalska, A., Stepinac, E., Richter, M., Kovacs, L., Pietras, Z., Puchinger, M., Dong, G., Dadlez, M. and Glover, D. M. (2021). The dimeric Golgi protein Gorab binds to Sas6 as a monomer to mediate centriole duplication. Elife 10. PubMed ID: 33704067
The duplication and 9-fold symmetry of the Drosophila centriole requires that the cartwheel molecule, Sas6, physically associates with Gorab, a trans-Golgi component. How Gorab achieves these disparate associations is unclear. This study used hydrogen-deuterium exchange mass spectrometry to define Gorab's interacting surfaces that mediate its sub-cellular localization. A core stabilization sequence within Gorab's C-terminal coiled-coil domain was identified that enables homodimerization, binding to Rab6, and thereby trans-Golgi localization. By contrast, part of the Gorab monomer's coiled-coil domain undergoes an anti-parallel interaction with a segment of the parallel coiled-coil dimer of Sas6. This stable hetero-trimeric complex can be visualized by electron microscopy. Mutation of a single leucine residue in Sas6's Gorab-binding domain generates a Sas6 variant with a 16-fold reduced binding affinity for Gorab that can not support centriole duplication. Thus Gorab dimers at the Golgi exist in equilibrium with Sas-6 associated monomers at the centriole to balance Gorab's dual role.

Friday, March 26th - Chromatin

Ulianov, S. V., Zakharova, V. V., Galitsyna, A. A., Kos, P. I., Polovnikov, K. E., Flyamer, I. M., Mikhaleva, E. A., Khrameeva, E. E., Germini, D., Logacheva, M. D., Gavrilov, A. A., Gorsky, A. S., Nechaev, S. K., Gelfand, M. S., Vassetzky, Y. S., Chertovich, A. V., Shevelyov, Y. Y. and Razin, S. V. (2021). Order and stochasticity in the folding of individual Drosophila genomes. Nat Commun 12(1): 41. PubMed ID: 33397980
Mammalian and Drosophila genomes are partitioned into topologically associating domains (TADs). Although this partitioning has been reported to be functionally relevant, it is unclear whether TADs represent true physical units located at the same genomic positions in each cell nucleus or emerge as an average of numerous alternative chromatin folding patterns in a cell population. This study used a single-nucleus Hi-C technique to construct high-resolution Hi-C maps in individual Drosophila genomes. These maps demonstrate chromatin compartmentalization at the megabase scale and partitioning of the genome into non-hierarchical TADs at the scale of 100 kb, which closely resembles the TAD profile in the bulk in situ Hi-C data. Over 40% of TAD boundaries are conserved between individual nuclei and possess a high level of active epigenetic marks. Polymer simulations demonstrate that chromatin folding is best described by the random walk model within TADs and is most suitably approximated by a crumpled globule build of Gaussian blobs at longer distances. Prominent cell-to-cell variability was observed in the long-range contacts between either active genome loci or between Polycomb-bound regions, suggesting an important contribution of stochastic processes to the formation of the Drosophila 3D genome.
Torosin, N. S., Anand, A., Golla, T. R., Cao, W. and Ellison, C. E. (2020). 3D genome evolution and reorganization in the Drosophila melanogaster species group. PLoS Genet 16(12): e1009229. PubMed ID: 33284803
Topologically associating domains, or TADs, are functional units that organize chromosomes into 3D structures of interacting chromatin. While the mechanisms of TAD formation have been well-studied, current knowledge on the patterns of TAD evolution across species is limited. Due to the integral role TADs play in gene regulation, their structure and organization is expected to be conserved during evolution. However, more recent research suggests that TAD structures diverge relatively rapidly. This study used Hi-C chromosome conformation capture to measure evolutionary conservation of whole TADs and TAD boundary elements between D. melanogaster and D. triauraria, two early-branching species from the melanogaster species group which diverged ∼15 million years ago. The majority of TADs were found to have been reorganized since the common ancestor of D. melanogaster and D. triauraria, via a combination of chromosomal rearrangements and gain/loss of TAD boundaries. TAD reorganization between these two species is associated with a localized effect on gene expression, near the site of disruption. By separating TADs into subtypes based on their chromatin state, it was found that different subtypes are evolving under different evolutionary forces. TADs enriched for broadly expressed, transcriptionally active genes are evolving rapidly, potentially due to positive selection, whereas TADs enriched for developmentally-regulated genes remain conserved, presumably due to their importance in restricting gene-regulatory element interactions. These results provide novel insight into the evolutionary dynamics of TADs and help to reconcile contradictory reports related to the evolutionary conservation of TADs and whether changes in TAD structure affect gene expression.
Tchurikov, N. A., Klushevskaya, E. S., Fedoseeva, D. M., Alembekov, I. R., Kravatskaya, G. I., Chechetkin, V. R., Kravatsky, Y. V. and Kretova, O. V. (2020). Dynamics of Whole-Genome Contacts of Nucleoli in Drosophila Cells Suggests a Role for rDNA Genes in Global Epigenetic Regulation. Cells 9(12). PubMed ID: 33287227
Chromosomes are organized into 3D structures that are important for the regulation of gene expression and differentiation. Important role in formation of inter-chromosome contacts play rDNA clusters that make up nucleoli. In the course of differentiation, heterochromatization of rDNA units in mouse cells is coupled with the repression or activation of different genes. Furthermore, the nucleoli of human cells shape the direct contacts with genes that are involved in differentiation and cancer. This study identified and categorized the genes located in the regions where rDNA clusters make frequent contacts. Using a 4C approach, this study demonstrates that in Drosophila S2 cells, rDNA clusters form contacts with genes that are involved in chromosome organization and differentiation. Heat shock treatment induces changes in the contacts between nucleoli and hundreds of genes controlling morphogenesis. Nucleoli form contacts with regions that are enriched with active or repressive histone marks and where small non-coding RNAs are mapped. These data indicate that rDNA contacts are involved in the repression and activation of gene expression and that rDNA clusters orchestrate large groups of Drosophila genes involved in differentiation.
Sun, Q., Perez-Rathke, A., Czajkowsky, D. M., Shao, Z. and Liang, J. (2021). High-resolution single-cell 3D-models of chromatin ensembles during Drosophila embryogenesis. Nat Commun 12(1): 205. PubMed ID: 33420075
Single-cell chromatin studies provide insights into how chromatin structure relates to functions of individual cells. However, balancing high-resolution and genome wide-coverage remains challenging. This study describes a computational method for the reconstruction of large 3D-ensembles of single-cell (sc) chromatin conformations from population Hi-C that was applied to study embryogenesis in Drosophila. With minimal assumptions of physical properties and without adjustable parameters, this method generates large ensembles of chromatin conformations via deep-sampling. This method identifies specific interactions, which constitute 5-6% of Hi-C frequencies, but surprisingly are sufficient to drive chromatin folding, giving rise to the observed Hi-C patterns. Modeled sc-chromatins quantify chromatin heterogeneity, revealing significant changes during embryogenesis. Furthermore, >50% of modeled sc-chromatin maintain topologically associating domains (TADs) in early embryos, when no population TADs are perceptible. Domain boundaries become fixated during development, with strong preference at binding-sites of insulator-complexes upon the midblastula transition. Overall, high-resolution 3D-ensembles of sc-chromatin conformations enable further in-depth interpretation of population Hi-C, improving understanding of the structure-function relationship of genome organization.
Donovan, D. A., Crandall, J. G., Truong, V. N., Vaaler, A. L., Bailey, T. B., Dinwiddie, D., Banks, O. G., McKnight, L. E. and McKnight, J. N. (2021). Basis of specificity for a conserved and promiscuous chromatin remodeling protein. Elife 10. PubMed ID: 33576335
Eukaryotic genomes are organized dynamically through the repositioning of nucleosomes. Isw2 is an enzyme that has been previously defined as a genome-wide, non-specific nucleosome spacing factor. This study shows that Isw2 instead acts as an obligately targeted nucleosome remodeler in vivo through physical interactions with sequence-specific factors. This study demonstrates that Isw2- recruiting factors use small and previously uncharacterized epitopes, which direct Isw2 activity through highly conserved acidic residues in the Isw2 accessory protein Itc1. This interaction orients Isw2 on target nucleosomes, allowing for precise nucleosome positioning at targeted loci. Finally, this study shows that these critical acidic residues have been lost in the Drosophila lineage, potentially explaining the inconsistently characterized function of Isw2-like proteins. Altogether, these data suggest an 'interacting barrier model' where Isw2 interacts with a sequence-specific factor to accurately and reproducibly position a single, targeted nucleosome to define the precise border of phased chromatin arrays.
Farago, A., Urmosi, A., Farkas, A. and Bodai, L. (2021). The histone replacement gene His4r is involved in heat stress induced chromatin rearrangement. Sci Rep 11(1): 4878. PubMed ID: 33649489
His4r is the only known variant of histone H4 in Drosophila. It is encoded by the His4r single-copy gene that is located outside of the histone gene cluster and expressed in a different pattern than H4, although the encoded polypeptides are identical. A null mutant (His4r(Δ42)) was generated which is homozygous viable and fertile without any apparent morphological defects. Heterozygous His4r(Δ42) is a mild suppressor of position-effect variegation, suggesting that His4r has a role in the formation or maintenance of condensed chromatin. Under standard conditions loss of His4r has a modest effect on gene expression. Upon heat-stress the induction of the Heat shock protein (HSP) genes Hsp27 and Hsp68 is stronger in His4r(Δ42) mutants with concordantly increased survival rate. Analysis of chromatin accessibility after heat shock at a Hsp27 regulatory region showed less condensed chromatin in the absence of His4r while there was no difference at the gene body. Interestingly, preconditioning before heat shock led to increased chromatin accessibility, HSP gene transcription and survival rate in control flies while it did not cause notable changes in His4r(Δ42). Thus, these results suggest that His4r might play a role in fine tuning chromatin structure at inducible gene promoters upon environmental stress conditions.

Thursday, March 25th - RNA and Transposons

Chi, W., Liu, W., Fu, W., Xia, S., Heckscher, E. S. and Zhuang, X. (2021). RNA-binding protein syncrip regulates starvation-induced hyperactivity in adult Drosophila. PLoS Genet 17(2): e1009396. PubMed ID: 33617535
How to respond to starvation determines fitness. One prominent behavioral response is increased locomotor activities upon starvation, also known as Starvation-Induced Hyperactivity (SIH). SIH is paradoxical as it promotes food seeking but also increases energy expenditure. Despite its importance in fitness, the genetic contributions to SIH as a behavioral trait remains unexplored. This stduy examined SIH in the Drosophila melanogaster Genetic Reference Panel (DGRP) and performed genome-wide association studies. 23 significant loci were identified, corresponding to 14 genes, significantly associated with SIH in adult Drosophila. Gene enrichment analyses indicated that genes encoding ion channels and mRNA binding proteins (RBPs) were most enriched in SIH. RBPs were especially interesting because they provide a potential mechanism to quickly change protein expression in response to environmental challenges. Using RNA interference, the role of syp was validated in regulating SIH. syp encodes Syncrip (Syp), an RBP. While ubiquitous knockdown of syp led to semi-lethality in adult flies, adult flies with neuron-specific syp knockdown were viable and exhibited decreased SIH. Using the Temporal and Regional Gene Expression Targeting (TARGET) system, the role of Syp was further confirmed in adult neurons in regulating SIH. To determine how syp is regulated by starvation, RNA-seq was performed using the heads of flies maintained under either food or starvation conditions. RNA-seq analyses revealed that syp was alternatively spliced under starvation while its expression level was unchanged. An alternatively-spliced-exon-specific knockout (KO) line was generated, and KO flies were found to show reduced SIH. Together, this study demonstrates a significant genetic contribution to SIH as a behavioral trait, identifies syp as a SIH gene, and highlights the significance of RBPs and post-transcriptional processes in the brain in regulating behavioral responses to starvation.
Yushkova, E. (2020). Involvement of DNA Repair Genes and System of Radiation-Induced Activation of Transposons in Formation of Transgenerational Effects. Front Genet 11: 596947. PubMed ID: 33329741
The study of the genetic basis of the manifestation of radiation-induced effects and their transgenerational inheritance makes it possible to identify the mechanisms of adaptation and possible effective strategies for the survival of organisms in response to chronic radioactive stress. One persistent hypothesis is that the activation of certain genes involved in cellular defense is a specific response of the cell to irradiation. There is also data indicating the important role of transposable elements in the formation of radiosensitivity/radioresistance of biological systems. This work studied the interaction of the systems of hobo transposon activity and DNA repair in the cell under conditions of chronic low-dose irradiation and its participation in the inheritance of radiation-induced transgenerational instability in Drosophila. The results showed a significant increase of sterility and locus-specific mutability, a decrease of survival, fertility and genome stability (an increase the frequency of dominant lethal mutations and DNA damage) in non-irradiated F(1)/F(2) offspring of irradiated parents with dysfunction of the mus304 gene which is responsible for excision and post-replicative recombination repair and repair of double-stranded DNA breaks. The combined action of dysfunction of the mus309 (Bloom syndrome helicase) gene and transpositional activity of hobo elements also led to the transgenerational effects of irradiation but only in the F(1) offspring. Dysfunction of the genes of other DNA repair systems (mus101 and mus210) showed no visible effects inherited from irradiated parents subjected to hobo transpositions. The mei-41 gene showed specificity in this type of interaction, which consists in its higher efficiency in sensing events induced by transpositional activity rather than irradiation.
Zhang, R., Zhao, X., Du, J., Wei, L. and Zhao, Z. (2021). Regulatory mechanism of daily sleep by miR-276a. FASEB J 35(1): e21222. PubMed ID: 33337563
MiRNAs have attracted more attention in recent years as regulators of sleep and circadian rhythms after their roles in circadian rhythm and sleep were discovered. This study explored the roles of the miR-276a on daily sleep in Drosophila melanogaster, and found a regulatory cycle for the miR-276a pathway, in which miR-276a, regulated by the core CLOCK/CYCLE (CLK/CYC) transcription factor upstream, regulates sleep via suppressing targets TIM and NPFR1. (a) Loss of miR-276a function makes the flies sleep more during both daytime and nighttime, while flies with gain of miR-276a function sleep less; (b) MiR-276a is widely expressed in the mushroom body (MB), the pars intercerebralis (PI) and some clock neurons lateral dorsal neurons (LNds), in which tim neurons is important for sleep regulation; (c) MiR-276a promoter is identified to locate in the 8th fragment (aFrag8) of the pre-miR-276a, and this fragment is directly activated and regulated by CLK/CYC; (4) MiR-276a is rhythmically oscillating in heads of the wild-type w(1118), but this oscillation disappears in the loss of function mutant clk(jrk) ; (5) The neuropeptide F receptor 1 (npfr1) was found to be a downstream target of miR-276a. These results clarify that the miR-276a is a very important factor for sleep regulation.
Bejarano, F., Chang, C. H., Sun, K., Hagen, J. W., Deng, W. M. and Lai, E. C. (2021). A comprehensive in vivo screen for anti-apoptotic miRNAs indicates broad capacities for oncogenic synergy. Dev Biol 475: 10-20. PubMed ID: 33662357
microRNAs (miRNAs) are ~21-22 nucleotide (nt) RNAs that mediate broad post-transcriptional regulatory networks. However, genetic analyses have shown that the phenotypic consequences of deleting individual miRNAs are generally far less overt compared to their misexpression. This suggests that miRNA deregulation may have broader phenotypic impacts during disease situations. This concept was explored in the Drosophila eye, by screening for miRNAs whose misexpression could modify the activity of pro-apoptotic factors. Via unbiased and comprehensive in vivo phenotypic assays, this study identified an unexpectedly large set of miRNA hits that can suppress the action of pro-apoptotic genes hid and grim. Secondary assays were used to validate that a subset of these miRNAs can inhibit irradiation-induced cell death. Since cancer cells might seek to evade apoptosis pathways, this situation was modeled by asking whether activation of anti-apoptotic miRNAs could serve as "second hits". Indeed, while clones of the lethal giant larvae (lgl) tumor suppressor are normally eliminated during larval development, this study found that diverse anti-apoptotic miRNAs mediate the survival of lgl mutant clones in third instar larvae. Notably, while certain anti-apoptotic miRNAs can target apoptotic factors, most of the screen hits lack obvious targets in the core apoptosis machinery. These data highlight how a genetic approach can reveal distinct and powerful activities of miRNAs in vivo, including unexpected functional synergies during disease or cancer-relevant settings.
Bush, K. M., Barber, K. R., Martinez, J. A., Tang, S. J. and Wairkar, Y. P. (2021). Drosophila model of anti-retroviral therapy induced peripheral neuropathy and nociceptive hypersensitivity. Biol Open 10(1). PubMed ID: 33504470
The success of antiretroviral therapy (ART) has improved the survival of HIV-infected patients significantly. However, significant numbers of patients on ART whose HIV disease is well controlled show peripheral sensory neuropathy (PSN), suggesting that ART may cause PSN. Although the nucleoside reverse transcriptase inhibitors (NRTIs), one of the vital components of ART, are thought to contribute to PSN, the mechanisms underlying the PSN induced by NRTIs are unclear. This study developed a Drosophila model of NRTI-induced PSN that recapitulates the salient features observed in patients undergoing ART: PSN and nociceptive hypersensitivity. Furthermore, the data demonstrate that pathways known to suppress PSN induced by chemotherapeutic drugs are ineffective in suppressing the PSN or nociception induced by NRTIs. Instead, it was found that increased dynamics of a peripheral sensory neuron may possibly underlie NRTI-induced PSN and nociception. This model provides a solid platform in which to investigate further mechanisms of ART-induced PSN and nociceptive hypersensitivity.
Eastwood, E. L., Jara, K. A., Bornelov, S., Munafo, M., Frantzis, V., Kneuss, E., Barbar, E. J., Czech, B. and Hannon, G. J. (2021). Dimerisation of the PICTS complex via LC8/Cut-up drives co-transcriptional transposon silencing in Drosophila. Elife 10. PubMed ID: 33538693
In animal gonads, the PIWI-interacting RNA (piRNA) pathway guards genome integrity in part through the co-transcriptional gene silencing of transposon insertions. In Drosophila ovaries, piRNA-loaded Piwi detects nascent transposon transcripts and instructs heterochromatin formation through the Panoramix-induced co-transcriptional silencing (PICTS) complex, containing Panoramix, Nxf2 and Nxt1. This study reports that the highly conserved dynein light chain LC8/Cut-up (Ctp) is an essential component of the PICTS complex. Loss of Ctp results in transposon de-repression and a reduction in repressive chromatin marks specifically at transposon loci. In turn, Ctp can enforce transcriptional silencing when artificially recruited to RNA and DNA reporters. This study showrf that Ctp drives dimerisation of the PICTS complex through its interaction with conserved motifs within Panoramix. Artificial dimerisation of Panoramix bypasses the necessity for its interaction with Ctp, demonstrating that conscription of a protein from a ubiquitous cellular machinery has fulfilled a fundamental requirement for a transposon silencing complex.

Wednesday, March 24th - Oogenesis

Tu, R., Duan, B., Song, X., Chen, S., Scott, A., Hall, K., Blanck, J., DeGraffenreid, D., Li, H., Perera, A., Haug, J. and Xie, T. (2020). Multiple Niche Compartments Orchestrate Stepwise Germline Stem Cell Progeny Differentiation. Curr Biol. PubMed ID: 33357404
The niche controls stem cell self-renewal and progenitor differentiation for maintaining adult tissue homeostasis in various organisms. However, it remains unclear whether the niche is compartmentalized to control stem cell self-renewal and stepwise progeny differentiation. In the Drosophila ovary, inner germarial sheath (IGS) cells form a niche for controlling germline stem cell (GSC) progeny differentiation. This study has identified four IGS subpopulations, which form linearly arranged niche compartments for controlling GSC maintenance and multi-step progeny differentiation. Single-cell analysis of the adult ovary has identified four IGS subpopulations (IGS1-IGS4), the identities and cellular locations of which have been further confirmed by fluorescent in situ hybridization. IGS1 and IGS2 physically interact with GSCs and mitotic cysts to control GSC maintenance and cyst formation, respectively, whereas IGS3 and IGS4 physically interact with 16-cell cysts to regulate meiosis, oocyte development, and cyst morphological change. Finally, one follicle cell progenitor population has also been transcriptionally defined for facilitating future studies on follicle stem cell regulation. Therefore, this study has structurally revealed that the niche is organized into multiple compartments for orchestrating stepwise adult stem cell development and has also provided useful resources and tools for further functional characterization of the niche in the future.
Beachum, A. N., Whitehead, K. M., McDonald, S. I., Phipps, D. N., Berghout, H. E. and Ables, E. T. (2021). Orphan nuclear receptor ftz-f1 (NR5A3) promotes egg chamber survival in the Drosophila ovary. G3 (Bethesda) 11(2). PubMed ID: 33693603
Gamete production in mammals and insects is controlled by cell signaling pathways that facilitate communication between germ cells and somatic cells. Nuclear receptor signaling is a key mediator of many aspects of reproduction, including gametogenesis. For example, the NR5A subfamily of nuclear receptors is essential for gonad development and sex steroid production in mammals. Despite the original identification of the NR5A subfamily in the model insect Drosophila melanogaster, it has been unclear whether Drosophila NR5A receptors directly control oocyte production. Ftz-f1 is expressed throughout the ovary, including in germline stem cells, germline cysts, and several populations of somatic cells. This study shows that ftz-f1 is required in follicle cells prior to stage 10 to promote egg chamber survival at the mid-oogenesis checkpoint. The data suggest that egg chamber death in the absence of ftz-f1 is due, at least in part, to failure of follicle cells to exit the mitotic cell cycle or failure to accumulate oocyte-specific factors in the germline. Taken together, these results show that, as in mammals, the NR5A subfamily promotes maximal reproductive output in Drosophila. These data underscore the importance of nuclear receptors in the control of reproduction and highlight the utility of Drosophila oogenesis as a key model for unraveling the complexity of nuclear receptor signaling in gametogenesis.
Chen, M. Y., Tayyeb, A. and Wang, Y. F. (2021). shrub is required for spermatogenesis of Drosophila melanogaster. Arch Insect Biochem Physiol: e21779. PubMed ID: 33660341
Shrub (CG8055) encodes the vps32/snf7 protein, a filament-forming subunit of the ESCRT (endosomal sorting complexes required for transport)-III complex involved in inward membrane budding. It was reported that shrub was required for abscission in female germline stem cells. This study shows that the expression level of shrub in the testis was significantly higher than that in the ovary of 1-day-old Drosophila melanogaster, suggesting a role in male reproduction. nosGal4 driver was used to knockdown shrub specifically in the fly testis and found that this resulted in a significantly lower paternal effect egg hatch rate relative to the control group. Immunofluorescence staining showed that shrub knockdown in fly testes caused an accumulation of early-stage germ cells and lack of spectrin caps. In the late stages (spermiogenesis), the control testis contained multiple compacted spermatid bundles and individualization complexes (ICs) consisting of actin cones, whereas there were scattered spermatid nuclei and only a few ICs with disorganized actin cones in the shrub knockdown testis. Finally, the control seminal vesicle was full of mature sperms with needle-like heads, but in shrub knockdown testis 75% of seminal vesicles had no mature sperms. Knockdown of shrub in fly testes led to upregulated expression of several cytoskeleton-associated genes, and an accumulation of ubiquitylated proteins. These results suggest that knockdown of shrub in fly testes might damage spermatogenesis by affecting transportability.

Diaz-Torres, A., Rosales-Nieves, A. E., Pearson, J. R., Santa-Cruz Mateos, C., Marin-Menguiano, M., Marshall, O. J., Brand, A. H. and Gonzalez-Reyes, A. (2021). Stem cell niche organization in the Drosophila ovary requires the ECM component Perlecan. Curr Biol. PubMed ID: 33621481
Stem cells reside in specialized microenvironments or niches that balance stem cell proliferation and differentiation. The extracellular matrix (ECM) is an essential component of most niches, because it controls niche homeostasis, provides physical support, and conveys extracellular signals. Basement membranes (BMs) are thin ECM sheets that are constituted mainly by Laminins, Perlecan, Collagen IV, and Entactin/Nidogen and surround epithelia and other tissues. Perlecans are secreted proteoglycans that interact with ECM proteins, ligands, receptors, and growth factors such as FGF, PDGF, VEGF, Hedgehog, and Wingless. Thus, Perlecans have structural and signaling functions through the binding, storage, or sequestering of specific ligands. This study used the Drosophila ovary to assess the importance of Perlecan in the functioning of a stem cell niche. Ovarioles in the adult ovary are enveloped by an ECM sheath and possess a tapered structure at their anterior apex termed the germarium. The anterior tip of the germarium hosts the germline niche, where two to four germline stem cells (GSCs) reside together with a few somatic cells: terminal filament cells (TFCs), cap cells (CpCs), and escort cells (ECs). This study reports that niche architecture in the developing gonad requires trol, that niche cells secrete an isoform-specific Perlecan-rich interstitial matrix, and that DE-cadherin-dependent stem cell-niche adhesion necessitates trol. Hence, this study provides evidence to support a structural role for Perlecan in germline niche establishment during larval stages and in the maintenance of a normal pool of stem cells in the adult niche.
Colonnetta, M. M., Lym, L. R., Wilkins, L., Kappes, G., Castro, E. A., Ryder, P. V., Schedl, P., Lerit, D. A. and Deshpande, G. (2021). Antagonism between germ cell-less and Torso receptor regulates transcriptional quiescence underlying germline/soma distinction. Elife 10. PubMed ID: 33459591
Transcriptional quiescence, an evolutionarily conserved trait, distinguishes the embryonic primordial germ cells (PGCs) from their somatic neighbors. In Drosophila melanogaster, PGCs from embryos maternally compromised for germ cell-less (gcl) misexpress somatic genes, possibly resulting in PGC loss. Recent studies documented a requirement for Gcl during proteolytic degradation of the terminal patterning determinant, Torso receptor. This study demonstrates that the somatic determinant of female fate, Sex-lethal (Sxl), is a biologically relevant transcriptional target of Gcl. Underscoring the significance of transcriptional silencing mediated by Gcl, ectopic expression of a degradation-resistant form of Torso (torso(Deg)) can activate Sxl transcription in PGCs, whereas simultaneous loss of torso-like (tsl) reinstates the quiescent status of gcl PGCs. Intriguingly, like gcl mutants, embryos derived from mothers expressing torso(Deg) in the germline display aberrant spreading of pole plasm RNAs, suggesting that mutual antagonism between Gcl and Torso ensures the controlled release of germ-plasm underlying the germline/soma distinction.
Shi, J., Jin, Z., Yu, Y., Zhang, Y., Yang, F., Huang, H., Cai, T. and Xi, R. (2020). A Progressive Somatic Cell Niche Regulates Germline Cyst Differentiation in the Drosophila Ovary. Curr Biol. PubMed ID: 33340458
In the germarium of the Drosophila ovary, developing germline cysts are surrounded by a population of somatic escort cells that are known to function as the niche cells for germline differentiation;(1) however, the underlying molecular mechanisms of this niche function remain poorly understood. Through single-cell gene expression profiling combined with genetic analyses, this study demonstrates that the escort cells can be spatially and functionally divided into two successive domains. The anterior escort cells (aECs) specifically produce ecdysone, which acts on the cystoblast to promote synchronous cell division, whereas the posterior escort cells (pECs) respond to ecdysone signaling and regulate soma-germline cell adhesion to promote the transition from 16-cell cyst-to-egg chamber formation. The patterning of the aEC and pEC domains is independent of the germline but is dependent on JAK/STAT signaling activity, which emanates from the posterior. Thus, a heterogeneous population of escort cells constitutes a stepwise niche environment to orchestrate cystoblast division and differentiation toward egg chamber formation.

Tuesday, March 23rd - Adult Physiology

Cheslock, A., Andersen, M. K. and MacMillan, H. A. (2021). Thermal acclimation alters Na(+)/K(+)-ATPase activity in a tissue-specific manner in Drosophila melanogaster. Comp Biochem Physiol A Mol Integr Physiol: 110934. PubMed ID: 33684554
Insects, like the model species Drosophila melanogaster, lose neuromuscular function and enter a state of paralysis (chill coma) at a population- and species-specific low temperature threshold that is decreased by cold acclimation. Entry into this coma is related to a spreading depolarization in the central nervous system, while recovery involves restoration of electrochemical gradients across muscle cell membranes. The Na(+)/K(+)-ATPase helps maintain ion balance and membrane potential in both the brain and hemolymph (surrounding muscles), and changes in thermal tolerance traits have therefore been hypothesized to be closely linked to variation in the expression and/or activity of this pump in multiple tissues. This study tested this hypothesis by measuring activity and thermal sensitivity of the Na(+)/K(+)-ATPase at the tagma-specific level (head, thorax and abdomen) in warm- (25 °C) and cold-acclimated (15 °C) flies by Na(+)/K(+)-ATPase activity at 15, 20, and 25 °C. Differences in pump activity were related to differences in chill coma temperature, spreading depolarization temperature, and thermal dependence of muscle cell polarization. Differences in pump activity and thermal sensitivity induced by cold acclimation varied in a tissue-specific manner: While cold-acclimated flies had decreased thermal sensitivity of Na(+)/K(+)-ATPase that maintains activity at low temperatures in the thorax (mainly muscle), activity instead decreased in the heads (mainly brain). It is argued that these changes may assist in maintenance of K(+) homeostasis and membrane potential across muscle membranes and discuss how reduced Na(+)/K(+)-ATPase activity in the brain may counterintuitively help insects delay coma onset in the cold.
Cormier, R. J., Strang, R., Menail, H., Touaibia, M. and Pichaud, N. (2021). Systemic and mitochondrial effects of metabolic inflexibility induced by high fat diet in Drosophila melanogaster. Insect Biochem Mol Biol: 103556. PubMed ID: 33626368
Metabolic inflexibility is a condition that occurs following a nutritional stress which causes blunted fuel switching at the mitochondrial level in response to hormonal and cellular signalling. Linked to obesity and obesity related disorders, chronic exposure to a high fat diet (HFD) in animal models has been extensively used to induce metabolic inflexibility and investigate the development of various metabolic diseases. However, many questions concerning the systemic and mitochondrial responses to metabolic inflexibility remain. This study investigated the global and mitochondrial variations following a 10-day exposure to a HFD in adult Drosophila melanogaster. The results show that following 10-day exposure to the HFD, mitochondrial respiration rates measured in isolated mitochondria at the level of complex I were decreased. This was associated with increased contributions of non-classical providers of electrons to the electron transport system (ETS) such as the proline dehydrogenase (ProDH) and the mitochondrial glycerol-3-phosphate dehydrogenase (mtG3PDH) alleviating complex I dysfunctions, as well as with increased ROS production per molecule of oxygen consumed. These results also show an accumulation of metabolites from multiple different metabolic pathways in whole adult Drosophila and a drastic shift in the lipid profile which translated into decreased proportion of saturated and monounsaturated fatty acids combined with an increased proportion of polyunsaturated fatty acids. Thus, these results demonstrate the various responses to the HFD treatment in adult Drosophila melanogaster that are hallmarks of the development of metabolic inflexibility and reinforce this organism as a suitable model for the study of metabolic disorders.
Cobb, T., Damschroder, D. and Wessells, R. (2021). Sestrin regulates acute chill coma recovery in Drosophila melanogaster. Insect Biochem Mol Biol: 103548. PubMed ID: 33549817
When chill-susceptible insects are exposed to low temperatures they enter a temporary state of paralysis referred to as a chill coma. The most well-studied physiological mechanism of chill coma onset and recovery involves regulation of ion homeostasis. Previous studies show that changes in metabolism may also underlie the ability to recovery quickly, but the roles of genes that regulate metabolic homeostasis in chill coma recovery time (CCRT) are not well understood. This study investigated the roles of Sestrin and Spargel (Drosophila homolog of PGC-1α), which are involved in metabolic homeostasis and substrate oxidation, on CCRT in Drosophila melanogaster. sestrin and spargel mutants have impaired CCRT. sestrin is required in the muscle and nervous system tissue for normal CCRT and spargel is required in muscle and adipose. On the basis that exercise induces sestrin and spargel, the interaction of cold and exercise was tested. Pre-treatment with one of these stressors does not consistently confer acute protection against the other. It is concluded that Sestrin and Spargel are important in the chill coma response, independent of their role in exercise.
Chakraborty, A., Sgro, C. M. and Mirth, C. K. (2020). Does local adaptation along a latitudinal cline shape plastic responses to combined thermal and nutritional stress?. Evolution 74(9): 2073-2087. PubMed ID: 33616935
Thermal and nutritional stress are commonly experienced by animals. This will become increasingly so with climate change. Whether populations can plastically respond to such changes will determine their survival. Plasticity can vary among populations depending on the extent of environmental heterogeneity. However, theory conflicts as to whether environmental heterogeneity should increase or decrease plasticity. Using three locally adapted populations of Drosophila melanogaster sampled from a latitudinal gradient, thus study investigated whether plastic responses to combinations of nutrition and temperature increase or decrease with latitude for four traits: egg-adult viability, egg-adult development time, and two body size traits. Employing nutritional geometry, larvae were reared on 25 diets varying in protein and carbohydrate content at two temperatures: 18° and 25°C. Plasticity varied among traits and across the three populations. Viability was highly canalized in all three populations. The tropical population showed the least plasticity for development time, the sub-tropical showed the highest plasticity for wing area, and the temperate population showed the highest plasticity for femur length. No evidence was found of latitudinal plasticity gradients in either direction. These data highlight that differences in thermal variation and resource predictability experienced by populations along a latitudinal cline are not sufficient to predict their plasticity.
Cai, Q., Ji, S., Li, M., Zheng, S., Zhou, X., Guo, H., Deng, S., Zhu, J., Li, D. and Xie, Z. (2021). Theaflavin-regulated Imd condensates control Drosophila intestinal homeostasis and aging. iScience 24(3): 102150. PubMed ID: 33665569
Black tea is the most widely consumed tea drink in the world and has consistently been reported to possess anti-aging benefits. However, whether theaflavins, one type of the characteristic phytochemicals in black tea extracts, are involved in regulating aging and lifespan in consumers remains largely unknown. This study shows that theaflavins play a beneficial role in preventing age-onset intestinal leakage and dysbiosis, thus delaying aging in Drosophila. Mechanistically, theaflavins regulate the condensate assembly of Imd to negatively govern the overactivation of Imd signals in fruit fly intestines. In addition, theaflavins prevent DSS-induced colitis in mice, suggesting theaflavins play a role in modulating intestinal integrity. Overall, this study reveals a molecular mechanism by which theaflavins regulate gut homeostasis likely through controlling Imd coalescence.
Chandrasekaran, S., Schneps, C. M., Dunleavy, R., Lin, C., DeOliveira, C. C., Ganguly, A. and Crane, B. R. (2021). Tuning flavin environment to detect and control light-induced conformational switching in Drosophila cryptochrome. Commun Biol 4(1): 249. PubMed ID: 33637846
Light-induction of an anionic semiquinone (SQ) flavin radical in Drosophila cryptochrome (dCRY) alters the dCRY conformation to promote binding and degradation of the circadian clock protein Timeless (TIM). Specific peptide ligation with sortase A attaches a nitroxide spin-probe to the dCRY C-terminal tail (CTT) while avoiding deleterious side reactions. Pulse dipolar electron-spin resonance spectroscopy from the CTT nitroxide to the SQ shows that flavin photoreduction shifts the CTT ~1 nm and increases its motion, without causing full displacement from the protein. dCRY engineered to form the neutral SQ serves as a dark-state proxy to reveal that the CTT remains docked when the flavin ring is reduced but uncharged. Substitutions of flavin-proximal His378 promote CTT undocking in the dark or diminish undocking in the light, consistent with molecular dynamics simulations and TIM degradation activity. The His378 variants inform on recognition motifs for dCRY cellular turnover and strategies for developing optogenetic tools.

Monday, March 22nd - Disease models

Towler, B. P., Pashler, A. L., Haime, H. J., Przybyl, K. M., Viegas, S. C., Matos, R. G., Morley, S. J., Arraiano, C. M. and Newbury, S. F. (2020). Dis3L2 regulates cell proliferation and tissue growth through a conserved mechanism. PLoS Genet 16(12): e1009297. PubMed ID: 33370287
Dis3L2 is a highly conserved 3'-5' exoribonuclease which is mutated in the human overgrowth disorders Perlman syndrome and Wilms' tumour of the kidney. Using Drosophila melanogaster as a model system, a new dis3L2 null mutant together with wild-type and nuclease-dead genetic lines in Drosophila to were generated demonstrate that the catalytic activity of Dis3L2 is required to control cell proliferation. To understand the cellular pathways regulated by Dis3L2 to control proliferation, RNA-seq was used on dis3L2 mutant wing discs to show that the imaginal disc growth factor Idgf2 is responsible for driving the wing overgrowth. IDGFs are conserved proteins homologous to human chitinase-like proteins such as CHI3L1/YKL-40 which are implicated in tissue regeneration as well as cancers including colon cancer and non-small cell lung cancer. This study also demonstrated that loss of DIS3L2 in human kidney HEK-293T cells results in cell proliferation, illustrating the conservation of this important cell proliferation pathway. Using these human cells, it was shown that loss of DIS3L2 results in an increase in the PI3-Kinase/AKT signalling pathway, which was subsequently shown to contribute towards the proliferation phenotype in Drosophila. This work therefore provides the first mechanistic explanation for DIS3L2-induced overgrowth in humans and flies and identifies an ancient proliferation pathway controlled by Dis3L2 to regulate cell proliferation and tissue growth.
Conte, M., Casas-Tinto, S. and Soler, J. (2021). Modeling invasion patterns in the glioblastoma battlefield. PLoS Comput Biol 17(1): e1008632. PubMed ID: 33513131
Glioblastoma is the most aggressive tumor of the central nervous system, due to its great infiltration capacity. Understanding the mechanisms that regulate the Glioblastoma invasion front is a major challenge with preeminent potential clinical relevances. In the infiltration front, the key features of tumor dynamics relate to biochemical and biomechanical aspects, which result in the extension of cellular protrusions known as tumor microtubes. The coordination of metalloproteases expression, extracellular matrix degradation, and integrin activity emerges as a leading mechanism that facilitates Glioblastoma expansion and infiltration in uncontaminated brain regions. This paper proposes a novel multidisciplinary approach, based on in vivo experiments in Drosophila and mathematical models, that describes the dynamics of active and inactive integrins in relation to matrix metalloprotease concentration and tumor density at the Glioblastoma invasion front. The mathematical model is based on a non-linear system of evolution equations in which the mechanisms leading chemotaxis, haptotaxis, and front dynamics compete with the movement induced by the saturated flux in porous media. This approach is able to capture the relative influences of the involved agents and reproduce the formation of patterns, which drive tumor front evolution. These patterns have the value of providing biomarker information that is related to the direction of the dynamical evolution of the front and based on static measures of proteins in several tumor samples. Furthermore, biomechanical elements, like the tissue porosity, are considered as indicators of the healthy tissue resistance to tumor progression.
Bolukbasi, E., Woodling, N. S., Ivanov, D. K., Adcott, J., Foley, A., Rajasingam, A., Gittings, L. M., Aleyakpo, B., Niccoli, T., Thornton, J. M. and Partridge, L. (2021). Cell type-specific modulation of healthspan by Forkhead family transcription factors in the nervous system. Proc Natl Acad Sci U S A 118(8). PubMed ID: 33593901
Reduced activity of insulin/insulin-like growth factor signaling (IIS) increases healthy lifespan among diverse animal species. Downstream of IIS, multiple evolutionarily conserved transcription factors (TFs) are required; however, distinct TFs are likely responsible for these effects in different tissues.this study asked which TFs can extend healthy lifespan within distinct cell types of the adult nervous system in Drosophila. Starting from published single-cell transcriptomic data, it is reported that forkhead (FKH) is endogenously expressed in neurons, whereas forkhead-box-O (FOXO) is expressed in glial cells. Accordingly, this study found that neuronal FKH and glial FOXO exert independent prolongevity effects. This study further explored the role of neuronal FKH in a model of Alzheimer's disease-associated neuronal dysfunction, where it as found that increased neuronal FKH preserves behavioral function and reduces ubiquitinated protein aggregation. Finally, using transcriptomic profiling, Atg17 was identified a member of the Atg1 autophagy initiation family, as one FKH-dependent target whose neuronal overexpression is sufficient to extend healthy lifespan. Taken together, these results underscore the importance of cell type-specific mapping of TF activity to preserve healthy function with age.
Cheng, Y., Pitoniak, A., Wang, J. and Bohmann, D. (2021). Preserving transcriptional stress responses as an anti-aging strategy. Aging Cell 20(2): e13297. PubMed ID: 33474790
The progressively increasing frailty, morbidity and mortality of aging organisms coincides with, and may be causally related to, their waning ability to adapt to environmental perturbations. Transcriptional responses to challenges, such as oxidative stress or pathogens, diminish with age. This effect is manifest in the declining function of the stress responsive transcription factor Nrf2. Protective gene expression programs that are controlled by the Drosophila Nrf2 homolog, CncC, support homeostasis and longevity. Age-associated chromatin changes make these genes inaccessible to CncC binding and render them inert to signal-dependent transcriptional activation in old animals. In a previous paper, it was reported that overexpression of the CncC dimerization partner Maf-S counteracts this degenerative effect and preserves organism fitness. Building on this work, this study shows that Maf-S overexpression prevents loss of chromatin accessibility and maintains gene responsiveness. Moreover, the same outcome, along with an extension of lifespan, can be achieved by inducing CncC target gene expression pharmacologically throughout adult life. Thus, pharmacological or dietary interventions that can preserve stress responsive gene expression may be feasible anti-aging strategies.
Talsness, D. M., Owings, K. G., Coelho, E., Mercenne, G., Pleinis, J. M., Partha, R., Hope, K. A., Zuberi, A. R., Clark, N. L., Lutz, C. M., Rodan, A. R. and Chow, C. Y. (2020). A Drosophila screen identifies NKCC1 as a modifier of NGLY1 deficiency. Elife 9. PubMed ID: 33315011
N-Glycanase 1 (NGLY1) is a cytoplasmic deglycosylating enzyme. Loss-of-function mutations in the NGLY1 gene cause NGLY1 deficiency, which is characterized by developmental delay, seizures, and a lack of sweat and tears. To model the phenotypic variability observed among patients, a Drosophila model of NGLY1 deficiency was crossed onto a panel of genetically diverse strains. The resulting progeny showed a phenotypic spectrum from 0 to 100% lethality. Association analysis on the lethality phenotype, as well as an evolutionary rate covariation analysis, generated lists of modifying genes, providing insight into NGLY1 function and disease. The top association hit was Ncc69 (human NKCC1/2), a conserved ion transporter. Analyses in NGLY1-/- mouse cells demonstrated that NKCC1 has an altered average molecular weight and reduced function. The misregulation of this ion transporter may explain the observed defects in secretory epithelium function in NGLY1 deficiency patients.
Yang, S., Tian, M. and Johnson, A. N. (2020). SARS-CoV-2 protein ORF3a is pathogenic in Drosophila and causes phenotypes associated with COVID-19 post-viral syndrome. bioRxiv. PubMed ID: 33398283
The Coronavirus Disease 2019 (COVID-19) pandemic has caused millions of deaths and will continue to exact incalculable tolls worldwide. While great strides have been made toward understanding and combating the mechanisms of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infection, relatively little is known about the individual SARS-CoV-2 proteins that contribute to pathogenicity during infection and that cause neurological sequela after viral clearance. Drosophila was used to develop an in vivo model that characterizes mechanisms of SARS-CoV-2 pathogenicit; SARS-CoV-2 ORF3a adversely affected longevity and motor function by inducing apoptosis and inflammation in the nervous system. Chloroquine alleviated ORF3a induced phenotypes in the CNS, arguing the Drosophila model is amenable to high throughput drug screening. This work provides novel insights into the pathogenic nature of SARS-CoV-2 in the nervous system that can be used to develop new treatment strategies for post-viral syndrome. In summary, SARS-CoV-2 ORF3a is pathogenic in the nervous system.ORF3a induces cell death, inflammation, and lysosome dysfunction.Chloroquine protects against ORF3a induced CNS distress and lysosome dysfunction.

Friday, March 19th - Adult development

Zhang, W., Reeves, G. R. and Tautz, D. (2021). Testing Implications of the Omnigenic Model for the Genetic Analysis of Loci Identified through Genome-wide Association. Curr Biol. PubMed ID: 33417882
Organismal phenotypes usually have a quantitative distribution, and their genetic architecture can be studied by genome-wide association (GWA) mapping approaches. In most of such studies, it has become clear that many genes of moderate or small effects contribute to the phenotype. Hence, the attention has turned toward the loci falling below the GWA cut-off, which may contribute to the phenotype through modifier interactions with a set of core genes, as proposed in the omnigenic model. One can thus predict that both moderate effect GWA-derived candidate genes and randomly chosen genes should have a similar likelihood to affect a given phenotype when they are analyzed via gene disruption assays. This hypothesis was tested by using an automated phenotyping system for Drosophila pupal phenotypes. First candidate genes for pupal length were identified in a GWA based on the Drosophila Genetic Reference Panel (DGRP), and most of these candidate genes were shown to be involved in the phenotype. Then genes below a GWA significance threshold were randomly chosen, and three-quarters of them had also an effect on the trait with comparable effect sizes as the GWA candidate genes. The effects of these knockout lines were further tested on an independent behavioral pupal trait (pupation site choice); a similar fraction had a significant effect as well. These data thus confirm the implication that a large number of genes can influence independent quantitative traits.
Akai, N., Ohsawa, S., Sando, Y. and Igaki, T. (2021). Epithelial cell-turnover ensures robust coordination of tissue growth in Drosophila ribosomal protein mutants. PLoS Genet 17(1): e1009300. PubMed ID: 33507966
Highly reproducible tissue development is achieved by robust, time-dependent coordination of cell proliferation and cell death. To study the mechanisms underlying robust tissue growth, this study analyzed the developmental process of wing imaginal discs in Drosophila Minute mutants, a series of heterozygous mutants for a ribosomal protein gene. Minute animals show significant developmental delay during the larval period but develop into essentially normal flies, suggesting there exists a mechanism ensuring robust tissue growth during abnormally prolonged developmental time. Surprisingly, this study found that both cell death and compensatory cell proliferation were dramatically increased in developing wing pouches of Minute animals. Blocking the cell-turnover by inhibiting cell death resulted in morphological defects, indicating the essential role of cell-turnover in Minute wing morphogenesis. These analyses showed that Minute wing discs elevate Wg expression and JNK-mediated Dilp8 expression that causes developmental delay, both of which are necessary for the induction of cell-turnover. Furthermore, forced increase in Wg expression together with developmental delay caused by ecdysone depletion induced cell-turnover in the wing pouches of non-Minute animals. These findings suggest a novel paradigm for robust coordination of tissue growth by cell-turnover, which is induced when developmental time axis is distorted.
Chu, W. C. and Hayashi, S. (2021). Mechano-chemical enforcement of tendon apical ECM into nano-filaments during Drosophila flight muscle development. Curr Biol. PubMed ID: 33545042
Contractile tension is critical for musculoskeletal system development and maintenance. In insects, the muscular force is transmitted to the exoskeleton through the tendon cells and tendon apical extracellular matrix (ECM). In Drosophila, tendon cells were found to secrete Dumpy (Dpy), a zona pellucida domain (ZPD) protein, to form the force-resistant filaments in the exuvial space, anchoring the tendon cells to the pupal cuticle. Dpy undergoes filamentous conversion in response to the tension increment during indirect flight muscle development. Another ZPD protein Quasimodo (Qsm) was found to protect the notum epidermis from collapsing under the muscle tension by enhancing the tensile strength of Dpy filaments. Qsm is co-transported with Dpy in the intracellular vesicles and diffuses into the exuvial space after secretion. Tissue-specific qsm expression rescued the qsm mutant phenotypes in distant tissues, suggesting Qsm can function in a long-range, non-cell-autonomous manner. In the cell culture assay, Qsm interacts with Dpy-ZPD and promotes secretion and polymerization of Dpy-ZPD. The roles of Qsm underlies the positive feedback mechanism of force-dependent organization of Dpy filaments, providing new insights into apical ECM remodeling through the unconventional interaction of ZPD proteins.
Zuber, R., Wang, Y., Gehring, N., Bartoszewski, S. and Moussian, B. (2020). Tweedle proteins form extracellular two-dimensional structures defining body and cell shape in Drosophila melanogaster. Open Biol 10(12): 200214. PubMed ID: 33292106
Tissue function and shape rely on the organization of the extracellular matrix (ECM) produced by the respective cells. Understanding of the underlying molecular mechanisms is limited. This study shows that extracellular Tweedle (Twdl) proteins in the fruit fly Drosophila melanogaster form two adjacent two-dimensional sheets underneath the cuticle surface and above a distinct layer of dityrosinylated and probably elastic proteins enwrapping the whole body. Dominant mutations in twdl genes cause ectopic spherical aggregation of Twdl proteins that recruit dityrosinylated proteins at their periphery within lower cuticle regions. These aggregates perturb parallel ridges at the surface of epidermal cells that have been demonstrated to be crucial for body shaping. In one scenario, hence, this disorientation of epidermal ridges may explain the squatty phenotype of twdl mutant larvae. In an alternative scenario, this phenotype may be due to the depletion of the dityrosinylated and elastic layer, and the consequent weakening of cuticle resistance against the internal hydrostatic pressure. According to Barlow's formula describing the distribution of internal pressure forces in pipes in dependence of pipe wall material properties, it follows that this reduction in turn causes lateral expansion at the expense of the antero-posterior elongation of the body.
Parker, J. and Struhl, G. (2020). Control of Drosophila wing size by morphogen range and hormonal gating. Proc Natl Acad Sci U S A 117(50): 31935-31944. PubMed ID: 33257577
The stereotyped dimensions of animal bodies and their component parts result from tight constraints on growth. Yet, the mechanisms that stop growth when organs reach the right size are unknown. Growth of the Drosophila wing-a classic paradigm-is governed by two morphogens, Decapentaplegic (Dpp, a BMP) and Wingless (Wg, a Wnt). Wing growth during larval life ceases when the primordium attains full size, concomitant with the larval-to-pupal molt orchestrated by the steroid hormone ecdysone. This study blocked the molt by genetically dampening ecdysone production, creating an experimental paradigm in which the wing stops growing at the correct size while the larva continues to feed and gain body mass. Under these conditions, wing growth is limited by the ranges of Dpp and Wg, and by ecdysone, which regulates the cellular response to their signaling activities. Further, evidence is presented that growth terminates because of the loss of two distinct modes of morphogen action: 1) maintenance of growth within the wing proper and 2) induced growth of surrounding "pre-wing" cells and their recruitment into the wing. These results provide a precedent for the control of organ size by morphogen range and the hormonal gating of morphogen action.
White, M. A., Bonfini, A., Wolfner, M. F. and Buchon, N. (2021). Drosophila melanogaster sex peptide regulates mated female midgut morphology and physiology. Proc Natl Acad Sci U S A 118(1). PubMed ID: 33443193
Drosophila melanogaster females experience a large shift in energy homeostasis after mating to compensate for nutrient investment in egg production. To cope with this change in metabolism, mated females undergo widespread physiological and behavioral changes, including increased food intake and altered digestive processes. The mechanisms by which the female digestive system responds to mating remain poorly characterized. This study demonstrates that the seminal fluid protein Sex Peptide (SP) is a key modulator of female post-mating midgut growth and gene expression. SP is both necessary and sufficient to trigger post-mating midgut growth in females under normal nutrient conditions, and likely acting via its receptor, Sex Peptide Receptor (SPR). Moreover, SP is responsible for almost the totality of midgut transcriptomic changes following mating, including up-regulation of protein and lipid metabolism genes and down-regulation of carbohydrate metabolism genes. These changes in metabolism may help supply the female with the nutrients required to sustain egg production. Thus, this study reports a role for SP in altering female physiology to enhance reproductive output: Namely, SP triggers the switch from virgin to mated midgut state.

Thursday, March 18th - Adult Physiology

Strilbytska, O. M., Zayachkivska, A., Koliada, A., Galeotti, F., Volpi, N., Storey, K. B., Vaiserman, A. and Lushchak, O. (2020). Anise Hyssop Agastache foeniculum Increases Lifespan, Stress Resistance, and Metabolism by Affecting Free Radical Processes in Drosophila. Front Physiol 11: 596729. PubMed ID: 33391017
Anise hyssop, Agastache foeniculum, is a widely used medicinal herb with known antioxidant properties. This study examined how dietary supplementation with dried A. foeniculum leaf powder affected physiological and metabolic traits as well as activities of antioxidant enzymes and markers of oxidative stress in Drosophila melanogaster. Dietary hyssop extended the lifespan in a sex and genotype independent manner over a broad range of concentrations up to 30 mg/ml. Dietary supplementation with the herb significantly increased fecundity, resistance to oxidative stress and starvation. Higher transcript levels of Drosophila insulin-like peptide (dilp2) and decreased dilp3 and dilp6 transcripts together with increased levels of glycogen and triacylglycerols support an alteration of insulin signaling by the plant extract. Increased enzymatic activities of superoxide dismutase and aconitase as well as elevated protein and low molecular mass thiols also supported an alteration of free radical process in flies treated with dietary A. foeniculum leaf powder. Thus, physiological and metabolic traits as well as free radical processed may be affected by active compounds detected in extracts of anise hyssop leaves and contribute to the increased lifespan and reproductive (egg-laying) activity observed.
Vincow, E. S., Thomas, R. E., Merrihew, G. E., MacCoss, M. J. and Pallanck, L. J. (2021). Slowed protein turnover in aging Drosophila reflects a shift in cellular priorities. J Gerontol A Biol Sci Med Sci. PubMed ID: 33453098
The accumulation of protein aggregates and dysfunctional organelles as organisms age has led to the hypothesis that aging involves general breakdown of protein quality control. This hypothesis was tested using a proteomic and informatic approach in the fruit fly Drosophila melanogaster. Turnover of most proteins was markedly slower in old flies. However, ribosomal and proteasomal proteins maintained high turnover rates, suggesting that the observed slowdowns in protein turnover might not be due to a global failure of quality control. As protein turnover reflects the balance of protein synthesis and degradation, whether decreases in synthesis or decreases in degradation would best explain the observed slowdowns in protein turnover was investigated. It was found that while many individual proteins in old flies showed slower turnover due to decreased degradation, an approximately equal number showed slower turnover due to decreased synthesis, and enrichment analyses revealed that translation machinery itself was less abundant. Mitochondrial complex I subunits and glycolytic enzymes were decreased in abundance as well, and proteins involved in glutamine-dependent anaplerosis were increased, suggesting that old flies modify energy production to limit oxidative damage. Together, these findings suggest that age-related proteostasis changes in Drosophila represent a coordinated adaptation rather than a systems collapse.
Walls, S., Diop, S., Birse, R., Elmen, L., Gan, Z., Kalvakuri, S., Pineda, S., Reddy, C., Taylor, E., Trinh, B., Vogler, G., Zarndt, R., McCulloch, A., Lee, P., Bhattacharya, S., Bodmer, R. and Ocorr, K. (2020). Prolonged Exposure to Microgravity Reduces Cardiac Contractility and Initiates Remodeling in Drosophila. Cell Rep 33(10): 108445. PubMed ID: 33242407
Understanding the effects of microgravity on human organs is crucial to exploration of low-earth orbit, the moon, and beyond. Drosophila can be sent to space in large numbers to examine the effects of microgravity on heart structure and function, which is fundamentally conserved from flies to humans. Flies reared in microgravity exhibit cardiac constriction with myofibrillar remodeling and diminished output. RNA sequencing (RNA-seq) in isolated hearts revealed reduced expression of sarcomeric/extracellular matrix (ECM) genes and dramatically increased proteasomal gene expression, consistent with the observed compromised, smaller hearts and suggesting abnormal proteostasis. This was examined further on a second flight in which dramatically elevated proteasome aggregates were found co-localizing with increased amyloid and polyQ deposits. Remarkably, in long-QT causing sei/hERG mutants, proteasomal gene expression at 1g, although less than the wild-type expression, was nevertheless increased in microgravity. Therefore, cardiac remodeling and proteostatic stress may be a fundamental response of heart muscle to microgravity.
Wang, L. J., Hsu, T., Lin, H. L. and Fu, C. Y. (2020). Drosophila MICOS knockdown impairs mitochondrial structure and function and promotes mitophagy in muscle tissue. Biol Open 9(12). PubMed ID: 33268479
The mitochondrial contact site and cristae organizing system (MICOS) is a multi-protein interaction hub that helps define mitochondrial ultrastructure. While the functional importance of MICOS is mostly characterized in yeast and mammalian cells in culture, the contributions of MICOS to tissue homeostasis in vivo remain further elucidation. This study examined how knocking down expression of Drosophila MICOS genes affects mitochondrial function and muscle tissue homeostasis. CG5903/MIC26-MIC27 was found to colocalize and function with Mitofilin/MIC60 and QIL1/MIC13 as a Drosophila MICOS component; knocking down expression of any of these three genes predictably altered mitochondrial morphology, causing loss of cristae junctions, and disruption of cristae packing. Furthermore, the knockdown flies exhibited low mitochondrial membrane potential, fusion/fission imbalances, increased mitophagy, and limited cell death. Reductions in climbing ability indicated deficits in muscle function. Knocking down MICOS genes also caused reduced mtDNA content and fragmented mitochondrial nucleoid structure in Drosophila. Together, these data demonstrate an essential role of Drosophila MICOS in maintaining proper homeostasis of mitochondrial structure and function to promote the function of muscle tissue.
Banerjee, S., Woods, C., Burnett, M., Park, S. J., Ja, W. W. and Curtiss, J. (2021). The Drosophila melanogaster Neprilysin Nepl15 is involved in lipid and carbohydrate storage. Sci Rep 11(1): 2099. PubMed ID: 33483521
The prototypical M13 peptidase, human Neprilysin, functions as a transmembrane "ectoenzyme" that cleaves neuropeptides that regulate e.g. glucose metabolism, and has been linked to type 2 diabetes. The M13 family has undergone a remarkable, and conserved, expansion in the Drosophila genus. This study describes the function of Drosophila melanogaster Neprilysin-like 15 (Nepl15). Nepl15 is likely to be a secreted protein, rather than a transmembrane protein. Nepl15 has changes in critical catalytic residues that are conserved across the Drosophila genus and likely renders the Nepl15 protein catalytically inactive. Nevertheless, a knockout of the Nepl15 gene reveals a reduction in triglyceride and glycogen storage, with the effects likely occurring during the larval feeding period. Conversely, flies overexpressing Nepl15 store more triglycerides and glycogen. Protein modeling suggests that Nepl15 is able to bind and sequester peptide targets of catalytically active Drosophila M13 family members, peptides that are conserved in humans and Drosophila, potentially providing a novel mechanism for regulating the activity of neuropeptides in the context of lipid and carbohydrate homeostasis.
Baumgartner, M. E., Dinan, M. P., Langton, P. F., Kucinski, I. and Piddini, E. (2021). Proteotoxic stress is a driver of the loser status and cell competition. Nat Cell Biol 23(2): 136-146. PubMed ID: 33495633
Cell competition allows winner cells to eliminate less fit loser cells in tissues. In Minute cell competition, cells with a heterozygous mutation in ribosome genes, such as RpS3(+/-) cells, are eliminated by wild-type cells. How cells are primed as losers is partially understood and it has been proposed that reduced translation underpins the loser status of ribosome mutant, or Minute, cells. Using Drosophila this study shows that reduced translation does not cause cell competition. Instead, proteotoxic stress was identified as the underlying cause of the loser status for Minute competition and competition induced by mahjong, an unrelated loser gene. RpS3(+/-) cells exhibit reduced autophagic and proteasomal flux, accumulate protein aggregates and can be rescued from competition by improving their proteostasis. Conversely, inducing proteotoxic stress is sufficient to turn otherwise wild-type cells into losers. Thus, it is proposed that tissues may preserve their health through a proteostasis-based mechanism of cell competition and cell selection.

Wednesday, March 17th - Signaling

Parra, A. S. and Johnston, C. A. (2020). Mud Loss Restricts Yki-Dependent Hyperplasia in Drosophila Epithelia. J Dev Biol 8(4). PubMed ID: 33322177
Tissue development demands precise control of cell proliferation and organization, which is achieved through multiple conserved signaling pathways and protein complexes in multicellular animals. Epithelia are a ubiquitous tissue type that provide diverse functions including physical protection, barrier formation, chemical exchange, and secretory activity. However, epithelial cells are also a common driver of tumorigenesis; thus, understanding the molecular mechanisms that control their growth dynamics is important in understanding not only developmental mechanisms but also disease. One prominent pathway that regulates epithelial growth is the conserved Hippo/Warts/Yorkie network. Hippo/Warts inactivation, or activating mutations in Yorkie that prevent its phosphorylation (e.g., Yki(S168A)), drive hyperplastic tissue growth. It has been shown that loss of Mushroom body defect (Mud), a microtubule-associated protein that contributes to mitotic spindle function, restricts Yki(S168A)-mediated growth in Drosophila imaginal wing disc epithelia. This study shows that Mud loss alters cell cycle progression and triggers apoptosis with accompanying Jun kinase (JNK) activation in Yki(S168A)-expressing discs. To identify additional molecular insights, RNAseq and differential gene expression profiling were performed. This analysis revealed that Mud knockdown in Yki(S168A)-expressing discs resulted in a significant downregulation in expression of core basement membrane (BM) and extracellular matrix (ECM) genes, including the type IV collagen gene viking. Furthermore, it was found that Yki(S168A)-expressing discs accumulated increased collagen protein, which was reduced following Mud knockdown. These results suggest that ECM/BM remodeling can limit untoward growth initiated by an important driver of tumor growth and highlight a potential regulatory link with cytoskeleton-associated genes.
Zhang, C., Seong, K. M., Sun, W., Mittapalli, O., Qiu, B., Clark, J. M. and Pittendrigh, B. R. (2021). The insulin signaling pathway in Drosophila melanogaster: A nexus revealing an "Achilles' heel" in DDT resistance. Pestic Biochem Physiol 171: 104727. PubMed ID: 33357549
Insecticide resistance is an ongoing challenge in agriculture and disease vector control. This study demonstrates a novel strategy to attenuate resistance. This study used genomics tools to target fundamental energy-associated pathways and identified a potential "Achilles' heel" for resistance, a resistance-associated protein that, upon inhibition, results in a substantial loss in the resistance phenotype. Specifically, the gene expression profiles and structural variations of the insulin/insulin-like growth factor signaling (IIS) pathway genes were compared in DDT-susceptible (91-C) and -resistant (91-R) Drosophila melanogaster (Drosophila) strains. A total of eight and seven IIS transcripts were up- and down-regulated, respectively, in 91-R compared to 91-C. A total of 114 nonsynonymous mutations were observed between 91-C and 91-R, of which 51.8% were fixed. Among the differentially expressed transcripts, phosphoenolpyruvate carboxykinase (PEPCK), down-regulated in 91-R, encoded the greatest number of amino acid changes, prompting the performance of PEPCK inhibitor-pesticide exposure bioassays. The inhibitor of PEPCK, hydrazine sulfate, resulted in a 161- to 218-fold decrease in the DDT resistance phenotype (91-R) and more than a 4- to 5-fold increase in susceptibility in 91-C. A second target protein, Glycogen synthase kinase 3β (GSK3β-PO), had one amino acid difference between 91-C and 91-R, and the corresponding transcript was also down-regulated in 91-R. A GSK3β-PO inhibitor, lithium chloride, likewise reduced the resistance but to a lesser extent than did hydrazine sulfate for PEPCK. This study has demonstrated the potential role of IIS genes in DDT resistance and the potential discovery of an "Achilles' heel" against pesticide resistance in this pathway.
Srivastava, D., de Toledo, M., Manchon, L., Tazi, J. and Juge, F. (2020). Modulation of Yorkie activity by alternative splicing is required for developmental stability. Embo j: e104895. PubMed ID: 33320356
The Hippo signaling pathway is a major regulator of organ growth, which controls the activity of the transcription coactivator Yorkie (Yki) in Drosophila and its homolog YAP in mammals. Both Yki and YAP proteins exist as alternatively spliced isoforms containing either one or two WW domains. The biological importance of this conserved alternative splicing event is unknown. This study identified the splicing factor B52 as a regulator of yki alternative splicing in Drosophila and shows that B52 modulates growth in part through modulation of yki alternative splicing. Yki isoforms differ by their transcriptional activity as well as their ability to bind and bridge PPxY motifs-containing partners, and can compete in vivo. Strikingly, flies in which yki alternative splicing has been abrogated, thus expressing only Yki2 isoform, exhibit fluctuating wing asymmetry, a signal of developmental instability. The results identify yki alternative splicing as a new level of modulation of the Hippo pathway, that is required for growth equilibration during development. This study provides the first demonstration that the process of alternative splicing contributes to developmental robustness.
Sharma, V., Mutsuddi, M. and Mukherjee, A. (2020). Deltex cooperates with TRAF6 to promote apoptosis and cell migration through Eiger-independent JNK activation in Drosophila. Cell Biol Int. PubMed ID: 33300258
JNK signaling is a highly conserved signaling pathway that regulates a broad spectrum of cellular processes including cell proliferation, migration, and apoptosis. The tumor necrosis factor receptor (TNFR) associated factor 6 (TRAF6) is an adaptor protein, which transduces the signal from TNFRs and Toll-like receptor/interleukin-1 receptor superfamily to induce a wide spectrum of cellular responses. TRAF6 also acts as the adaptor protein that mediates Eiger/JNK signaling in Drosophila. In a genetic interaction study, deltex (Dx) was identified as a novel interactor of TRAF6. Dx is well known to regulate Notch signaling in a context-dependent manner. The data suggest that combinatorial action of Dx and TRAF6 enhances the Dx-induced wing nicking phenotype by inducing caspase-mediated cell death. Co-expression of Dx and TRAF6 also results in enhanced invasive behavior and perturbs the normal morphology of cells. The cooperative action of Dx and TRAF6 is attributed to JNK activation, which also leads to ectopic wingless (Wg) and decapentaplegic (Dpp) expression. The results also reveal that the endocytic pathway component Rab7 may play a pivotal role in the regulation of Dx-TRAF6-mediated activation of JNK signaling. This study presents the fact that Dx and TRAF6 together activate JNK signaling in an Eiger-independent mechanism.
Sawyer, J. K., Kabiri, Z., Montague, R. A., Allen, S. R., Stewart, R., Paramore, S. V., Cohen, E., Zaribafzadeh, H., Counter, C. M. and Fox, D. T. (2020). Exploiting codon usage identifies intensity-specific modifiers of Ras/MAPK signaling in vivo. PLoS Genet 16(12): e1009228. PubMed ID: 33296356
Signal transduction pathways are intricately fine-tuned to accomplish diverse biological processes. An example is the conserved Ras/mitogen-activated-protein-kinase (MAPK) pathway, which exhibits context-dependent signaling output dynamics and regulation. By altering codon usage as a novel platform to control signaling output, the Drosophila genome was screened for modifiers specific to either weak or strong Ras-driven eye phenotypes. This screen enriched for regions of the genome not previously connected with Ras phenotypic modification. The underlying gene from one modifier mapped to the ribosomal gene RpS21. In multiple contexts, it was shown that RpS21 preferentially influences weak Ras/MAPK signaling outputs. These data show that codon usage manipulation can identify new, output-specific signaling regulators, and identify RpS21 as an in vivo Ras/MAPK phenotypic regulator.
Basak, B., Krishnan, H. and Padinjat, R. (2021). Interdomain interactions regulate the localization of a lipid transfer protein at ER-PM contact sites. Biol Open. PubMed ID: 33597200
During phospholipase C-β (PLC-β) signalling in Drosophila photoreceptors, the phosphatidylinositol transfer protein (PITP) RDGB, is required for lipid transfer at endoplasmic reticulum (ER)-plasma membrane (PM) contact sites (MCS). Depletion of RDGB or its mis-localization away from the ER-PM MCS results in multiple defects in photoreceptor function. Previously, the interaction between the FFAT motif of RDGB and the integral ER protein dVAP-A was shown to be essential for accurate localization to ER-PM MCS. This study reports that the FFAT/dVAP-A interaction alone is insufficient to localize RDGB accurately; this also requires the function of the C-terminal domains, DDHD and LNS2. Mutations in each of these domains results in mis-localization of RDGB leading to loss of function. While the LNS2 domain is necessary, it is not sufficient for the correct localization of RDGB, which also requires the C-terminal DDHD domain. The function of the DDHD domain is mediated through an intramolecular interaction with the LNS2 domain. Thus, interactions between the additional domains in a multi-domain PITP together lead to accurate localization at the MCS and signalling function.

Tuesday, March 16th - Adult neural development and function

Zhang, Y., Ng, R., Neville, M. C., Goodwin, S. F. and Su, C. Y. (2020). Distinct Roles and Synergistic Function of Fru(M) Isoforms in Drosophila Olfactory Receptor Neurons. Cell Rep 33(11): 108516. PubMed ID: 33326795
Sexual dimorphism in Drosophila courtship circuits requires the male-specific transcription factor fru(M), which is alternatively spliced to encode the Fru(MA), Fru(MB), and Fru(MC) isoforms. Most ffru(M)-positive neurons express multiple variants; however, the functional significance of their co-expression remains undetermined. Do co-expressed isoforms each play unique roles to jointly regulate dimorphism? By focusing on fru(M)-positive olfactory receptor neurons (ORNs), this study shows that Fru(MB) and Fru(MC) are both required for males' age-dependent sensitization to aphrodisiac olfactory cues in a cell-autonomous manner. Interestingly, Fru(MB) expression is upregulated with age in Or47b and Ir84a ORNs, and its overexpression mimics the effect of age in elevating olfactory responses. Mechanistically, Fru(MB) and Fru(MC) synergistically mediate response sensitization through cooperation of their respective downstream effectors, namely, PPK25 and PPK23, which are both required for forming a functional amplification channel in ORNs. Together, these results provide critical mechanistic insight into how co-expressed Fru(M) isoforms jointly coordinate dimorphic neurophysiology.
Wu, C., Boisclair Lachance, J. F., Ludwig, M. Z. and Rebay, I. (2020). A context-dependent bifurcation in the Pointed transcriptional effector network contributes specificity and robustness to retinal cell fate acquisition. PLoS Genet 16(11): e1009216. PubMed ID: 33253156
Recruitment of R1-R7 photoreceptor fates requires reiterative receptor tyrosine kinase / mitogen activated protein kinase (MAPK) signaling mediated by the transcriptional effector Pointed (Pnt). However the overall signaling levels experienced by R2-R5 cells are distinct from those experienced by R1, R6 and R7. A relay mechanism between two Pnt isoforms initiated by MAPK activation directs the universal transcriptional response. This study asked how the generic Pnt response is tailored to these two rounds of photoreceptor fate transitions. During R2-R5 specification PntP2 was found to be coexpressed with a closely related but previously uncharacterized isoform, PntP3. Under otherwise wild type conditions, R2-R5 fate specification is robust to loss of either PntP2 or PntP3, and the two activate pntP1 redundantly; however under conditions of reduced MAPK activity, both are required. Mechanistically, the data suggest that intrinsic activity differences between PntP2 and PntP3, combined with positive and unexpected negative transcriptional auto- and cross-regulation, buffer first-round fates against conditions of compromised RTK signaling. In contrast, in a mechanism that may be adaptive to the stronger signaling environment used to specify R1, R6 and R7 fates, the Pnt network resets to a simpler topology in which PntP2 uniquely activates pntP1 and auto-activates its own transcription. It is proposed that differences in expression patterns, transcriptional activities and regulatory interactions between Pnt isoforms together facilitate context-appropriate cell fate specification in different signaling environments.
Zhao, Z., Zhao, X., He, T., Wu, X., Lv, P., Zhu, A. J. and Du, J. (2021). Epigenetic regulator Stuxnet modulates octopamine effect on sleep through a Stuxnet-Polycomb-Octbeta2R cascade. EMBO Rep: e47910. PubMed ID: 33410264
Sleep homeostasis is crucial for sleep regulation. The role of epigenetic regulation in sleep homeostasis is unestablished. Previous studies showed that octopamine is important for sleep homeostasis. However, the regulatory mechanism of octopamine reception in sleep is unknown. This study identified an epigenetic regulatory cascade (Stuxnet-Polycomb-Octβ2R) that modulates the octopamine receptor in Drosophila. stuxnet positively regulates Octβ2R through repression of Polycomb in the ellipsoid body of the adult fly brain and Octβ2R is one of the major receptors mediating octopamine function in sleep homeostasis. In response to octopamine, Octβ2R transcription is inhibited as a result of stuxnet downregulation. This feedback through the Stuxnet-Polycomb-Octβ2R cascade is crucial for sleep homeostasis regulation. This study demonstrates a Stuxnet-Polycomb-Octβ2R-mediated epigenetic regulatory mechanism for octopamine reception, thus providing an example of epigenetic regulation of sleep homeostasis.
Arguello, J. R., Abuin, L., Armida, J., Mika, K., Chai, P. C. and Benton, R. (2021). Targeted molecular profiling of rare olfactory sensory neurons identifies fate, wiring and functional determinants. Elife 10. PubMed ID: 33666172
Determining the molecular properties of neurons is essential to understand their development, function and evolution. Using Targeted DamID (TaDa), RNA polymerase II occupancy and chromatin accessibility were characterized in selected Ionotropic receptor (Ir)-expressing olfactory sensory neurons in Drosophila. Although individual populations represent a minute fraction of cells, TaDa is sufficiently sensitive and specific to identify the expected receptor genes. Unique Ir expression is not consistently associated with differences in chromatin accessibility, but rather to distinct transcription factor profiles. Genes that are heterogeneously-expressed across populations are enriched for neurodevelopmental factors, and functions for the POU-domain protein Pdm3 were identified as a genetic switch of Ir neuron fate, and the atypical cadherin Flamingo in segregation of neurons into discrete glomeruli. Together this study reveals the effectiveness of TaDa in profiling rare neural populations, identifies new roles for a transcription factor and a neuronal guidance molecule, and provides valuable datasets for future exploration.
Xie, Q., Brbic, M., Horns, F., Kolluru, S. S., Jones, R. C., Li, J., Reddy, A. R., Xie, A., Kohani, S., Li, Z., McLaughlin, C. N., Li, T., Xu, C., Vacek, D., Luginbuhl, D. J., Leskovec, J., Quake, S. R., Luo, L. and Li, H. (2021). Temporal evolution of single-cell transcriptomes of Drosophila olfactory projection neurons. Elife 10. PubMed ID: 33427646
Neurons undergo substantial morphological and functional changes during development to form precise synaptic connections and acquire specific physiological properties. What are the underlying transcriptomic bases? This study obtained the single-cell transcriptomes of Drosophila olfactory projection neurons (PNs) at four developmental stages. The identity of 21 transcriptomic clusters corresponding to 20 PN types was decoded and methods were developed to match transcriptomic clusters representing the same PN type across development. PN transcriptomes were found to reflect unique biological processes unfolding at each stage-neurite growth and pruning during metamorphosis at an early pupal stage; peaked transcriptomic diversity during olfactory circuit assembly at mid-pupal stages; and neuronal signaling in adults. At early developmental stages, PN types with adjacent birth order share similar transcriptomes. Together, this work reveals principles of cellular diversity during brain development and provides a resource for future studies of neural development in PNs and other neuronal types.
Barber, A. F., Fong, S. Y., Kolesnik, A., Fetchko, M. and Sehgal, A. (2021). Drosophila clock cells use multiple mechanisms to transmit time-of-day signals in the brain. Proc Natl Acad Sci U S A 118(10). PubMed ID: 33658368
Regulation of circadian behavior and physiology by the Drosophila brain clock requires communication from central clock neurons to downstream output regions, but the mechanism by which clock cells regulate downstream targets is not known. This study shows that the pars intercerebralis (PI), previously identified as a target of the morning cells in the clock network, also receives input from evening cells. Morning and evening clock neurons were determined to have time-of-day-dependent connectivity to the PI, which is regulated by specific peptides as well as by fast neurotransmitters. Interestingly, PI cells that secrete the peptide DH44, and control rest:activity rhythms, are inhibited by clock inputs while insulin-producing cells (IPCs) are activated, indicating that the same clock cells can use different mechanisms to drive cycling in output neurons. Inputs of morning cells to IPCs are relevant for the circadian rhythm of feeding, reinforcing the role of the PI as a circadian relay that controls multiple behavioral outputs. These findings provide mechanisms by which clock neurons signal to nonclock cells to drive rhythms of behavior.

Monday, March 15th - Methods

Motiwala, Z., Darne, P., Prabhune, A., Inamdar, M. S. and Kulkarni, K. (2021). Expression, Purification and Crystallization of Asrij, A Novel Scaffold Transmembrane Protein. J Membr Biol. PubMed ID: 33433647
Asrij/OCIAD1 is a scaffold transmembrane protein belonging to the Ovarian Carcinoma Immunoreactive Antigen Domain containing protein family. In Drosophila and mouse models, Asrij localizes at the endosomal and mitochondrial membrane and is shown to regulate the stemness of hematopoietic stem cells. Interaction of Asrij with ADP Ribosylation Factor 1 (Arf1) is shown to be crucial for hematopoietic niche function and prohemocyte maintenance. This study reports the heterologous expression, standardization of detergents and purification methodologies for crystallization of Asrij/OCIAD1. To probe the activity of bacterially expressed Asrij, a protein complementation assay was developed, and it was conclusively shown that Asrij and Arf1 physically interact. Further, it was found that sophorolipids improve the solubility and monodispersibility of Asrij. Hence, it is proposed that sophorolipids could be novel additives for stabilization of membrane proteins. This is the first study detailing methodology for the production and crystallization of a heterologously expressed scaffold membrane protein and will be widely applicable to understand membrane protein structure and function.
Saikumar, J., Kim, J., Byrns, C. N., Hemphill, M., Meaney, D. F. and Bonini, N. M. (2021). Inducing different severities of traumatic brain injury in Drosophila using a piezoelectric actuator. Nat Protoc 16(1): 263-282. PubMed ID: 33277631
Drosophila models have been instrumental in providing insights into molecular mechanisms of neurodegeneration, with wide application to human disease. The brain degeneration associated with traumatic brain injury (TBI) has been modeled in Drosophila using devices that inflict trauma on multiple parts of the fly body, including the head. However, the injuries produced by these models are not specific in location and are inconsistent between individual animals. Recently, a device was developed that can be used to inflict controlled head injury to flies, resulting in physiological responses that are remarkably similar to those observed in humans with TBI. This protocol describes the construction, calibration and use of the Drosophila TBI (dTBI) device, a platform that employs a piezoelectric actuator to reproducibly deliver a force in order to briefly compress the fly head against a metal surface. The extent of head compression can be controlled through an electrical circuit, allowing the operator to set different levels of injury. The entire device can be assembled and calibrated in under a week. The device components and the necessary electrical tools are readily available and cost ~$800. The dTBI device can be used to harness the power of Drosophila genetics and perform large-scale genetic or pharmacological screens, using a 7-d post-injury survival curve to identify modifiers of injury.
Messal, H. A., Almagro, J., Zaw Thin, M., Tedeschi, A., Ciccarelli, A., Blackie, L., Anderson, K. I., Miguel-Aliaga, I., van Rheenen, J. and Behrens, A. (2021). Antigen retrieval and clearing for whole-organ immunofluorescence by FLASH. Nat Protoc 16(1): 239-262. PubMed ID: 33247285
Advances in light-sheet and confocal microscopy now allow imaging of cleared large biological tissue samples and enable the 3D appreciation of cell and protein localization in their native organ environment. However, the sample preparations for such imaging are often onerous, and their capability for antigen detection is limited. This paper describes FLASH (fast light-microscopic analysis of antibody-stained whole organs), a simple, rapid, fully customizable technique for molecular phenotyping of intact tissue volumes. FLASH utilizes non-degradative epitope recovery and membrane solubilization to enable the detection of a multitude of membranous, cytoplasmic and nuclear antigens in whole mouse organs and embryos, human biopsies, organoids and Drosophila. Retrieval and immunolabeling of epithelial markers, an obstacle for previous clearing techniques, can be achieved with FLASH. Upon volumetric imaging, FLASH-processed samples preserve their architecture and integrity and can be paraffin-embedded for subsequent histopathological analysis. The technique can be performed by scientists trained in light microscopy and yields results in less than one week.
Murawski, C., Pulver, S. R. and Gather, M. C. (2020). Segment-specific optogenetic stimulation in Drosophila melanogaster with linear arrays of organic light-emitting diodes. Nat Commun 11(1): 6248. PubMed ID: 33288763
Optogenetics allows light-driven, non-contact control of neural systems, but light delivery remains challenging, in particular when fine spatial control of light is required to achieve local specificity. This study employed organic light-emitting diodes (OLEDs) that are micropatterned into linear arrays to obtain precise optogenetic control in Drosophila melanogaster larvae expressing the light-gated activator CsChrimson and the inhibitor GtACR2 within their peripheral sensory system. This method allows confinement of light stimuli to within individual abdominal segments, which facilitates the study of larval behaviour in response to local sensory input. Controlled triggering of specific crawling modes was demonstrated, and it was found that targeted neurostimulation in abdominal segments switches the direction of crawling. More broadly, this work demonstrates how OLEDs can provide tailored patterns of light for photo-stimulation of neuronal networks, with future implications ranging from mapping neuronal connectivity in cultures to targeted photo-stimulation with pixelated OLED implants in vivo.
Nguyen, T. H., Vicidomini, R., Choudhury, S. D., Coon, S. L., Iben, J., Brody, T. and Serpe, M. (2021). Single-Cell RNA Sequencing Analysis of the Drosophila Larval Ventral Cord. Curr Protoc 1(2): e38. PubMed ID: 33620770
The fly's small brain and complex behavior has been instrumental in mapping neuronal circuits and elucidating the neural basis of behavior. Recent advances in single-cell RNA sequencing (scRNA-seq) have further enhanced appreciation and understanding of neuronal diversity in a fly brain. However, due to the small size of the fly brain and its constituent cells, scRNA-seq methodologies require a few adaptations. This paper describes a set of protocols optimized for scRNA-seq analysis of the Drosophila larval ventral nerve cord, starting from tissue dissection and cell dissociation to cDNA library preparation, sequencing, and data analysis. This workflow was applied to three separate samples, and the technical challenges associated with successful application of scRNA-seq to studies on neuronal diversity is detailed. An accompanying article presents a custom multistage analysis pipeline that integrates modules contained in different R packages to ensure high-flexibility, high-quality RNA-seq data analysis. These protocols are developed for Drosophila larval ventral nerve cord, but could easily be adapted to other tissues and model organisms.
Vicidomini, R., Nguyen, T. H., Choudhury, S. D., Brody, T. and Serpe, M. (2021). Assembly and Exploration of a Single Cell Atlas of the Drosophila Larval Ventral Cord. Identification of Rare Cell Types. Curr Protoc 1(2): e37. PubMed ID: 33600085
Single-cell RNA sequencing provides a new approach to an old problem: how to study cellular diversity in complex biological systems. This powerful tool has been instrumental in profiling different cell types and investigating, at the single-cell level, cell states, functions, and responses. However, mining these data requires new analytical and statistical methods for high-dimensional analyses that must be customized and adapted to specific goals. This paper presents a custom multistage analysis pipeline which integrates modules contained in different R packages to ensure flexible, high-quality RNA-seq data analysis. This workflow is described step by step, providing the codes, explaining the rationale for each function, and discussing the results and the limitations. This pipeline was applied to analyze different datasets of Drosophila larval ventral cords, identifying and describing rare cell types, such as astrocytes and neuroendocrine cells. This multistage analysis pipeline can be easily implemented by both novice and experienced scientists interested in neuronal and/or cellular diversity beyond the Drosophila model system.
Trivedi, D., Cm, V., Bisht, K., Janardan, V., Pandit, A., Basak, B., H, S., Ramesh, N. and Raghu, P. (2020). A genome engineering resource to uncover principles of cellular organization and tissue architecture by lipid signaling. Elife 9. PubMed ID: 33320085
Phosphoinositides (PI) are key regulators of cellular organization in eukaryotes and genes that tune PI signaling are implicated in human disease mechanisms. Biochemical analyses and studies in cultured cells have identified a large number of proteins that can mediate PI signaling. However, the role of such proteins in regulating cellular processes in vivo and development in metazoans remains to be understood. This study describes a set of CRISPR-based genome engineering tools that allow the manipulation of each of these proteins with spatial and temporal control during metazoan development. The use of these reagents was demonstrated to deplete a set of 103 proteins individually in the Drosophila eye and identify several new molecules that control eye development. This work demonstrates the power of this resource in uncovering the molecular basis of tissue homeostasis during normal development and in human disease biology.
Amamoto, R., Garcia, M. D., West, E. R., Choi, J., Lapan, S. W., Lane, E. A., Perrimon, N. and Cepko, C. L. (2020). Probe-Seq: Method for RNA Sequencing of Specific Cell Types from Animal Tissue. Bio Protoc 10(18): e3749. PubMed ID: 33659409
Most organs and tissues are composed of many types of cells. To characterize cellular state, various transcription profiling approaches are currently available, including whole-tissue bulk RNA sequencing, single cell RNA sequencing (scRNA-Seq), and cell type-specific RNA sequencing. What is missing in this repertoire is a simple, versatile method for bulk transcriptional profiling of cell types for which cell type-specific genetic markers or antibodies are not readily available. Therefore Probe-Seq, which uses hybridization of gene-specific probes to RNA markers for isolation of specific types of cells, was developed to enable downstream FACS isolation and bulk RNA sequencing. This method can enable isolation and profiling of specific cell types from mouse retina, frozen human retina, Drosophila midgut, and developing chick retina, suggesting that it is likely useful for most organisms.

Friday, March 12th - Enhancers and transcriptional regulation

Salomone, J., Qin, S., Fufa, T. D., Cain, B., Farrow, E., Guan, B., Hufnagel, R. B., Nakafuku, M., Lim, H. W., Campbell, K. and Gebelein, B. (2021). Conserved Gsx2/Ind homeodomain monomer versus homodimer DNA binding defines regulatory outcomes in flies and mice.. Genes Dev 35(1-2): 157-174. PubMed ID: 33334823
How homeodomain proteins gain sufficient specificity to control different cell fates has been a long-standing problem in developmental biology. The conserved Gsx homeodomain proteins (ind in Drosophila) regulate specific aspects of neural development in animals from flies to mammals, and yet they belong to a large transcription factor family that bind nearly identical DNA sequences in vitro. This study showa that the mouse and fly Gsx factors unexpectedly gain DNA binding specificity by forming cooperative homodimers on precisely spaced and oriented DNA sites. High-resolution genomic binding assays revealed that Gsx2 binds both monomer and homodimer sites in the developing mouse ventral telencephalon. Importantly, reporter assays showed that Gsx2 mediates opposing outcomes in a DNA binding site-dependent manner: Monomer Gsx2 binding represses transcription, whereas homodimer binding stimulates gene expression. In Drosophila, the Gsx homolog, Ind, similarly represses or stimulates transcription in a site-dependent manner via an autoregulatory enhancer containing a combination of monomer and homodimer sites. Integrating these findings, this study tested a model showing how the homodimer to monomer site ratio and the Gsx protein levels defines gene up-regulation versus down-regulation. Altogether, these data serve as a new paradigm for how cooperative homeodomain transcription factor binding can increase target specificity and alter regulatory outcomes.
Mazina, M. Y., Kovalenko, E. V. and Vorobyeva, N. E. (2021). The negative elongation factor NELF promotes induced transcriptional response of Drosophila ecdysone-dependent genes. Sci Rep 11(1): 172. PubMed ID: 33420323
For many years it was believed that promoter-proximal RNA-polymerase II (Pol II) pausing manages the transcription of genes in Drosophila development by controlling spatiotemporal properties of their activation and repression. But the exact proteins that cooperate to stall Pol II in promoter-proximal regions of developmental genes are still largely unknown. The current work describes the molecular mechanism employed by the Negative ELongation Factor (NELF) to control the Pol II pause at genes whose transcription is induced by 20-hydroxyecdysone (20E). According to the current data, the NELF complex is recruited to the promoters and enhancers of 20E-dependent genes. Its presence at the regulatory sites of 20E-dependent genes correlates with observed interaction between the NELF-A subunit and the ecdysone receptor (EcR). NELF depletion causes a significant decrease in transcription induced by 20E, which is associated with the disruption of Pol II elongation complexes. A considerable reduction in the promoter-bound level of the Spt5 subunit of transcription elongation factor DSIF was observed at the 20E-dependent genes upon NELF depletion. It is presumed that an important function of NELF is to participate in stabilizing the Pol II-DSIF complex, resulting in a significant impact on transcription of its target genes. In order to directly link NELF to regulation of 20E-dependent genes in development, this study shows the presence of NELF at the promoters of 20E-dependent genes during their active transcription in both embryogenesis and metamorphosis. This study also demonstrates that 20E-dependent promoters, while temporarily inactive at the larval stage, preserve a Pol II paused state and bind NELF complex.
Soverna, A. F., Rodriguez, N. C., Korgaonkar, A., Hasson, E., Stern, D. L. and Frankel, N. (2020). Cis-regulatory variation in the shavenbaby gene underlies intraspecific phenotypic variation, mirroring interspecific divergence in the same trait. Evolution. PubMed ID: 33314059
Despite considerable progress in recent decades in dissecting the genetic causes of natural morphological variation, there is limited understanding of how variation within species ultimately contributes to species differences. Patterning was studyied of the non-sensory hairs, commonly known as "trichomes," on the dorsal cuticle of first-instar larvae of Drosophila. Most Drosophila species produce a dense lawn of dorsal trichomes, but a subset of these trichomes were lost in D. sechellia and D. ezoana due entirely to regulatory evolution of the shavenbaby (svb) gene. This study describes intraspecific variation in dorsal trichome patterns of first-instar larvae of D. virilis that is similar to the trichome pattern variation identified previously between species. A single large effect QTL, which includes svb, explains most of the trichome number difference between two D. virilis strains and that svb expression correlates with the trichome difference between strains. This QTL does not explain the entire difference between strains, implying that additional loci contribute to variation in trichome numbers. Thus, the genetic architecture of intraspecific variation exhibits similarities and differences with interspecific variation that may reflect differences in long-term and short-term evolutionary processes.
Yang, Q., Lyu, X., Zhao, F. and Liu, Y. (2021). Effects of codon usage on gene expression are promoter context dependent. Nucleic Acids Res. PubMed ID: 33410890
Codon usage bias is a universal feature of all genomes. Although codon usage has been shown to regulate mRNA and protein levels by influencing mRNA decay and transcription in eukaryotes, little or no genome-wide correlations between codon usage and mRNA levels are detected in mammalian cells, raising doubt on the significance of codon usage effect on gene expression. This study shows that gene-specific regulation reduces the genome-wide codon usage and mRNA correlations: Constitutively expressed genes exhibit much higher genome-wide correlations than differentially expressed genes from fungi to human cells. Using Drosophila S2 cells as a model system, this study showed that the effect of codon usage on mRNA expression level is promoter-dependent. Regions downstream of the core promoters of differentially expressed genes can repress the codon usage effects on mRNA expression. An element in the Hsp70 promoter was identified to be necessary and sufficient for this inhibitory effect. The promoter-dependent codon usage effects on mRNA levels are regulated at the transcriptional level through modulation of histone modifications, nucleosome densities and premature termination. Together, these results demonstrate that promoters play a major role in determining whether codon usage influences gene expression and further establish the transcription-dependent codon usage effects on gene expression.
Zhao, X., Li, Y., Zhao, Z. and Du, J. (2021). Extra sex combs buffers sleep-related stresses through regulating Heat shock proteins. FASEB J 35(1): e21190. PubMed ID: 33220007
The impact of global warming on the life of the earth is increasingly concerned. Previous studies indicated that temperature changes have a serious impact on insect sleep. Sleep is critical for animals as it has many important physiological functions. It is of great significance to study the regulation mechanism of temperature-induced sleep changes for understanding the impact of global warming on insects. More importantly, understanding how these pressures regulate sleep can provide insights into improving sleep. This study found that extra sex combs (ESC) are a regulatory factor in this process. The data showed that ESC was an upstream negative regulatory factor of Heat shock proteins (Hsps), and it could regulate sleep in mushroom and ellipsoid of Drosophila. ESC mutation exaggerates the sleep change caused by temperature, while buffering the shortening of life caused by sleep deprivation. These phenotypes can be rescued by Hsps mutants. Therefore, it is concluded that the ESC buffers sleep-related stresses through regulating Hsps.
Zrimec, J., Borlin, C. S., Buric, F., Muhammad, A. S., Chen, R., Siewers, V., Verendel, V., Nielsen, J., Topel, M. and Zelezniak, A. (2020). Deep learning suggests that gene expression is encoded in all parts of a co-evolving interacting gene regulatory structure. Nat Commun 11(1): 6141. PubMed ID: 33262328
Understanding the genetic regulatory code governing gene expression is an important challenge in molecular biology. However, how individual coding and non-coding regions of the gene regulatory structure interact and contribute to mRNA expression levels remains unclear. This study applied deep learning on over 20,000 mRNA datasets to examine the genetic regulatory code controlling mRNA abundance in 7 model organisms ranging from bacteria to Human. In all organisms, it was possible to predict mRNA abundance directly from DNA sequence, with up to 82% of the variation of transcript levels encoded in the gene regulatory structure. By searching for DNA regulatory motifs across the gene regulatory structure, this study found that motif interactions could explain the whole dynamic range of mRNA levels. Co-evolution across coding and non-coding regions suggests that it is not single motifs or regions, but the entire gene regulatory structure and specific combination of regulatory elements that define gene expression levels.

Thursday, March 11th - Cytoskeleton and junctions

Pietra, S., Ng, K., Lawrence, P. A. and Casal, J. (2020). Planar cell polarity in the larval epidermis of Drosophila and the role of microtubules. Open Biol 10(12): 200290. PubMed ID: 33295841
This study investigated planar cell polarity (PCP) in the Drosophila larval epidermis. The intricate pattern of denticles depends on only one system of PCP, the Dachsous/Fat system. Dachsous molecules in one cell bind to Fat molecules in a neighbour cell to make intercellular bridges. The disposition and orientation of these Dachsous-Fat bridges allows each cell to compare two neighbours and point its denticles towards the neighbour with the most Dachsous. Measurements of the amount of Dachsous reveal a peak at the back of the anterior compartment of each segment. Localization of Dachs and orientation of ectopic denticles help reveal the polarity of every cell. Whether these findings support the gradient model of Dachsous activity is discussed. Several groups have proposed that Dachsous and Fat fix the direction of PCP via oriented microtubules that transport PCP proteins to one side of the cell. This proposition was tested in the larval cells; most microtubules grow perpendicularly to the axis of PCP. No meaningful bias was found in the polarity of microtubules aligned close to that axis. Published data from the pupal abdomen was reexamined, and no evidence was found supporting the hypothesis that microtubular orientation draws the arrow of PCP.
Sallee, J. L., Crawford, J. M., Singh, V. and Kiehart, D. P. (2021). Mutations in Drosophila crinkled/Myosin VIIA disrupt denticle morphogenesis. Dev Biol 470: 121-135. PubMed ID: 33248112
One protein necessary for stereocilia formation, Myosin VIIA, is an actin - based motor protein conserved throughout phylogeny. In Drosophila, severe mutations in the MyoVIIA homolog crinkled (ck) are "semi-lethal". Such survivors show morphological defects related to actin bundling in hairs and bristles. To better understand ck/MyoVIIA's function in bundled - actin structures, dominant female sterile approaches were used to analyze the loss of maternal and zygotic (M/Z) ck/MyoVIIA in the morphogenesis of denticles, small actin - based projections on the ventral epidermis of Drosophila embryos. M/Z ck mutants displayed severe defects in denticle morphology - actin filaments initiated in the correct location, but failed to elongate and bundle to form normal projections. Using deletion mutant constructs, it was demonstrated that both of the C - terminal MyTH4 and FERM domains are necessary for proper denticle formation. Furthermore, it was shown that ck/MyoVIIA interacts genetically with dusky - like (dyl), a member of the ZPD family of proteins that links the extracellular matrix to the plasma membrane, and when mutated also disrupts normal denticle formation. Loss of either protein alone does not alter the localization of the other; however, loss of the two proteins together dramatically enhances the defects in denticle shape observed when either protein alone was absent. These data indicate that ck/MyoVIIA plays a key role in the formation and/or organization of actin filament bundles, which drive proper shape of cellular projections.
Riparbelli, M. G., Persico, V. and Callaini, G. (2020). The Microtubule Cytoskeleton during the Early Drosophila Spermiogenesis. Cells 9(12). PubMed ID: 33327573
Sperm elongation and nuclear shaping in Drosophila largely depends on the microtubule cytoskeleton that in early spermatids has centrosomal and non-centrosomal origins. This study reports an additional γ-tubulin focus localized on the anterior pole of the nucleus in correspondence of the apical end of the perinuclear microtubules that run within the dense complex. The perinuclear microtubules are nucleated by the pericentriolar material, or centriole adjunct, that surrounds the basal body and are retained to play a major role in nuclear shaping. However, it was found that both the perinuclear microtubules and the dense complex are present in spermatids lacking centrioles. Therefore, the basal body or the centriole adjunct seem to be dispensable for the organization and assembly of these structures. These observations shed light on a novel localization of γ-tubulin and open a new scenario on the distribution of the microtubules and the organization of the dense complex during early Drosophila spermiogenesis.
Sui, L. and Dahmann, C. (2020). Increased lateral tension is sufficient for epithelial folding in Drosophila. Development 147(23). PubMed ID: 33277300
The folding of epithelial sheets is important for tissues, organs and embryos to attain their proper shapes. Epithelial folding requires subcellular modulations of mechanical forces in cells. Fold formation has mainly been attributed to mechanical force generation at apical cell sides, but several studies indicate a role of mechanical tension at lateral cell sides in this process. However, whether lateral tension increase is sufficient to drive epithelial folding remains unclear. This study used optogenetics to locally increase mechanical force generation at apical, lateral or basal sides of epithelial Drosophila wing disc cells, an important model for studying morphogenesis. Optogenetic recruitment of RhoGEF2 to apical, lateral or basal cell sides leads to local accumulation of F-actin and increase in mechanical tension. Increased lateral tension, but not increased apical or basal tension, results in sizeable fold formation. These results stress the diversification of folding mechanisms between different tissues and highlight the importance of lateral tension increase for epithelial folding.
Metivier, M., Gallaud, E., Thomas, A., Pascal, A., Gagne, J. P., Poirier, G. G., Chretien, D., Gibeaux, R., Richard-Parpaillon, L., Benaud, C. and Giet, R. (2020). Drosophila Tubulin-Specific Chaperone E Recruits Tubulin around Chromatin to Promote Mitotic Spindle Assembly. Curr Biol. PubMed ID: 33259793
Proper assembly of mitotic spindles requires microtubule nucleation not only at the centrosomes but also around chromatin. This study found that the Drosophila tubulin-specific chaperone dTBCE is required for the enrichment of tubulin in the nuclear space after nuclear envelope breakdown and for subsequent promotion of spindle microtubule nucleation. These events depend on the CAP-Gly motif found in dTBCE and are regulated by Ran and lamin proteins. These data suggest that during early mitosis, dTBCE and nuclear pore proteins become enriched in the nucleus, where they interact with the Ran GTPase to promote dynamic tubulin enrichment. It is proposed that this novel mechanism enhances microtubule nucleation around chromatin, thereby facilitating mitotic spindle assembly.
Yu, H. H. and Zallen, J. A. (2020). Abl and Canoe/Afadin mediate mechanotransduction at tricellular junctions. Science 370(6520). PubMed ID: 33243859
Epithelial structure is generated by the dynamic reorganization of cells in response to mechanical forces. Adherens junctions transmit forces between cells, but how cells sense and respond to these forces in vivo is not well understood. This study identified a mechanotransduction pathway involving the Abl tyrosine kinase and Canoe/Afadin that stabilizes cell adhesion under tension at tricellular junctions in the Drosophila embryo. Canoe is recruited to tricellular junctions in response to actomyosin contractility, and this mechanosensitivity requires Abl-dependent phosphorylation of a conserved tyrosine in the Canoe actin-binding domain. Preventing Canoe tyrosine phosphorylation destabilizes tricellular adhesion, and anchoring Canoe at tricellular junctions independently of mechanical inputs aberrantly stabilizes adhesion, arresting cell rearrangement. These results identify a force-responsive mechanism that stabilizes tricellular adhesion under tension during epithelial remodeling.

Wednesday, March 10th - Adult neural development and function

Wong, J. Y. H., Wan, B. A., Bland, T., Montagnese, M., McLachlan, A. D., O'Kane, C. J., Zhang, S. W. and Masuda-Nakagawa, L. M. (2021). Octopaminergic neurons have multiple targets in Drosophila larval mushroom body calyx and can modulate behavioral odor discrimination. Learn Mem 28(2): 53-71. PubMed ID: 33452115
Discrimination of sensory signals is essential for an organism to form and retrieve memories of relevance in a given behavioral context. Sensory representations are modified dynamically by changes in behavioral state, facilitating context-dependent selection of behavior, through signals carried by noradrenergic input in mammals, or octopamine (OA) in insects. To understand the circuit mechanisms of this signaling, this study characterized the function of two OA neurons, sVUM1 neurons, that originate in the subesophageal zone (SEZ) and target the input region of the memory center, the mushroom body (MB) calyx, in larval Drosophila. sVUM1 neurons were found to target multiple neurons, including olfactory projection neurons (PNs), the inhibitory neuron APL, and a pair of extrinsic output neurons, but relatively few mushroom body intrinsic neurons, Kenyon cells. PN terminals carried the OA receptor Oamb, a Drosophila α1-adrenergic receptor ortholog. Using an odor discrimination learning paradigm, this study showed that optogenetic activation of OA neurons compromised discrimination of similar odors but not learning ability. These results suggest that sVUM1 neurons modify odor representations via multiple extrinsic inputs at the sensory input area to the MB olfactory learning circuit.
Tainton-Heap, L. A. L., Kirszenblat, L. C., Notaras, E. T., Grabowska, M. J., Jeans, R., Feng, K., Shaw, P. J. and van Swinderen, B. (2020). A Paradoxical Kind of Sleep in Drosophila melanogaster. Curr Biol. PubMed ID: 33238155
The dynamic nature of sleep in many animals suggests distinct stages that serve different functions. Genetic sleep induction methods in animal models provide a powerful way to disambiguate these stages and functions, although behavioral methods alone are insufficient to accurately identify what kind of sleep is being engaged. In Drosophila, activation of the dorsal fan-shaped body (dFB) promotes sleep, but it remains unclear what kind of sleep this is, how the rest of the fly brain is behaving, or if any specific sleep functions are being achieved. This study developed a method to record calcium activity from thousands of neurons across a volume of the fly brain during spontaneous sleep and compared this to dFB-induced sleep. Spontaneous sleep was found to typically transition from an active "wake-like" stage to a less active stage. In contrast, optogenetic activation of the dFB promotes sustained wake-like levels of neural activity even though flies become unresponsive to mechanical stimuli. When flies were probed with salient visual stimuli, it was found that the activity of visually responsive neurons in the central brain was blocked by transient dFB activation, confirming an acute disconnect from the external environment. Prolonged optogenetic dFB activation nevertheless achieved a key sleep function by correcting visual attention defects brought on by sleep deprivation. These results suggest that dFB activation promotes a distinct form of sleep in Drosophila, where brain activity appears similar to wakefulness, but responsiveness to external sensory stimuli is profoundly suppressed.
Wang, K., Wang, F., Forknall, N., Yang, T., Patrick, C., Parekh, R. and Dickson, B. J. (2020). Neural circuit mechanisms of sexual receptivity in Drosophila females. Nature. PubMed ID: 33239786
Choosing a mate is one of the most consequential decisions a female will make during her lifetime. A female fly signals her willingness to mate by opening her vaginal plates, allowing a courting male to copulate. Vaginal plate opening (VPO) occurs in response to the male courtship song and is dependent on the mating status of the female. How these exteroceptive (song) and interoceptive (mating status) inputs are integrated to regulate VPO remains unknown. This study characterize the neural circuitry that implements mating decisions in the brain of female Drosophila melanogaster. VPO was shown to be controlled by a pair of female-specific descending neurons (vpoDNs). The vpoDNs receive excitatory input from auditory neurons (vpoENs), which are tuned to specific features of the D. melanogaster song, and from pC1 neurons, which encode the mating status of the female. The song responses of vpoDNs, but not vpoENs, are attenuated upon mating, accounting for the reduced receptivity of mated females. This modulation is mediated by pC1 neurons. The vpoDNs thus directly integrate the external and internal signals that control the mating decisions of Drosophila females.
Worpenberg, L., Paolantoni, C., Longhi, S., Mulorz, M. M., Lence, T., Wessels, H. H., Dassi, E., Aiello, G., Sutandy, F. X. R., Scheibe, M., Edupuganti, R. R., Busch, A., Mockel, M. M., Vermeulen, M., Butter, F., Konig, J., Notarangelo, M., Ohler, U., Dieterich, C., Quattrone, A., Soldano, A. and Roignant, J. Y. (2021). Ythdf is a N6-methyladenosine reader that modulates Fmr1 target mRNA selection and restricts axonal growth in Drosophila. Embo J: e104975. PubMed ID: 33428246
N6-methyladenosine (m(6) A) regulates a variety of physiological processes through modulation of RNA metabolism. This modification is particularly enriched in the nervous system of several species, and its dysregulation has been associated with neurodevelopmental defects and neural dysfunctions. In Drosophila, loss of m(6) A alters fly behavior, albeit the underlying molecular mechanism and the role of m(6) A during nervous system development have remained elusive. This study finds that impairment of the m(6) A pathway leads to axonal overgrowth and misguidance at larval neuromuscular junctions as well as in the adult mushroom bodies. Ythdf was identified as the main m(6) A reader in the nervous system, being required to limit axonal growth. Mechanistically, this study showed that the m(6) A reader Ythdf directly interacts with Fmr1, the fly homolog of Fragile X mental retardation RNA binding protein (FMRP), to inhibit the translation of key transcripts involved in axonal growth regulation. Altogether, this study demonstrates that the m(6) A pathway controls development of the nervous system and modulates Fmr1 target transcript selection.
Thornquist, S. C., Pitsch, M. J., Auth, C. S. and Crickmore, M. A. (2021). Biochemical evidence accumulates across neurons to drive a network-level eruption. Mol Cell. PubMed ID: 33453167
Neural network computations are usually assumed to emerge from patterns of fast electrical activity. Challenging this view, this study shows that a male fly's decision to persist in mating hinges on a biochemical computation that enables processing over minutes to hours. Each neuron in a recurrent network contains slightly different internal molecular estimates of mating progress. Protein kinase A (PKA) activity contrasts this internal measurement with input from the other neurons to represent accumulated evidence that the goal of the network has been achieved. When consensus is reached, PKA pushes the network toward a large-scale and synchronized burst of calcium influx that is called an eruption. Eruptions transform continuous deliberation within the network into an all-or-nothing output, after which the male will no longer sacrifice his life to continue mating. In this case, biochemical activity, invisible to most large-scale recording techniques, is the key computational currency directing behavior and motivational state.
Wu, Y., Funato, Y., Meschi, E., Jovanoski, K., Miki, H. and Waddell, S. (2020). Magnesium efflux from Drosophila Kenyon Cells is critical for normal and diet-enhanced long-term memory. Elife 9. PubMed ID: 33242000
Dietary magnesium (Mg(2+)) supplementation can enhance memory in young and aged rats. Memory-enhancing capacity was largely ascribed to increases in hippocampal synaptic density and elevated expression of the NR2B subunit of the NMDA-type glutamate receptor. This study shows that Mg(2+) feeding also enhances long-term memory in Drosophila. Normal and Mg(2+) enhanced fly memory appears independent of NMDA receptors in the mushroom body and instead requires expression of a conserved CNNM-type Mg(2+)-efflux transporter encoded by the unextended (uex) gene. UEX contains a putative cyclic nucleotide-binding homology domain and its mutation separates a vital role for uex from a function in memory. Moreover, UEX localization in mushroom body Kenyon Cells is altered in memory defective flies harboring mutations in cAMP-related genes. Functional imaging suggests that UEX-dependent efflux is required for slow rhythmic maintenance of Kenyon Cell Mg(2+). It is proposed that regulated neuronal Mg(2+) efflux is critical for normal and Mg(2+) enhanced memory.

Tuesday, March 9th - Evolution

Go, A. C. and Civetta, A. (2020). Hybrid Incompatibilities and Transgressive Gene Expression Between Two Closely Related Subspecies of Drosophila. Front Genet 11: 599292. PubMed ID: 33362859
Genome-wide assays of expression between species and their hybrids have identified genes that become either over- or underexpressed relative to the parental species (i.e., transgressive). Transgressive expression in hybrids is of interest because it highlights possible changes in gene regulation linked to hybrid dysfunction. Previous studies in Drosophila that used long-diverged species pairs with complete or nearly complete isolation (i.e., full sterility and partial inviability of hybrids) and high-levels of genome misregulation have found correlations between expression and coding sequence divergence. The work highlighted the possible effects of directional selection driving sequence divergence and transgressive expression. Whether the same is true for taxa at early stages of divergence that have only achieved partial isolation remains untested. This study reanalyzed previously published genome expression data and available genome sequence reads from a pair of partially isolated subspecies of Drosophila to compare expression and sequence divergence. A significant correlation was found in rates of expression and sequence evolution, but no support for directional selection driving transgressive expression in hybrids. Most transgressive genes in hybrids show no differential expression between parental subspecies. SNP data was used to explore the role of stabilizing selection through compensatory mutations. This study also examined possible misregulation through cascade effects that could be driven by interacting gene networks or co-option of off-target cis-regulatory elements.
Sureka, R. and Mishra, R. (2021). Identification of Evolutionarily Conserved Nuclear Matrix Proteins and Their Prokaryotic Origins. J Proteome Res 20(1): 518-530. PubMed ID: 33289389
Compared to prokaryotic cells, a typical eukaryotic cell is much more complex along with its endomembrane system and membrane-bound organelles. Although the endosymbiosis theories convincingly explain the evolution of membrane-bound organelles such as mitochondria and chloroplasts, very little is understood about the evolutionary origins of the nucleus, the defining feature of eukaryotes. Most studies on nuclear evolution have not been able to take into consideration the underlying structural framework of the nucleus, attributed to the nuclear matrix (NuMat), a ribonucleoproteinaceous structure. This can largely be attributed to the lack of annotation of its core components. Since NuMat has been shown to provide a structural platform for facilitating a variety of nuclear functions such as replication, transcription, and splicing, it is important to identify its protein components to better understand these processes. In this study, this issue was addressed using the developing embryos of Drosophila melanogaster and Danio rerio; 362 core NuMat proteins were identified that are conserved between the two organisms. These results were compared with publicly available Mus musculus NuMat dataset and Homo sapiens cellular localization dataset to define the core homologous NuMat proteins consisting of 252 proteins. Of them, 86 protein groups have originated from pre-existing proteins in prokaryotes. While 36 were conserved across all eukaryotic supergroups, 14 new proteins evolved before the evolution of the last eukaryotic common ancestor and together, these 50 proteins out of the 252 core conserved NuMat proteins are conserved across all eukaryotes, indicating their indispensable nature for nuclear function for over 1.5 billion years of eukaryotic history. This analysis paves the way to understand the evolution of the complex internal nuclear architecture and its functions.
Frazee, S. R., Harper, A. R., Afkhami, M., Wood, M. L., McCrory, J. C. and Masly, J. P. (2021). Interspecific introgression reveals a role of male genital morphology during the evolution of reproductive isolation in Drosophila. Evolution. PubMed ID: 33433903
Rapid divergence in genital structures among nascent species has been posited to be an early-evolving cause of reproductive isolation, although evidence supporting this idea as a widespread phenomenon remains mixed. Using a collection of interspecific introgression lines between two Drosophila species that diverged ∼240,000 years ago, this study tested the hypothesis that even modest divergence in genital morphology can result in substantial fitness losses. The reproductive consequences were studied of variation in the male epandrial posterior lobes between Drosophila mauritiana and D. sechellia; divergence in posterior lobe morphology has significant fitness costs on several pre-fertilization and post-copulatory reproductive measures. Males with divergent posterior lobe morphology also significantly reduced the life span of their mates. Interestingly, one of the consequences of genital divergence was decreased oviposition and fertilization, which suggests that a sensory bias for posterior lobe morphology could exist in females, and thus posterior lobe morphology may be the target of cryptic female choice in these species. These results provide evidence that divergence in genitalia can in fact give rise to substantial reproductive isolation early during species divergence, and they also reveal novel reproductive functions of the external male genitalia in Drosophila.
Maggu, K., Ahlawat, N., Geeta Arun, M., Meena, A. and Prasad, N. G. (2020). Divergence of responses to variable socio-sexual environments in laboratory populations of Drosophila melanogaster evolving under altered operational sex ratios. Evolution. PubMed ID: 33319380
Post-copulatory sexual selection (PSS) is an important selective force that determines fitness in polyandrous species. PSS can be intense in some cases and can drive the evolution of remarkable ejaculate properties. In males, investment in ejaculate plays an important role in the outcome of PSS. Thus, males are expected to adaptively tailor their ejaculate according to the perceived competition in their vicinity. Plastic responses in ejaculate investment to variation in intrasexual competition are disparate and widespread in males. This study investigated the evolution of plasticity in reproductive traits using Drosophila melanogaster populations evolving for more than 150 generations under male- or female-biased sex ratios. When exposed to different numbers of competitors early in their life, males from these two regimes responded differently in terms of their copulation duration and sperm competitive ability. In addition, the effect of this early life experience wore off at different rates in males of male-biased and female-biased regimes with increasing time from the removal of competitive cues. Furthermore, this study finds that males change their reproductive strategies depending upon the identity of rival males. Together, these results provide evidence of the evolution of male reproductive investment that depends on socio-sexual cues experienced early in life.
Kapopoulou, A., Kapun, M., Pieper, B., Pavlidis, P., Wilches, R., Duchen, P., Stephan, W. and Laurent, S. (2020). Demographic analyses of a new sample of haploid genomes from a Swedish population of Drosophila melanogaster. Sci Rep 10(1): 22415. PubMed ID: 33376238
European and African natural populations of Drosophila melanogaster have been the focus of several studies aiming at inferring demographic and adaptive processes based on genetic variation data. However, in these analyses little attention has been given to gene flow between African and European samples. This paper presents a dataset consisting of 14 fully sequenced haploid genomes sampled from a natural population from the northern species range (Umea, Sweden). This new data was co-analyzed with an African population to compare the likelihood of several competing demographic scenarios for European and African populations and shows that gene flow improves the fit of demographic models to data.
Souto-Maior, C., Serrano Negron, Y. L. and Harbison, S. T. (2020). Natural selection on sleep duration in Drosophila melanogaster. Sci Rep 10(1): 20652. PubMed ID: 33244154
Sleep is ubiquitous across animal species, but why it persists is not well understood. This study observed natural selection act on Drosophila sleep by relaxing bi-directional artificial selection for extreme sleep duration for 62 generations. When artificial selection was suspended, sleep increased in populations previously selected for short sleep. Likewise, sleep decreased in populations previously selected for long sleep when artificial selection was relaxed. The corresponding changes were measured in the allele frequencies of genomic variants responding to artificial selection. The allele frequencies of these variants reversed course in response to relaxed selection, and for short sleepers, the changes exceeded allele frequency changes that would be expected under random genetic drift. These observations suggest that the variants are causal polymorphisms for sleep duration responding to natural selection pressure. These polymorphisms may therefore pinpoint the most important regions of the genome maintaining variation in sleep duration.

Monday, March 8th - Behavior

Peralta-Rincon, J. R., Aoulad, F. Z., Prado, A. and Edelaar, P. (2020). Phenotype-dependent habitat choice is too weak to cause assortative mating between Drosophila melanogaster strains differing in light sensitivity. PLoS One 15(10): e0234223. PubMed ID: 33057335
Matching habitat choice is gaining attention as a mechanism for maintaining biodiversity and driving speciation. It revolves around the idea that individuals select the habitat in which they perceive to obtain greater fitness based on a prior evaluation of their local performance across heterogeneous environments. This results in individuals with similar ecologically relevant traits converging to the same patches, and hence it could indirectly cause assortative mating when mating occurs in those patches. White-eyed mutants of Drosophila fruit flies have a series of disadvantages compared to wild type flies, including a poorer performance under bright light. It has been previously reported that, when given a choice, wild type Drosophila simulans preferred a brightly lit habitat while white-eyed mutants occupied a dimly lit one. This spatial segregation allowed the eye color polymorphism to be maintained for several generations, whereas normally it is quickly replaced by the wild type. This study compared the habitat choice decisions of white-eyed and wild type flies in another species, D. melanogaster. Groups of flies were released in a light gradient, and their departure and settlement behavior was recorded. Departure depended on sex and phenotype, but not on the light conditions of the release point. Settlement depended on sex, and on the interaction between phenotype and light conditions of the point of settlement. Nonetheless, simulations showed that this differential habitat use by the phenotypes would only cause a minimal degree of assortative mating in this species.
Kumar, S., Tunc, I., Tansey, T. R., Pirooznia, M. and Harbison, S. T. (2020). Identification of Genes Contributing to a Long Circadian Period in Drosophila Melanogaster. J Biol Rhythms: 748730420975946. PubMed ID: 33274675
The endogenous circadian period of animals and humans is typically very close to 24 h. Individuals with much longer circadian periods have been observed, however, and in the case of humans, these deviations have health implications. Previously, a line of Drosophila was observed with a very long average period of 31.3 h for locomotor activity behavior. Preliminary mapping indicated that the long period did not map to known canonical clock genes but instead mapped to multiple chromosomes. Using RNA-Seq, the whole transcriptome of fly heads from this line was surveyed across time and compared with a wild-type control. A three-way generalized linear model revealed that approximately two-thirds of the genes were expressed differentially among the two genotypes, while only one quarter of the genes varied across time. Using these results, algorithms were applied to search for genes that oscillated over 24 h, identifying genes not previously known to cycle. 166 differentially expressed genes were identified that overlapped with a previous Genome-wide Association Study (GWAS) of circadian behavior, strongly implicating them in the long-period phenotype. Mutations were tested in 45 of these genes for their effect on the circadian period. Mutations in Alk, alph, CG10089, CG42540, CG6034, Kairos (CG6123), CG8768, klg, Lar, sick, and tinc had significant effects on the circadian period, with seven of these mutations increasing the circadian period of locomotor activity behavior. Genetic rescue of mutant Kairos restored the circadian period to wild-type levels, suggesting it has a critical role in determining period length in constant darkness.
Ozturk-Çolak, A., Inami, S., Buchler, J. R., McClanahan, P. D., Cruz, A., Fang-Yen, C. and Koh, K. (2020). Sleep Induction by Mechanosensory Stimulation in Drosophila. Cell Rep 33(9): 108462. PubMed ID: 33264620
People tend to fall asleep when gently rocked or vibrated. Experimental studies have shown that rocking promotes sleep in humans and mice. However, the mechanisms underlying the phenomenon are not well understood. A habituation model proposes that habituation, a form of non-associative learning, mediates sleep induction by monotonous stimulation. This study showed that gentle vibration promotes sleep in Drosophila in part through habituation. Vibration-induced sleep (VIS) leads to increased homeostatic sleep credit and reduced arousability, and can be suppressed by heightened arousal or reduced GABA signaling. Multiple mechanosensory organs mediate VIS, and the magnitude of VIS depends on vibration frequency and genetic background. Sleep induction improves over successive blocks of vibration. Furthermore, training with continuous vibration does not generalize to intermittent vibration, demonstrating stimulus specificity, a characteristic of habituation. These findings suggest that habituation plays a significant role in sleep induction by vibration.
Simon, J. C. and Heberlein, U. (2020). Social hierarchy is established and maintained with distinct acts of aggression in male Drosophila melanogaster. J Exp Biol 223(Pt 24). PubMed ID: 33268534
Social interactions pivot on an animal's experiences, internal states and feedback from others. This complexity drives the need for precise descriptions of behavior to dissect the fine detail of its genetic and neural circuit bases. In laboratory assays, male Drosophila melanogaster reliably exhibit aggression, and its extent is generally measured by scoring lunges, a feature of aggression in which one male quickly thrusts onto his opponent. This study introduces an explicit approach to identify both the onset and reversals in hierarchical status between opponents and observe that distinct aggressive acts reproducibly precede, concur or follow the establishment of dominance. Lunges were found to be insufficient for establishing dominance. Rather, lunges appear to reflect the dominant state of a male and help in maintaining his social status. Lastly, this study characterized the recurring and escalating structure of aggression that emerges through subsequent reversals in dominance. Collectively, this work provides a framework for studying the complexity of agonistic interactions in male flies, enabling its neurogenetic basis to be understood with precision.
Teles-de-Freitas, R., Rivas, G. B. S., Peixoto, A. A. and Bruno, R. V. (2020). The Summer Is Coming: nocte and timeless Genes Are Influenced by Temperature Cycles and May Affect Aedes aegypti Locomotor Activity. Front Physiol 11: 614722. PubMed ID: 33424639
Mosquitoes exhibit activity rhythms, crucial for the transmission of pathogens, under the control of a circadian clock. Aedes aegypti is one of the world's leading vectors. For decades, several studies have linked the rise in ambient temperature with the increase in their activity. This study identified candidate genes whose expression is influenced by temperature cycles and may affect Aedes locomotor activity. timeless completely lost its rhythmic expression in light/dark, with out-of-phase temperature cycles, and by RNAi mediated knockdown of nocte, an important gene for Drosophila circadian synchronization by temperature cycles. Thus, timeless and nocte are important genes for synchronization by temperature cycles in Aedes aegypti. To reinforce these findings, the gradual temperature fluctuations that were as close as possible to daily temperature variations in Brazil were simulated in the laboratory. It was observed that the activity and the expression of the molecular circadian clock of Ae. aegypti differs significantly from that of mosquitoes subjected to constant or rectangular abrupt changes in temperature. It is suggested that for understanding the circadian behavior of Aedes with possible implications for intervention strategies, the seminatural paradigm needs to replace the traditional laboratory study.
He, J., Tan, A. M. X., Ng, S. Y., Rui, M. and Yu, F. (2021). Cannabinoids modulate food preference and consumption in Drosophila melanogaster. Sci Rep 11(1): 4709. PubMed ID: 33633260
Cannabinoids have an important role in regulating feeding behaviors via cannabinoid receptors in mammals. Cannabinoids also exhibit potential therapeutic functions in Drosophila melanogaster, or fruit fly that lacks cannabinoid receptors. However, it remains unclear whether cannabinoids affect food consumption and metabolism in a cannabinoid receptors-independent manner in flies. This study systematically investigated pharmacological functions of various cannabinoids in modulating food preference and consumption in flies. Flies display preferences for consuming cannabinoids, independent of two important sensory regulators Poxn and Orco. Interestingly, phyto- and endo- cannabinoids exhibit an inhibitory effect on food intake. Unexpectedly, the non-selective CB1 receptor antagonist AM251 attenuates the suppression of food intake by endocannabinoids. Moreover, the endocannabinoid anandamide (AEA) and its metabolite inhibit food intake and promote resistance to starvation, possibly through reduced lipid metabolism. Thus, this study has provided insights into a pharmacological role of cannabinoids in feeding behaviors using an adult Drosophila model.

Friday, March 5th - Disease Models

Sarkar, S., Murphy, M. A., Dammer, E. B., Olsen, A. L., Rangaraju, S., Fraenkel, E. and Feany, M. B. (2020). Comparative proteomic analysis highlights metabolic dysfunction in α-synucleinopathy. NPJ Parkinsons Dis 6(1): 40. PubMed ID: 33311497
The synaptic protein α-synuclein is linked through genetics and neuropathology to the pathogenesis of Parkinson's disease and related disorders. However, the mechanisms by which α-synuclein influences disease onset and progression are incompletely understood. To identify pathogenic pathways and therapeutic targets proteomic analysis was performed in a highly penetrant new Drosophila model of α-synucleinopathy. 476 significantly upregulated and 563 significantly downregulated proteins were identified in heads from α-synucleinopathy model flies compared to controls. Multiple complementary analyses was used to identify and prioritize genes and pathways within the large set of differentially expressed proteins for functional studies. Gene Ontology enrichment analysis was performed, the proteomic changes were integrated with human Parkinson's disease genetic studies, and the α-synucleinopathy proteome was compared with that of tauopathy model flies, which are relevant to Alzheimer's disease and related disorders. These approaches identified GTP cyclohydrolase (GCH1) and folate metabolism as candidate mediators of α-synuclein neurotoxicity. In functional validation studies, it was found that the knockdown of Drosophila Gch1 enhanced locomotor deficits in α-synuclein transgenic flies, while folate supplementation protected from α-synuclein toxicity. This integrative analysis suggested that mitochondrial dysfunction was a common downstream mediator of neurodegeneration. Accordingly, Gch1 knockdown enhanced metabolic dysfunction in α-synuclein transgenic fly brains while folate supplementation partially normalized brain bioenergetics. An integrative approach was outlined and implemented to identify and validate potential therapeutic pathways using comparative proteomics and genetics and capitalizing on the facile genetic and pharmacological tools available in Drosophila.
Quintero-Rivera, F., Eno, C. C., Sutanto, C., Jones, K. L., Nowaczyk, M. J. M., Wong, D., Earl, D., Mirzaa, G., Beck, A. and Martinez-Agosto, J. A. (2021). 5q35 duplication presents with psychiatric and undergrowth phenotypes mediated by NSD1 overexpression and mTOR signaling downregulation. Hum Genet. PubMed ID: 33389145
Nuclear receptor binding SET domain protein 1, NSD1, encodes a histone methyltransferase H3K36. NSD1 is responsible for the phenotype of the reciprocal 5q35.2q35.3 microdeletion-microduplication syndromes. This study expanded the phenotype and demonstrated the functional role of NSD1 (Drosophila homolog: NSD) in microduplication 5q35 syndrome. Through an international collaboration, this study reports nine new patients, contributing to the emerging phenotype, highlighting psychiatric phenotypes in older affected individuals. Focusing specifically on the undergrowth phenotype, the effects of Mes-4/NSD overexpression were modeled in Drosophila melanogaster. The individuals (including a family) from diverse backgrounds with duplications ranging in size from 0.6 to 4.5 Mb, have a consistent undergrowth phenotype. Mes-4 overexpression in the developing wing causes undergrowth, increased H3K36 methylation, and increased apoptosis. Altering the levels of insulin receptor (IR) rescues the apoptosis and the wing undergrowth phenotype, suggesting changes in mTOR pathway signaling. Leucine supplementation rescued Mes-4/NSD induced cell death, demonstrating decreased mTOR signaling caused by NSD1. Given that this study shows mTOR inhibition as a likely mechanism and amelioration of the phenotype by leucine supplementation in a fly model, it is suggested further studies should evaluate the therapeutic potential of leucine or branched chain amino acids as an adjunct possible treatment to ameliorate human growth and psychiatric phenotypes, and inclusion of 5q35-microduplication as part of the differential diagnosis for children and adults with delayed bone age, short stature, microcephaly, developmental delay, and psychiatric phenotypes is proposed.
Savola, E., Montgomery, C., Waldron, F. M., Monteith, K. M., Vale, P. and Walling, C. (2020). Testing evolutionary explanations for the lifespan benefit of dietary restriction in fruit flies (Drosophila melanogaster). Evolution. PubMed ID: 33320333
Dietary restriction (DR), limiting calories or specific nutrients without malnutrition, extends lifespan across diverse taxa. Traditionally, this lifespan extension has been explained as a result of diet-mediated changes in the trade-off between lifespan and reproduction, with survival favored when resources are scarce. However, a recently proposed alternative suggests that the selective benefit of the response to DR is the maintenance of reproduction. This hypothesis predicts that lifespan extension is a side effect of benign laboratory conditions, and DR individuals would be frailer and unable to deal with additional stressors, and thus lifespan extension should disappear under more stressful conditions. This was tested by rearing outbred female fruit flies (Drosophila melanogaster) on 10 different protein:carbohydrate diets. Flies were either infected with a bacterial pathogen (Pseudomonas entomophila), injured with a sterile pinprick, or unstressed. Lifespan, fecundity, and measures of aging were monitored. DR extended lifespan and reduced reproduction irrespective of injury and infection. Infected flies on lower protein diets had particularly poor survival. Exposure to infection and injury did not substantially alter the relationship between diet and aging patterns. These results do not provide support for lifespan extension under DR being a side effect of benign laboratory conditions.
Park, J., Choi, H., Kim, Y. D., Kim, S. H., Kim, Y., Gwon, Y., Lee, D. Y., Park, S. H., Heo, W. D. and Jung, Y. K. (2021). Aberrant role of ALK in tau proteinopathy through autophagosomal dysregulation. Mol Psychiatry. PubMed ID: 33452442
Proteinopathy in neurodegenerative diseases is typically characterized by deteriorating activity of specific protein aggregates. In tauopathies, including Alzheimer's disease (AD), tau protein abnormally accumulates and induces dysfunction of the affected neurons. This study reports that anaplastic lymphoma kinase (ALK), a receptor tyrosine kinase, is crucial for the tau-mediated AD pathology. ALK caused abnormal accumulation of highly phosphorylated tau in the somatodendritic region of neurons through its tyrosine kinase activity. ALK-induced LC3-positive axon swelling and loss of spine density, leading to tau-dependent neuronal degeneration. Notably, ALK activation in neurons impaired Stx17-dependent autophagosome maturation and this defect was reversed by a dominant-negative Grb2. In a Drosophila melanogaster model, transgenic flies neuronally expressing active Drosophila Alk exhibited the aggravated tau rough eye phenotype with retinal degeneration and shortened lifespan. In contrast, expression of kinase-dead Alk blocked these phenotypes. ALK levels were significantly elevated in the brains of AD patients showing autophagosomal defects. Injection of an ALK.Fc-lentivirus exacerbated memory impairment in 3xTg-AD mice. Conversely, pharmacologic inhibition of ALK activity with inhibitors reversed the memory impairment and tau accumulation in both 3xTg-AD and tauC3 (caspase-cleaved tau) transgenic mice. Together, it is proposed that aberrantly activated ALK is a bona fide mediator of tau proteinopathy that disrupts autophagosome maturation and causes tau accumulation and aggregation, leading to neuronal dysfunction in AD.
Prifti, E., Tsakiri, E. N., Vourkou, E., Stamatakis, G., Samiotaki, M. and Papanikolopoulou, K. (2020). The two Cysteines of Tau protein are functionally distinct and contribute differentially to its pathogenicity in vivo. J Neurosci. PubMed ID: 33334867
Although Tau accumulation is clearly linked to pathogenesis in Alzheimer's disease (AD) and other Tauopathies, the mechanism that initiates the aggregation of this highly soluble protein in vivo remains largely unanswered. Interestingly, in vitro Tau can be induced to form fibrillar filaments by oxidation of its two cysteine residues, generating an intermolecular disulfide bond that promotes dimerization and fibrillization. The recently solved structures of Tau filaments revealed that the two cysteine residues are not structurally equivalent since Cys-322 is incorporated into the core of the fibril whereas Cys-291 projects away from the core to form the fuzzy coat. This study examined whether mutation of these cysteines to alanine affects differentially Tau mediated toxicity and dysfunction in the well-established Drosophila Tauopathy model. Experiments were conducted with both sexes, or with either sex. Each cysteine residue contributes differentially to Tau stability, phosphorylation status, aggregation propensity, resistance to stress, learning and memory. Importantly, this work uncovers a critical role of Cys-322 in determining Tau toxicity and dysfunction.
Park, J. H., Chung, C. G., Park, S. S., Lee, D., Kim, K. M., Jeong, Y., Kim, E. S., Cho, J. H., Jeon, Y. M., Shen, C. J., Kim, H. J., Hwang, D. and Lee, S. B. (2020). Cytosolic calcium regulates cytoplasmic accumulation of TDP-43 through Calpain-A and Importin alpha3. Elife 9. PubMed ID: 33305734
Cytoplasmic accumulation of TDP-43 in motor neurons is the most prominent pathological feature in amyotrophic lateral sclerosis (ALS). A feedback cycle between nucleocytoplasmic transport (NCT) defect and TDP-43 aggregation was shown to contribute to accumulation of TDP-43 in the cytoplasm. However, little is known about cellular factors that can control the activity of NCT, thereby affecting TDP-43 accumulation in the cytoplasm. This study identified via FRAP and optogenetics cytosolic calcium as a key cellular factor controlling NCT of TDP-43. Dynamic and reversible changes in TDP-43 localization were observed in Drosophila sensory neurons during development. Genetic and immunohistochemical analyses identified the cytosolic calcium-Calpain-A-Importin α3 pathway as a regulatory mechanism underlying NCT of TDP-43. In C9orf72 ALS fly models, upregulation of the pathway activity by increasing cytosolic calcium reduced cytoplasmic accumulation of TDP-43 and mitigated behavioral defects. Together, these results suggest the calcium-Calpain-A-Importin α3 pathway as a potential therapeutic target of ALS.

Thursday, March 4th - Adult physiology

Rajpurohit, S., Vrkoslav, V., Hanus, R., Gibbs, A. G., Cvacka, J. and Schmidt, P. S. (2021). Post-eclosion temperature effects on insect cuticular hydrocarbon profiles. Ecol Evol 11(1): 352-364. PubMed ID: 33437434
The insect cuticle is the interface between internal homeostasis and the often harsh external environment. Cuticular hydrocarbons (CHCs) are key constituents of this hard cuticle and are associated with a variety of functions including stress response and communication. CHC production and deposition on the insect cuticle vary among natural populations and are affected by developmental temperature; however, little is known about CHC plasticity in response to the environment experienced following eclosion, during which time the insect cuticle undergoes several crucial changes. This study targeted this crucial to important phase and studied post-eclosion temperature effects on CHC profiles in two natural populations of Drosophila melanogaster. A forty-eight hour post-eclosion exposure to three different temperatures (18, 25, and 30°C) significantly affected CHCs in both ancestral African and more recently derived North American populations of D. melanogaster. A clear shift from shorter to longer CHCs chain length was observed with increasing temperature, and the effects of post-eclosion temperature varied across populations and between sexes. The quantitative differences in CHCs were associated with variation in desiccation tolerance among populations. Surprisingly, no significant differences in water loss rate was detected between African and North American populations. Overall, these results demonstrate strong genetic and plasticity effects in CHC profiles in response to environmental temperatures experienced at the adult stage as well as associations with desiccation tolerance, which is crucial in understanding holometabolan responses to stress.
Santalla, M., Pagola, L., Gomez, I., Balcazar, D., Valverde, C. A. and Ferrero, P. (2021). Smoking flies: Testing the effect of tobacco cigarettes on heart function of Drosophila melanogaster. Biol Open. PubMed ID: 33431431
Studies about the relationship between substances consumed by humans and their impact on health, in animal models have been a challenge due to differences between species in the animal kingdom. However, the homology of certain genes has allowed extrapolating certain knowledge obtained in animals. Drosophila melanogaster, studied for decades, has been widely used as model for human diseases as well as to study responses associated with the consumption of several substances. This work explores the impact of tobacco consumption on a model of "smoking flies". These experiments were designed to provide information about the effects of tobacco consumption on cardiac physiology. Intracellular calcium handling, a phenomenon underlying cardiac contraction and relaxation, was assessed. Flies chronically exposed to tobacco smoke exhibited an increased heart rate and alterations in the dynamics of the transient increase of intracellular calcium in myocardial cells. These effects were also evident under acute exposure to nicotine of the heart, in a semi-intact preparation. Moreover, the alpha 1 and alpha 7 subunits of the nicotinic receptors are involved in the heart response to tobacco and nicotine under chronic (in the intact fly) as well as acute exposure (in the semi-intact preparation). The present data would help to understand the implication of the intracellular cardiac pathways affected by nicotine on the heart tissue. Based on the probed genetic and physiological similarity between the fly and human heart, cardiac effects exerted by tobacco smoke in Drosophila would help to know the impact of it in the human heart. Additionally, it may also provide information on how nicotine-like substances, e.g. neonicotinoids used as insecticides, affect cardiac function.
Silva, V., Palacios-Munoz, A., Okray, Z., Adair, K. L., Waddell, S., Douglas, A. E. and Ewer, J. (2020). The impact of the gut microbiome on memory and sleep in Drosophila. J Exp Biol. PubMed ID: 33376141
The gut microbiome has been proposed to influence diverse behavioral traits of animals, although the experimental evidence is limited and often contradictory. This study made use of the tractability of Drosophila melanogaster for both behavioral analyses and microbiome studies to test how elimination of microorganisms affects a number of behavioral traits. Relative to conventional flies (i.e., with unaltered microbiome), microbiologically-sterile (axenic) flies displayed a moderate reduction in memory performance in olfactory appetitive conditioning and courtship assays. The microbiological status of the flies had small or no effect on anxiety-like behavior (centrophobism) or circadian rhythmicity of locomotor activity, but axenic flies tended to sleep for longer and displayed reduced sleep rebound after sleep deprivation. The latter effects were robust for most tests conducted on both wildtype Canton S and w (1118) strains, as well for tests using an isogenized panel of flies with mutations in the period gene, which causes altered circadian rhythmicity. Interestingly, the effect of absence of microbiota on a few behavioral features, most notably instantaneous locomotor activity speed, varied among wild-type strains. Taken together, these findings demonstrate that the microbiome can have subtle but significant effects on specific aspects of Drosophila behavior, some of which are dependent on genetic background.
Praggastis, S. A., Lam, G., Horner, M. A., Nam, H. J. and Thummel, C. S. (2020). The Drosophila E78 nuclear receptor regulates dietary triglyceride uptake and systemic lipid levels. Dev Dyn. PubMed ID: 33368768
Lipid levels are maintained by balancing lipid uptake, synthesis, and mobilization. Although many studies have focused on the control of lipid synthesis and mobilization, less is known about the regulation of lipid digestion and uptake. This study show that the Drosophila E78A nuclear receptor plays a central role in intestinal lipid homeostasis through regulation of the CG17192 digestive lipase. E78A mutant adults fail to maintain proper systemic lipid levels following eclosion, with this effect largely restricted to the intestine. Transcriptional profiling by RNA-seq revealed a candidate gene for mediating this effect, encoding the predicted adult intestinal lipase CG17192. Intestine-specific disruption of CG17192 results in reduced lipid levels similar to that seen in E78A mutants. In addition, dietary supplementation with free fatty acids, or intestine-specific expression of either E78A or CG17192, is sufficient to restore lipid levels in E78A mutant adults. These studies support the model that E78A is a central regulator of adult lipid homeostasis through its effects on CG17192 expression and lipid digestion. This work also provides new insights into the control of intestinal lipid uptake and demonstrate that nuclear receptors can play an important role in these pathways.
Shi, D., Han, T., Chu, X., Lu, H., Yang, X., Zi, T., Zhao, Y., Wang, X., Liu, Z., Ruan, J., Liu, X., Ning, H., Wang, M., Tian, Z., Wei, W., Sun, Y., Li, Y., Guo, R., Wang, Y., Ling, F., Guan, Y., Shen, D., Niu, Y., Li, Y. and Sun, C. (2021). An isocaloric moderately high-fat diet extends lifespan in male rats and Drosophila. Cell Metab. PubMed ID: 33440166
The health effect of dietary fat has been one of the most vexing issues in the field of nutrition. Few animal studies have examined the impact of high-fat diets on lifespan by controlling energy intake. This study found that compared to a normal diet, an isocaloric moderately high-fat diet (IHF) significantly prolonged lifespan by decreasing the profiles of free fatty acids (FFAs) in serum and multiple tissues via downregulating FFA anabolism and upregulating catabolism pathways in rats and flies. Proteomics analysis in rats identified PPRC1 as a key protein that was significantly upregulated by nearly 2-fold by IHF, and among the FFAs, only palmitic acid (PA) was robustly and negatively associated with the expression of PPRC1. Using PPRC1 transgenic RNAi/overexpression flies and in vitro experiments, IHF was demonstrated to significantly reduced PA, which could upregulate PPRC1 through PPARG, resulting in improvements in oxidative stress and inflammation and prolonging the lifespan.
Qi, W., Wang, G. and Wang, L. (2020). A novel satiety sensor detects circulating glucose and suppresses food consumption via insulin-producing cells in Drosophila. Cell Res. PubMed ID: 33273704
Sensing satiety is a crucial survival skill for all animal species including human. Despite the discovery of numerous neuromodulators that regulate food intake in Drosophila, the mechanism of satiety sensing remains largely elusive. This study investigated how neuropeptidergic circuitry conveyed satiety state to influence flies' food consumption. Drosophila tackykinin (DTK) and its receptor TAKR99D were identified in an RNAi screening as feeding suppressors. Two pairs of DTK(+) neurons in the fly brain could be activated by elevated D-glucose in the hemolymph and imposed a suppressive effect on feeding. These DTK(+) neurons formed a two-synapse circuitry targeting insulin-producing cells, a well-known feeding suppressor, via TAKR99D(+) neurons, and this circuitry could be rapidly activated during food ingestion and cease feeding. Taken together, this study identified a novel satiety sensor in the fly brain that could detect specific circulating nutrients and in turn modulate feeding, shedding light on the neural regulation of energy homeostasis.

Wednesday, March 3rd - RNA and Transposons

Perez-Lluch, S., Klein, C. C., Breschi, A., Ruiz-Romero, M., Abad, A., Palumbo, E., Bekish, L., Arnan, C. and Guigo, R. (2020). bsAS, an antisense long non-coding RNA, essential for correct wing development through regulation of blistered/DSRF isoform usage. PLoS Genet 16(12): e1009245. PubMed ID: 33370262
Natural Antisense Transcripts (NATs) are long non-coding RNAs (lncRNAs) that overlap coding genes in the opposite strand. NATs roles have been related to gene regulation through different mechanisms, including post-transcriptional RNA processing. With the aim to identify NATs with potential regulatory function during fly development, RNA-Seq data was generated in Drosophila developing tissues and found bsAS, one of the most highly expressed lncRNAs in the fly wing. bsAS is antisense to bs/DSRF, a gene involved in wing development and neural processes. bsAS plays a crucial role in the tissue specific regulation of the expression of the bs/DSRF isoforms. This regulation is essential for the correct determination of cell fate during Drosophila development, as bsAS knockouts show highly aberrant phenotypes. Regulation of bs isoform usage by bsAS is mediated by specific physical interactions between the promoters of these two genes, which suggests a regulatory mechanism involving the collision of RNA polymerases transcribing in opposite directions. Evolutionary analysis suggests that bsAS NAT emerged simultaneously to the long-short isoform structure of bs, preceding the emergence of wings in insects.
Krzywinska, E., Ferretti, L., Li, J., Li, J. C., Chen, C. H. and Krzywinski, J. (2021). femaleless Controls Sex Determination and Dosage Compensation Pathways in Females of Anopheles Mosquitoes. Curr Biol. PubMed ID: 33417880
Analyses of orthologs of the Drosophila genes identified in non-drosophilid taxa revealed that evolution of sex determination pathways is consistent with a bottom-up mode, where only the terminal genes within the pathway are well conserved. doublesex (dsx), occupying a bottom-most position and encoding sex-specific proteins orchestrating downstream sexual differentiation processes, is an ancient sex-determining gene present in all studied species. With the exception of lepidopterans, its female-specific splicing is known to be regulated by transformer (tra) and its co-factor transformer-2 (tra2). This study shows that in the African malaria mosquito Anopheles gambiae, a gene, which likely arose in the Anopheles lineage and which was called femaleless (fle), controls sex determination in females by regulating splicing of dsx and fruitless (fru; another terminal gene within a branch of the sex determination pathway). Moreover, fle represents a novel molecular link between the sex determination and dosage compensation pathways. It is necessary to suppress activation of dosage compensation in females, as demonstrated by the significant upregulation of the female X chromosome genes and a correlated female-specific lethality, but no negative effect on males, in response to fle knockdown. This unexpected property, combined with a high level of conservation in sequence and function in anopheline mosquitoes, makes fle an excellent target for genetic control of all major vectors of human malaria.
Onishi, R., Sato, K., Murano, K., Negishi, L., Siomi, H. and Siomi, M. C. (2020). Piwi suppresses transcription of Brahma-dependent transposons via Maelstrom in ovarian somatic cells. Sci Adv 6(50). PubMed ID: 33310860
Drosophila Piwi associates with PIWI-interacting RNAs (piRNAs) and represses transposons transcriptionally through heterochromatinization; however, this process is poorly understood. This study identified Brahma (Brm), the core adenosine triphosphatase of the SWI/SNF chromatin remodeling complex, as a new Piwi interactor and showed Brm involvement in activating transcription of Piwi-targeted transposons before silencing. Bioinformatic analyses indicated that Piwi, once bound to target RNAs, reduced the occupancies of SWI/SNF and RNA polymerase II (Pol II) on target loci, abrogating transcription. Artificial piRNA-driven targeting of Piwi to RNA transcripts enhanced repression of Brm-dependent reporters compared with Brm-independent reporters. This was dependent on Piwi cofactors, Gtsf1/Asterix (Gtsf1), Panoramix/Silencio (Panx), and Maelstrom (Mael), but not Eggless/dSetdb (Egg)-mediated H3K9me3 deposition. The λN-box B-mediated tethering of Mael to reporters repressed Brm-dependent genes in the absence of Piwi, Panx, and Gtsf1. It is proposed that Piwi, via Mael, can rapidly suppress transcription of Brm-dependent genes to facilitate heterochromatin formation.
Saint-Leandre, B., Christopher, C. and Levine, M. T. (2020). Adaptive evolution of an essential telomere protein restricts telomeric retrotransposons. Elife 9. PubMed ID: 33350936
Essential, conserved cellular processes depend not only on essential, strictly conserved proteins but also on essential proteins that evolve rapidly. To probe this poorly understood paradox, this study exploited the rapidly evolving Drosophila telomere-binding protein, cav/HOAP, which protects chromosomes from lethal end-to-end fusions. The D. melanogaster HOAP was replaced with a highly diverged version from its close relative, D. yakuba. The D. yakuba HOAP ('HOAP[yak]') localizes to D. melanogaster telomeres and protects D. melanogaster chromosomes from fusions. However, HOAP[yak] fails to rescue a previously uncharacterized HOAP function: silencing of the specialized telomeric retrotransposons that, instead of telomerase, maintain chromosome length in Drosophila. Whole genome sequencing and cytogenetics of experimentally evolved populations revealed that HOAP[yak] triggers telomeric retrotransposon proliferation, resulting in aberrantly long telomeres. This evolution-generated, separation-of-function allele resolves the paradoxical observation that a fast-evolving essential gene directs an essential, strictly conserved function: telomeric retrotransposon containment, not end-protection, requires evolutionary innovation at HOAP.
Ma, D., Przybylski, D., Abruzzi, K. C., Schlichting, M., Li, Q., Long, X. and Rosbash, M. (2021). A transcriptomic taxonomy of Drosophila circadian neurons around the clock. Elife 10. PubMed ID: 33438579
Many different functions are regulated by circadian rhythms, including those orchestrated by discrete clock neurons within animal brains. To comprehensively characterize and assign cell identity to the 75 pairs of Drosophila circadian neurons, this study optimized a single cell RNA sequencing method and assayed clock neuron gene expression at different times of day. The data identify at least 17 clock neuron categories with striking spatial regulation of gene expression. Transcription factor regulation is prominent and likely contributes to the robust circadian oscillation of many transcripts, including those that encode cell-surface proteins previously shown to be important for cell recognition and synapse formation during development. The many other clock-regulated genes also constitute an important resource for future mechanistic and functional studies between clock neurons and/or for temporal signaling to circuits elsewhere in the fly brain.
Shibata, T., Nagano, K., Ueyama, M., Ninomiya, K., Hirose, T., Nagai, Y., Ishikawa, K., Kawai, G. and Nakatani, K. (2021). Small molecule targeting r(UGGAA)(n) disrupts RNA foci and alleviates disease phenotype in Drosophila model. Nat Commun 12(1): 236. PubMed ID: 33431896
Synthetic small molecules modulating RNA structure and function have therapeutic potential for RNA diseases. This paper reports the discovery that naphthyridine carbamate dimer (NCD) targets disease-causing r(UGGAA)(n) repeat RNAs in spinocerebellar ataxia type 31 (SCA31). Structural analysis of the NCD-UGGAA/UGGAA complex by nuclear magnetic resonance (NMR) spectroscopy clarifies the mode of binding that recognizes four guanines in the UGGAA/UGGAA pentad by hydrogen bonding with four naphthyridine moieties of two NCD molecules. Biological studies show that NCD disrupts naturally occurring RNA foci built on r(UGGAA)(n) repeat RNA known as nuclear stress bodies (nSBs) by interfering with RNA-protein interactions resulting in the suppression of nSB-mediated splicing events. Feeding NCD to larvae of the Drosophila model of SCA31 alleviates the disease phenotype induced by toxic r(UGGAA)(n) repeat RNA. These studies demonstrate that small molecules targeting toxic repeat RNAs are a promising chemical tool for studies on repeat expansion diseases.

Tuesday March 2nd - Adult Neural Development and Function

Li, F., Lindsey, J. W., Marin, E. C., Otto, N., Dreher, M., Dempsey, G., Stark, I., Bates, A. S., Pleijzier, M. W., Schlegel, P., Nern, A., Takemura, S. Y., Eckstein, N., Yang, T., Francis, A., Braun, A., Parekh, R., Costa, M., Scheffer, L. K., Aso, Y., Jefferis, G. S., Abbott, L. F., Litwin-Kumar, A., Waddell, S. and Rubin, G. M. (2020). The connectome of the adult Drosophila mushroom body provides insights into function. Elife 9. PubMed ID: 33315010
Making inferences about the computations performed by neuronal circuits from synapse-level connectivity maps is an emerging opportunity in neuroscience. The mushroom body (MB) is well positioned for developing and testing such an approach due to its conserved neuronal architecture, recently completed dense connectome, and extensive prior experimental studies of its roles in learning, memory and activity regulation. This study identified new components of the MB circuit in Drosophila, including extensive visual input and MB output neurons (MBONs) with direct connections to descending neurons. Unexpected structure was found in sensory inputs, in the transfer of information about different sensory modalities to MBONs, and in the modulation of that transfer by dopaminergic neurons (DANs). Insights are provided into the circuitry used to integrate MB outputs, connectivity between the MB and the central complex and inputs to DANs, including feedback from MBONs. The results provide a foundation for further theoretical and experimental work.
Kwon, Y., Lee, J. and Chung, Y. D. (2020). Sub-Ciliary Segregation of Two Drosophila Transient Receptor Potential Channels Begins at the Initial Stage of Their Pre-Ciliary Trafficking. Mol Cells 43(12): 1002-1010. PubMed ID: 33323559
Cilia are important eukaryotic cellular compartments required for diverse biological functions. Recent studies have revealed that protein targeting into the proper ciliary subcompartments is essential for ciliary function. In Drosophila chordotonal cilium, where mechano-electric transduction occurs, two transient receptor potential (TRP) superfamily ion channels, TRPV and TRPN, are restricted to the proximal and distal subcompartments, respectively. To understand the mechanisms underlying the sub-ciliary segregation of the two TRPs, their localization was analyzed under various conditions. In developing chordotonal cilia, TRPN was directly targeted to the ciliary tip from the beginning of its appearance and was retained in the distal subcompartment throughout development, whereas the ciliary localization of TRPV was considerably delayed. Lack of intraflagella transport-related proteins affected TRPV from the initial stage of its pre-ciliary trafficking, whereas it affected TRPN from the ciliary entry stage. The ectopic expression of the two TRP channels in both ciliated and nonciliated cells revealed their intrinsic properties related to their localization. Taken together, these results suggest that subciliary segregation of the two TRP channels relies on their distinct intrinsic properties, and begins at the initial stage of their pre-ciliary trafficking.
Nanda, S., Bhattacharjee, S., Cox, D. N. and Ascoli, G. A. (2020). Distinct Relations of Microtubules and Actin Filaments with Dendritic Architecture. iScience 23(12): 101865. PubMed ID: 33319182
Microtubules (MTs) and F-actin (F-act) have long been recognized as key regulators of dendritic morphology. Nevertheless, precisely ascertaining their distinct influences on dendritic trees have been hampered until now by the lack of direct, arbor-wide cytoskeletal quantification. This study paired live confocal imaging of fluorescently labeled dendritic arborization (da) neurons in Drosophila larvae with complete multi-signal neural tracing to separately measure MTs and F-act. Dendritic arbor length is highly interrelated with local MT quantity, whereas local F-act enrichment is associated with dendritic branching. Computational simulation of arbor structure solely constrained by experimentally observed subcellular distributions of these cytoskeletal components generated synthetic morphological and molecular patterns statistically equivalent to those of real da neurons, corroborating the efficacy of local MT and F-act in describing dendritic architecture. The analysis and modeling outcomes hold true for the simplest (class I), most complex (class IV), and genetically altered (Formin3 overexpression) da neuron types.
Pacheco, D. A., Thiberge, S. Y., Pnevmatikakis, E. and Murthy, M. (2021). Auditory activity is diverse and widespread throughout the central brain of Drosophila. Nat Neurosci 24(1): 93-104. PubMed ID: 33230320
Sensory pathways are typically studied by starting at receptor neurons and following postsynaptic neurons into the brain. However, this leads to a bias in analyses of activity toward the earliest layers of processing. This study presents new methods for volumetric neural imaging with precise across-brain registration to characterize auditory activity throughout the entire central brain of Drosophila and make comparisons across trials, individuals and sexes. Auditory activity was found to be present in most central brain regions and in neurons responsive to other modalities. Auditory responses are temporally diverse, but the majority of activity is tuned to courtship song features. Auditory responses are stereotyped across trials and animals in early mechanosensory regions, becoming more variable at higher layers of the putative pathway, and this variability is largely independent of ongoing movements. This study highlights the power of using an unbiased, brain-wide approach for mapping the functional organization of sensory activity.
Palazzo, O., Rass, M. and Brembs, B. (2020). Identification of FoxP circuits involved in locomotion and object fixation in Drosophila. Open Biol 10(12): 200295. PubMed ID: 33321059
The FoxP family of transcription factors is necessary for operant self-learning, an evolutionary conserved form of motor learning. The expression pattern, molecular function and mechanisms of action of the Drosophila FoxP orthologue remain to be elucidated. By editing the genomic locus of FoxP with CRISPR/Cas9, this study found that the three different FoxP isoforms are expressed in neurons, but not in glia and that not all neurons express all isoforms. Furthermore, FoxP expression in, e.g. the protocerebral bridge, the fan-shaped body and in motor neurons, but not in the mushroom bodies. Finally, it was discovered that FoxP expression was detected during development, but not adulthood, is required for normal locomotion and landmark fixation in walking flies. While FoxP expression in the protocerebral bridge and motor neurons is involved in locomotion and landmark fixation, the FoxP gene can be excised from dorsal cluster neurons and mushroom-body Kenyon cells without affecting these behaviours.
Phelps, J. S., Hildebrand, D. G. C., Graham, B. J., Kuan, A. T., Thomas, L. A., Nguyen, T. M., Buhmann, J., Azevedo, A. W., Sustar, A., Agrawal, S., Liu, M., Shanny, B. L., Funke, J., Tuthill, J. C. and Lee, W. A. (2021). Reconstruction of motor control circuits in adult Drosophila using automated transmission electron microscopy. Cell. PubMed ID: 33400916
To investigate circuit mechanisms underlying locomotor behavior, this study used serial-section electron microscopy (EM) to acquire a synapse-resolution dataset containing the ventral nerve cord (VNC) of an adult female Drosophila melanogaster. To generate this dataset, GridTape, a technology that combines automated serial-section collection with automated high-throughput transmission EM, was developed. Using this dataset, neuronal networks were developed that control leg and wing movements by reconstructing all 507 motor neurons that control the limbs. Z specific class of leg sensory neurons synapses directly onto motor neurons with the largest-caliber axons on both sides of the body, representing a unique pathway for fast limb control. Open access is provided to the dataset and reconstructions were registered to a standard atlas to permit matching of cells between EM and light microscopy data. GridTape instrumentation designs and software are provided to make large-scale EM more accessible and affordable to the scientific community.

Monday, March 1st - Adult Physiology

Lerch, S., Zuber, R., Gehring, N., Wang, Y., Eckel, B., Klass, K. D., Lehmann, F. O. and Moussian, B. (2020). Resilin matrix distribution, variability and function in Drosophila. BMC Biol 18(1): 195. PubMed ID: 33317537
Elasticity prevents fatigue of tissues that are extensively and repeatedly deformed. Resilin is a resilient and elastic extracellular protein matrix in joints and hinges of insects. For its mechanical properties, Resilin is extensively analysed and applied in biomaterial and biomedical sciences. However, there is only indirect evidence for Resilin distribution and function in an insect. Commonly, the presence of dityrosines that covalently link Resilin protein monomers (Pro-Resilin), which are responsible for its mechanical properties and fluoresce upon UV excitation, has been considered to reflect Resilin incidence. Using a GFP-tagged Resilin version, Resilin was directly identified in pliable regions of the Drosophila body, some of which were not described before. Interestingly, the amounts of dityrosines are not proportional to the amounts of Resilin in different areas of the fly body, arguing that the mechanical properties of Resilin matrices vary according to their need. For a functional analysis of Resilin matrices, applying the RNA interference and Crispr/Cas9 techniques, flies were generated with reduced or eliminated Resilin function, respectively. These flies are flightless but capable of locomotion and viable suggesting that other proteins may partially compensate for Resilin function. Indeed, localizations of the potentially elastic protein Cpr56F and Resilin occasionally coincide. Thus, Resilin-matrices are composite in the way that varying amounts of different elastic proteins and dityrosinylation define material properties. Understanding the biology of Resilin will have an impact on Resilin-based biomaterial and biomedical sciences.
Min, K. W., Jang, T. and Lee, K. P. (2021). Thermal and nutritional environments during development exert different effects on adult reproductive success in Drosophila melanogaster. Ecol Evol 11(1): 443-457. PubMed ID: 33437441
Environments experienced during development have long-lasting consequences for adult performance and fitness. The "environmental matching" hypothesis predicts that individuals perform best when adult and developmental environments match whereas the "silver spoon" hypothesis expects that fitness is higher in individuals developed under favorable environments regardless of adult environments. This study compared how the temperature and nutrition of larval environment would affect adult survivorship and reproductive success in the fruit fly, Drosophila melanogaster. The aspect of nutrition focused on in this study was the dietary protein-to-carbohydrate (P:C) ratio. The impact of low developmental and adult temperature was to improve adult survivorship. High P:C diet had a negative effect on adult survivorship when ingested during the adult stage, but had a positive effect when ingested during development. No matter whether adult and developmental environments matched or not, females raised in warm and protein-enriched environments produced more eggs than those raised in cool and protein-limiting environments, suggesting the presence of a significant silver spoon effect of larval temperature and nutrition. The effect of larval temperature on adult egg production was weak but persisted across the early adult stage whereas that of larval nutrition was initially strong but diminished rapidly after day 5 posteclosion. Egg production after day 5 was strongly influenced by the P:C ratio of the adult diet, indicating that the diet contributing mainly to reproduction had shifted from larval to adult diet. These results highlight the importance of thermal and nutritional histories in shaping organismal performance and fitness and also demonstrate how the silver spoon effects of these aspects of environmental histories differ fundamentally in their nature, strength, and persistence.
Morris, O., Deng, H., Tam, C. and Jasper, H. (2020). Warburg-like metabolic reprogramming in aging intestinal stem cells contributes to tissue hyperplasia. Cell Rep 33(8): 108423. PubMed ID: 33238124
In many tissues, stem cell (SC) proliferation is dynamically adjusted to regenerative needs. How SCs adapt their metabolism to meet the demands of proliferation and how changes in such adaptive mechanisms contribute to age-related dysfunction remain poorly understood. This study identified mitochondrial Ca(2+) uptake as a central coordinator of SC metabolism. Live imaging of genetically encodasensors in intestinal SCs (ISCs) of Drosophila reveals that mitochondrial Ca(2+) uptake transiently adapts electron transport chain flux to match energetic demand upon proliferative activation. This tight metabolic adaptation is lost in ISCs of old flies, as declines in mitochondrial Ca(2+) uptake promote a "Warburg-like" metabolic reprogramming toward aerobic glycolysis. This switch mimics metabolic reprogramming by the oncogene Ras(V12) and enhances ISC hyperplasia. These data identify a critical mechanism for metabolic adaptation of tissue SCs and reveal how its decline sets aging SCs on a metabolic trajectory reminiscent of that seen upon oncogenic transformation.
Parkhitko, A. A., Ramesh, D., Wang, L., Leshchiner, D., Filine, E., Binari, R., Olsen, A. L., Asara, J. M., Cracan, V., Rabinowitz, J. D., Brockmann, A. and Perrimon, N. (2020). Downregulation of the tyrosine degradation pathway extends Drosophila lifespan. Elife 9. PubMed ID: 33319750
To identify metabolic pathways associated with aging, age-dependent changes in the metabolomes of long-lived Drosophila were examined. Among the metabolites that changed, levels of tyrosine were increased with age in long-lived flies. The levels of enzymes in the tyrosine degradation pathway increase with age in wild-type flies. Whole-body and neuronal-specific downregulation of enzymes in the tyrosine degradation pathway significantly extends Drosophila lifespan, causes alterations of metabolites associated with increased lifespan, and upregulates the levels of tyrosine-derived neuromediators. Moreover, feeding wild-type flies with tyrosine increased their lifespan. Mechanistically, it was shown that suppression of ETC complex I drives the upregulation of enzymes in the tyrosine degradation pathway, an effect that can be rescued by tigecycline, an FDA-approved drug that specifically suppresses mitochondrial translation. In addition, tyrosine supplementation partially rescued lifespan of flies with ETC complex I suppression. Altogether, this study highlights the tyrosine degradation pathway as a regulator of longevity.
Ma, Z. and Freeman, M. R. (2020). TrpML-mediated astrocyte microdomain Ca(2+) transients regulate astrocyte-tracheal interactions. Elife 9. PubMed ID: 33284108
Astrocytes exhibit spatially-restricted near-membrane microdomain Ca(2+)transients in their fine processes. How these transients are generated and regulate brain function in vivo remains unclear. This study shows that Drosophila astrocytes exhibit spontaneous, activity-independent microdomain Ca(2+) transients in their fine processes. Astrocyte microdomain Ca(2+) transients are mediated by the TRP channel TrpML, stimulated by reactive oxygen species (ROS), and can be enhanced in frequency by the neurotransmitter tyramine via the TyrRII receptor. Interestingly, many astrocyte microdomain Ca(2+) transients are closely associated with tracheal elements, which dynamically extend filopodia throughout the central nervous system (CNS) to deliver O(2) and regulate gas exchange. Many astrocyte microdomain Ca(2+) transients are spatio-temporally correlated with the initiation of tracheal filopodial retraction. Loss of TrpML leads to increased tracheal filopodial numbers, growth, and increased CNS ROS. It is proposed that local ROS production can activate astrocyte microdomain Ca(2+) transients through TrpML, and that a subset of these microdomain transients promotes tracheal filopodial retraction and in turn modulate CNS gas exchange.
Marguerite, N. T., Bernard, J., Harrison, D. A., Harris, D. and Cooper, R. L. (2021). Effect of Temperature on Heart Rate for Phaenicia sericata and Drosophila melanogaster with Altered Expression of the TrpA1 Receptors. Insects 12(1). PubMed ID: 33418937
The transient receptor potential (TrpA-ankyrin) receptor has been linked to pathological conditions in cardiac function in mammals. To better understand the function of the TrpA1 in regulation of the heart, a Drosophila melanogaster model was used to express TrpA1 in heart and body wall muscles. Heartbeat of in intact larvae as well as hearts in situ, devoid of hormonal and neural input, indicate that strong over-expression of TrpA1 in larvae at 30 or 37 °C stopped the heart from beating, but in a diastolic state. Cardiac function recovered upon cooling after short exposure to high temperature. Parental control larvae (UAS-TrpA1) increased heart rate transiently at 30 and 37 °C but slowed at 37 °C within 3 min for in-situ preparations, while in-vivo larvae maintained a constant heart rate. The in-situ preparations maintained an elevated rate at 30 °C. The heartbeat in the TrpA1-expressing strains could not be revived at 37 °C with serotonin. Thus, TrpA1 activation may have allowed enough Ca(2+) influx to activate K((Ca)) channels into a form of diastolic stasis. TrpA1 activation in body wall muscle confirmed a depolarization of membrane. In contrast, blowfly Phaenicia sericata larvae increased heartbeat at 30 and 37 °C, demonstrating greater cardiac thermotolerance.
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