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Current papers in developmental biology and gene function


Friday, April 30th, 2021 - Signal Transduction

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Ramanujam, P. L., Mehrotra, S., Kumar, R. P., Verma, S., Deshpande, G., Mishra, R. K. and Galande, S. (2021). Global chromatin organizer SATB1 acts as a context-dependent regulator of the Wnt/Wg target genes. Sci Rep 11(1): 3385. PubMed ID: 33564000
Special AT-rich binding protein-1 (SATB1) integrates higher-order chromatin architecture with gene regulation, thereby regulating multiple signaling pathways. In mammalian cells SATB1 directly interacts with β-catenin and regulates the expression of Wnt targets by binding to their promoters. Whether SATB1 regulates Wnt/wg signaling by recruitment of β-catenin and/or its interactions with other components remains elusive. Since Wnt/Wg signaling is conserved from invertebrates to humans, this study investigated SATB1 functions in regulation of Wnt/Wg signaling by using mammalian cell-lines and Drosophila. This study presents evidence that in mammalian cells, SATB1 interacts with Dishevelled, an upstream component of the Wnt/Wg pathway. Conversely, ectopic expression of full-length human SATB1 but not that of its N- or C-terminal domains in the eye imaginal discs and salivary glands of third instar Drosophila larvae increased the expression of Wnt/Wg pathway antagonists and suppressed phenotypes associated with activated Wnt/Wg pathway. These data argue that ectopically-provided SATB1 presumably modulates Wnt/Wg signaling by acting as negative regulator in Drosophila. Interestingly, comparison of SATB1 with PDZ- and homeo-domain containing Drosophila protein Defective Proventriculus suggests that both proteins exhibit limited functional similarity in the regulation of Wnt/Wg signaling in Drosophila. Collectively, these findings indicate that regulation of Wnt/Wg pathway by SATB1 is context-dependent.
Rogers, E. M., Allred, S. C. and Peifer, M. (2021). Abelson kinase's intrinsically disordered region plays essential roles in protein function and protein stability. Cell Commun Signal 19(1): 27. PubMed ID: 33627133
The non-receptor tyrosine kinase Abelson (Abl) is a key player in oncogenesis. Drosophila offers a superb model for studying Abl's normal function, because, unlike mammals, there is only a single fly Abl family member. Attention turned to one of Abl's least understood features-the long intrinsically-disordered region (IDR) linking Abl's kinase and F-actin binding domains. The past decade revealed unexpected, important roles for IDRs in diverse cell functions, as sites of posttranslational modifications, mediating multivalent interactions and enabling assembly of biomolecular condensates via phase separation. Previous work deleting conserved regions in Abl's IDR revealed an important role for a PXXP motif, but did not identify any other essential regions. This study extended this analysis by deleting the entire IDR, and asking whether AblΔIDR rescues the diverse roles of Abl in viability and embryonic morphogenesis in Drosophila. This revealed that the IDR is essential for embryonic and adult viability for cell shape changes and cytoskeletal regulation during embryonic morphogenesis, and, most surprisingly, revealed a role in modulating protein stability. These data provide new insights into the role of the IDR in an important signaling protein, the non-receptor kinase Abl, suggesting that it is essential for all aspects of protein function during embryogenesis, and revealing a role in protein stability. These data will stimulate new explorations of the mechanisms by which the IDR regulates Abl stability and function, both in Drosophila and also in mammals. They also will stimulate further interest in the broader roles IDRs play in diverse signaling proteins.
Rehman, N. and Varghese, J. (2021). Larval nutrition influences adult fat stores and starvation resistance in Drosophila. PLoS One 16(2): e0247175. PubMed ID: 33606785
Insulin plays a major role in connecting nutrient availability to energy homeostasis by regulating metabolic pathways. Defects in insulin signalling is the primary cause for diabetes, obesity and various metabolic disorders. Nutritional status during growth and developmental stages play a crucial role in determining adult size, fecundity and ageing. However, the association between developmental nutrition and adult metabolic disorders has not been fully explored. This study addresses the effects of nutrient status during the larval growth phase on adult metabolism in Drosophila. Restricted food supply in larvae led to higher fat reserves and starvation resistance in mature adult flies, which is attributed to low insulin signalling. A lesser amount of stored fat was mobilised during early adult stages and during acute starvation, which accounts for the metabolic effects. Furthermore, larval diet influenced the expression of fat mobilisation genes brummer and lipid storage droplet-2 in adult flies, which led to the metabolic phenotypes reported in this study. Thus, the restricted nutrient environment in developing larvae led to adaptive changes that entrain the adult flies for scarce food availability.
Rodrigues, D., Renaud, Y., VijayRaghavan, K., Waltzer, L. and Inamdar, M. S. (2021). Differential activation of JAK-STAT signaling reveals functional compartmentalization in Drosophila blood progenitors.. Elife 10. PubMed ID: 33594977
Blood cells arise from diverse pools of stem and progenitor cells. Understanding progenitor heterogeneity is a major challenge. The Drosophila larval lymph gland is a well-studied model to understand blood progenitor maintenance and recapitulates several aspects of vertebrate hematopoiesis. However in-depth analysis has focused on the anterior lobe progenitors (AP), ignoring the posterior progenitors (PP) from the posterior lobes. Using in situ expression mapping and developmental and transcriptome analysis, this study revealed PP heterogeneity and identified molecular-genetic tools to study this abundant progenitor population. Functional analysis shows that PP resist differentiation upon immune challenge, in a JAK-STAT-dependent manner. Upon wasp parasitism, AP downregulate JAK-STAT signaling and form lamellocytes. In contrast, this study shows that PP activate STAT92E and remain undifferentiated, promoting survival. Stat92E knockdown or genetically reducing JAK-STAT signaling permits PP lamellocyte differentiation. How heterogeneity and compartmentalization allow functional segregation in response to systemic cues and could be widely applicable is discussed.
Lu, J., Dong, W., Tao, Y. and Hong, Y. (2021). Electrostatic plasma membrane targeting contributes to Dlg function in cell polarity and tumorigenesis. Development. PubMed ID: 33688074
Discs large (Dlg) is an essential polarity protein and a tumor suppressor originally characterized in Drosophila but is also well conserved in vertebrates. Like the majority of polarity proteins, plasma membrane (PM)/cortical localization of Dlg is required for its function in polarity and tumorigenesis, but the exact mechanisms targeting Dlg to PM remain to be fully elucidated. This study shows that, similar to the recently discovered polybasic polarity proteins such as Lgl and aPKC, Dlg also contains a positively charged polybasic domain that electrostatically binds the PM phosphoinositides PI4P and PI(4,5)P(2). Electrostatic targeting by the polybasic domain contributes significantly to the PM localization of Dlg in follicular and early embryonic epithelial cells, and is crucial for Dlg to regulate both polarity and tumorigenesis. The electrostatic PM targeting of Dlg is controlled by a potential phosphorylation-dependent allosteric regulation of its polybasic domain, and is specifically enhanced by the interactions between Dlg and another basolateral polarity protein and tumor suppressor Scrib. These studies highlight an increasingly significant role of electrostatic PM targeting of polarity proteins in regulating cell polarity.
Romao, D., Muzzopappa, M., Barrio, L. and Milan, M. (2021). The Upd3 cytokine couples inflammation to maturation defects in Drosophila. Curr Biol. PubMed ID: 33609452
Developmental transitions, such as puberty or metamorphosis, are tightly controlled by steroid hormones and can be delayed by the appearance of growth abnormalities, developmental tumors, or inflammatory disorders such as inflammatory bowel disease or cystic fibrosis. This study used a highly inflammatory epithelial model of malignant transformation in Drosophila to unravel the role of Upd3-a cytokine with homology to interleukin-6-and the JAK/STAT signaling pathway in coupling inflammation to a delay in metamorphosis. Evidence is presented that Upd3 produced by malignant and nearby cell populations signals to the prothoracic gland-an endocrine tissue primarily dedicated to the production of the steroid hormone ecdysone-to activate JAK/STAT and bantam microRNA (miRNA) and to delay metamorphosis. Upd cytokines produced by the tumor site contribute to increasing the systemic levels of Upd3 by amplifying its expression levels in a cell-autonomous manner and by inducing Upd3 expression in neighboring tissues in a non-autonomous manner, culminating in a major systemic response to prevent larvae from initiating pupa transition. These results identify a new regulatory network impacting on ecdysone biosynthesis and provide new insights into the potential role of inflammatory cytokines and the JAK/STAT signaling pathway in coupling inflammation to delays in puberty.

Thursday, April 29th - Embryonic Development and Gene Function

Ponrathnam, T., Saini, R., Banu, S. and Mishra, R. K. (2021). Drosophila Hox genes induce melanized pseudo-tumors when misexpressed in hemocytes. Sci Rep 11(1): 1838. PubMed ID: 33469139
Hox genes are early determinants of cell identity along the anterior-posterior body axis across bilaterians. Several late non-homeotic functions of Hox genes have emerged in a variety of processes involved in organogenesis in several organisms, including mammals. Several studies have reported the misexpression of Hox genes in a variety of malignancies including acute myeloid leukemia. The Hox genes Dfd, Ubx, abd-A and Abd-B were overexpressed via the UAS-Gal4 system using Cg-Gal4, Lsp2-Gal4, He-Gal4 and HmlD3-Gal4 as specific drivers. Genetic interaction was tested by bringing overexpression lines in heterozygous mutant backgrounds of Polycomb and trithorax group factors. Larvae were visually scored for melanized bodies. Circulating hemocytes were quantified and tested for differentiation. Pupal lethality was assessed. Expression of Dfd, Ubx and abd-A, but not Abd-B in the hematopoietic compartment of Drosophila led to the appearance of circulating melanized bodies, an increase in cell number, cell-autonomous proliferation, and differentiation of hemocytes. Pupal lethality and melanized pseudo-tumors were suppressed in Psc1 and esc2 backgrounds while polycomb group member mutations Pc1 and Su(z)123 and trithorax group member mutation TrlR85 enhanced the phenotype. Dfd, Ubx and abd-A are leukemogenic. Mutations in Polycomb and trithorax group members modulate the leukemogenic phenotype. This RNAseq of Cg-Gal4 > UAS-abd-A hemocytes may contain genes important to Hox gene induced leukemias.
Lenz, J., Liefke, R., Funk, J., Shoup, S., Nist, A., Stiewe, T., Schulz, R., Tokusumi, Y., Albert, L., Raifer, H., Forstemann, K., Vazquez, O., Tokusumi, T., Fossett, N. and Brehm, A. (2021). Ush regulates hemocyte-specific gene expression, fatty acid metabolism and cell cycle progression and cooperates with dNuRD to orchestrate hematopoiesis. PLoS Genet 17(2): e1009318. PubMed ID: 33600407
U-shaped (Ush), a multi-zinc finger protein, maintains the multipotency of stem cell-like hemocyte progenitors during Drosophila hematopoiesis. Using genomewide approaches this study has revealed that Ush binds to promoters and enhancers and that it controls the expression of three gene classes that encode proteins relevant to stem cell-like functions and differentiation: cell cycle regulators, key metabolic enzymes and proteins conferring specific functions of differentiated hemocytes. Complementary biochemical approaches were employed to characterise the molecular mechanisms of Ush-mediated gene regulation. Distinct Ush isoforms were uncovered, one of which binds the Nucleosome Remodeling and Deacetylation (NuRD) complex (see HDAC1) using an evolutionary conserved peptide motif. Remarkably, the Ush/NuRD complex specifically contributes to the repression of lineage-specific genes but does not impact the expression of cell cycle regulators or metabolic genes. This reveals a mechanism that enables specific and concerted modulation of functionally related portions of a wider gene expression programme. Finally, genetic assays were used to demonstrate that Ush and NuRD regulate enhancer activity during hemocyte differentiation in vivo and that both cooperate to suppress the differentiation of lamellocytes, a highly specialised blood cell type. These findings reveal that Ush coordinates proliferation, metabolism and cell type-specific activities by isoform-specific cooperation with an epigenetic regulator.
Wu, Z., Pang, N., Zhang, Y., Chen, H., Peng, Y., Fu, J. and Wei, Q. (2020). CEP290 is essential for the initiation of ciliary transition zone assembly. PLoS Biol 18(12): e3001034. PubMed ID: 33370260
Cilia play critical roles during embryonic development and adult homeostasis. Dysfunction of cilia leads to various human genetic diseases, including many caused by defects in transition zones (TZs), the "gates" of cilia. The evolutionarily conserved TZ component centrosomal protein 290 (CEP290) is the most frequently mutated human ciliopathy gene, but its roles in ciliogenesis are not completely understood. This study reports that CEP290 plays an essential role in the initiation of TZ assembly in Drosophila. Mechanistically, the N-terminus of CEP290 directly recruits DAZ interacting zinc finger protein 1 (DZIP1), which then recruits Chibby (CBY) and Rab8 to promote early ciliary membrane formation. Complete deletion of CEP290 blocks ciliogenesis at the initiation stage of TZ assembly, which can be mimicked by DZIP1 deletion mutants. Remarkably, expression of the N-terminus of CEP290 alone restores the TZ localization of DZIP1 and subsequently ameliorates the defects in TZ assembly initiation in cep290 mutants. Our results link CEP290 to DZIP1-CBY/Rab8 module and uncover a previously uncharacterized important function of CEP290 in the coordination of early ciliary membrane formation and TZ assembly.
Kump, A. J., Panta, M., Schwab, K. R., Inlow, M. H. and Ahmad, S. M. (2021). The Drosophila Forkhead/Fox transcription factor Jumeau mediates specific cardiac progenitor cell divisions by regulating expression of the kinesin Nebbish. Sci Rep 11(1): 3221. PubMed ID: 33547352
The Drosophila Forkhead gene jumeau (jumu) controls three categories of cardiac progenitor cell division-asymmetric, symmetric, and cell division at an earlier stage-by regulating Polo kinase activity, and mediates the latter two categories in concert with the TF Myb. Those observations raised the question of whether other jumu-regulated genes also mediate all three categories of cardiac progenitor cell division or a subset thereof. By comparing microarray-based expression profiles of wild-type and jumu loss-of-function mesodermal cells, nebbish (neb), a kinesin-encoding gene activated by jumu, was identified. Phenotypic analysis shows that neb is required for only two categories of jumu-regulated cardiac progenitor cell division: symmetric and cell division at an earlier stage. Synergistic genetic interactions between neb, jumu, Myb, and polo and the rescue of jumu mutations by ectopic cardiac mesoderm-specific expression of neb demonstrate that neb is an integral component of a jumu-regulated subnetwork mediating cardiac progenitor cell divisions. These results emphasize the central role of Fox TFs in cardiogenesis and illustrate how a single TF can utilize different combinations of other regulators and downstream effectors to control distinct developmental processes.
Lindblad, J. L., Tare, M., Amcheslavsky, A., Shields, A. and Bergmann, A. (2021). Non-apoptotic enteroblast-specific role of the initiator caspase Dronc for development and homeostasis of the Drosophila intestine. Sci Rep 11(1): 2645. PubMed ID: 33514791
The initiator caspase Dronc is the only CARD-domain containing caspase in Drosophila and is essential for apoptosis. This study reports that homozygous dronc mutant adult animals are short-lived due to the presence of a poorly developed, defective and leaky intestine. Interestingly, this mutant phenotype can be significantly rescued by enteroblast-specific expression of dronc(+) in dronc mutant animals, suggesting that proper Dronc function specifically in enteroblasts, one of four cell types in the intestine, is critical for normal development of the intestine. Furthermore, enteroblast-specific knockdown of dronc in adult intestines triggers hyperplasia and differentiation defects. These enteroblast-specific functions of Dronc do not require the apoptotic pathway and thus occur in a non-apoptotic manner. In summary, this study demonstrated that an apoptotic initiator caspase has a very critical non-apoptotic function for normal development and for the control of the cell lineage in the adult midgut and therefore for proper physiology and homeostasis.
King, T. R., Kramer, J., Cheng, Y. S., Swope, D. and Kramer, S. G. (2021). Enabled/VASP is required to mediate proper sealing of opposing cardioblasts during Drosophila dorsal vessel formation. Dev Dyn. PubMed ID: 33587326
The Drosophila dorsal vessel (DV) is comprised of two opposing rows of cardioblasts (CBs) that migrate toward the dorsal midline during development. While approaching the midline, CBs change shape, enabling dorsal and ventral attachments with their contralateral partners to create a linear tube with a central lumen. Previous studies demonstrated DV closure occurs via a "buttoning" mechanism where specific CBs advance ahead of their lateral neighbors, and attach creating transient holes, which eventually seal. This study investigate the role of the actin-regulatory protein Enabled (Ena) in DV closure. Loss of Ena results in DV cell shape and alignment defects. Live analysis of DV formation in ena mutants shows a reduction in CB leading edge protrusion length and gaps in the DV between contralateral CB pairs. These gaps occur primarily between a specific genetic subtype of CBs, which express the transcription factor Seven-up (Svp) and form the ostia inflow tracts of the heart. In WT embryos these gaps between Svp(+) CBs are observed transiently during the final stages of DV closure. These data suggest that Ena modulates the actin cytoskeleton in order to facilitate the complete sealing of the DV during the final stages of cardiac tube formation.

Wednesday, April 28th - Synapse and Vesicles

Harrell, E. R., Pimentel, D. and Miesenbock, G. (2021). Changes in presynaptic gene expression during homeostatic compensation at a central synapse. J Neurosci. PubMed ID: 33608385
Homeostatic matching of pre- and postsynaptic function has been observed in many species and neural structures, but whether transcriptional changes contribute to this form of trans-synaptic coordination remains unknown. To identify genes whose expression is altered in presynaptic neurons as a result of perturbing postsynaptic excitability, a transcriptomics-friendly, temperature-inducible Kir2.1-based activity clamp was applied at the first synaptic relay of the Drosophila olfactory system, a central synapse known to exhibit trans-synaptic homeostatic matching. Twelve hours after adult-onset suppression of activity in postsynaptic antennal lobe projection neurons of males and females, changes were detected in the expression of many genes in the third antennal segment, which houses the somata of presynaptic olfactory receptor neurons. These changes affected genes with roles in synaptic vesicle release and synaptic remodeling, including several implicated in homeostatic plasticity at the neuromuscular junction. At 48 hours and beyond, the transcriptional landscape tilted toward protein synthesis, folding, and degradation; energy metabolism; and cellular stress defenses, indicating that the system had been pushed to its homeostatic limits. This analysis suggests that similar homeostatic machinery operates at peripheral and central synapses and identifies many of its components. The presynaptic transcriptional response to genetically targeted postsynaptic perturbations could be exploited for the construction of novel connectivity tracing tools.
Ramesh, N., Escher, M. J. F., Mampell, M. M., Bohme, M. A., Gotz, T. W. B., Goel, P., Matkovic, T., Petzoldt, A. G., Dickman, D. and Sigrist, S. J. (2021). Antagonistic interactions between two Neuroligins coordinate pre- and postsynaptic assembly. Curr Biol. PubMed ID: 33651992
As a result of developmental synapse formation, the presynaptic neurotransmitter release machinery becomes accurately matched with postsynaptic neurotransmitter receptors. Trans-synaptic signaling is executed through cell adhesion proteins such as Neurexin::Neuroligin pairs but also through diffusible and cytoplasmic signals. How exactly pre-post coordination is ensured in vivo remains largely enigmatic. This study identified a "molecular choreography" coordinating pre- with postsynaptic assembly during the developmental formation of Drosophila neuromuscular synapses. Two presynaptic Neurexin-binding scaffold proteins, Syd-1 and Spinophilin (Spn), spatio-temporally coordinated pre-post assembly in conjunction with two postsynaptically operating, antagonistic Neuroligin species: Nlg1 and Nlg2. The Spn/Nlg2 module promoted active zone (AZ) maturation by driving the accumulation of AZ scaffold proteins critical for synaptic vesicle release. Simultaneously, these regulators restricted postsynaptic glutamate receptor incorporation. Both functions of the Spn/Nlg2 module were directly antagonized by Syd-1/Nlg1. Nlg1 and Nlg2 also had divergent effects on Nrx-1 in vivo motility. Concerning diffusible signals, Spn and Syd-1 antagonistically controlled the levels of Munc13-family protein Unc13B at nascent AZs, whose release function facilitated glutamate receptor incorporation at assembling postsynaptic specializations. As a result, this study provides direct in vivo evidence illustrating how a highly regulative and interleaved communication between cell adhesion protein signaling complexes and diffusible signals allows for a precise coordination of pre- with postsynaptic assembly. It will be interesting to analyze whether this logic also transfers to plasticity processes.
Ma, C. J. and Brill, J. A. (2021). Endosomal Rab GTPases regulate secretory granule maturation in Drosophila larval salivary glands. Commun Integr Biol 14(1): 15-20. PubMed ID: 33628358
Secretory granules (SGs) are organelles responsible for regulated exocytosis of biologically active molecules in professional secretory cells. Maturation of SGs is a crucial process in which cargoes of SGs are processed and activated, allowing them to exert their function upon secretion. Nonetheless, the intracellular trafficking pathways required for SG maturation are not well defined. An RNA interference (RNAi) screen was performed in Drosophila larval salivary glands to identify trafficking components needed for SG maturation. From the screen, several Rab GTPases (Rabs) were identified that affect SG maturation. Expression of constitutively active (CA) and dominant-negative (DN) forms narrowed down the Rabs important for this process to Rab5, Rab9 and Rab11. However, none of these Rabs localizes to the limiting membrane of SGs. In contrast, examination of endogenously YFP-tagged Rabs (YRabs) in larval salivary glands revealed that YRab1 and YRab6 localize to the limiting membrane of immature SGs (iSGs) and SGs. These findings provide new insights into how Rab GTPases contribute to the process of SG maturation.
Kamemura, K., Chen, C. A., Okumura, M., Miura, M. and Chihara, T. (2021). Amyotrophic lateral sclerosis-associated VAP33 is required for maintaining neuronal dendrite morphology and organelle distribution in Drosophila. Genes Cells. PubMed ID: 33548103
VAMP-associated protein (VAP; see Drosophila Vap33) is an endoplasmic reticulum (ER) membrane protein that functions as a tethering protein at the membrane contact sites between the ER and various intracellular organelles. Mutations such as P56S in human VAPB cause neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). However, VAP functions in neurons are poorly understood. This study utilized Drosophila olfactory projection neurons with a mosaic analysis with a repressible cell marker (MARCM) to analyze the neuronal function of VAP33, a Drosophila ortholog of human VAPB. In vap33 null mutant clones, the dendrites of projection neurons exhibited defects in the maintenance of their morphology. The subcellular localization of the Golgi apparatus and mitochondria were also abnormal. These results indicate that Vap33 is required for neuronal morphology and organelle distribution. Additionally, to examine the impact of ALS-associated mutations in neurons, human VAPB-P56S was overexpressed in vap33 null mutant clones (mosaic rescue experiments) and found that, in aged flies, human VAPB-P56S expression caused mislocalization of Bruchpilot, a presynaptic protein. These results implied that synaptic protein localization and ER quality control may be affected by disease mutations. This study provides insights into the physiological and pathological functions of VAP in neurons.
Kawaguchi, K., Yamamoto-Hino, M., Matsuyama, N., Suzuki, E. and Goto, S. (2021). Subunits of the GPI transamidase complex localize to the endoplasmic reticulum and nuclear envelope in Drosophila. FEBS Lett. PubMed ID: 33496978
A total of 10-20% of plasma membrane proteins are anchored by glycosylphosphatidylinositol (GPI). GPI is attached to proteins by GPI transamidase (GPI-T), which contains five subunits named PIGK, PIGS, PIGT, PIGU, and GPAA1. It was previously reported that PIGT localizes near the nucleus in Drosophila. However, localizations of the other four subunits remain unknown. This study shows that a catalytic subunit of GPI-T, PIGK, mainly localizes to the endoplasmic reticulum (ER), while the other four subunits localize to the nuclear envelope (NE) and ER. The NE/ER localization ratio of PIGS differs between cell types and developmental stages. These results suggest that GPI-T catalyzes GPI attachment in the ER and the other four subunits may have other unknown functions in the NE.
Johnson, A. E., Orr, B. O., Fetter, R. D., Moughamian, A. J., Primeaux, L. A., Geier, E. G., Yokoyama, J. S., Miller, B. L. and Davis, G. W. (2021). SVIP is a molecular determinant of lysosomal dynamic stability, neurodegeneration and lifespan. Nat Commun 12(1): 513. PubMed ID: 33479240
Missense mutations in Valosin-Containing Protein (VCP) are linked to diverse degenerative diseases including IBMPFD, amyotrophic lateral sclerosis (ALS), muscular dystrophy and Parkinson's disease. This study characterize a VCP-binding co-factor (SVIP) that specifically recruits VCP to lysosomes. SVIP is essential for lysosomal dynamic stability and autophagosomal-lysosomal fusion. SVIP mutations cause muscle wasting and neuromuscular degeneration while muscle-specific SVIP over-expression increases lysosomal abundance and is sufficient to extend lifespan in a context, stress-dependent manner. Multiple links between SVIP and VCP-dependent disease were established in a Drosophila model system. A biochemical screen identifies a disease-causing VCP mutation that prevents SVIP binding. Conversely, over-expression of an SVIP mutation that prevents VCP binding is deleterious. Finally, a human SVIP mutation was identified and the pathogenicity of this mutation was confirmed in the Drosophila model. A model is proposed for VCP disease based on the differential, co-factor-dependent recruitment of VCP to intracellular organelles.

Tuesday, April 27th - Cytoskeleton

Sarkar, S., Olsen, A. L., Sygnecka, K., Lohr, K. M. and Feany, M. B. (2021). alpha-synuclein impairs autophagosome maturation through abnormal actin stabilization. PLoS Genet 17(2): e1009359. PubMed ID: 33556113
Vesicular trafficking defects, particularly those in the autophagolysosomal system, have been strongly implicated in the pathogenesis of Parkinson's disease and related α-synucleinopathies. However, mechanisms mediating dysfunction of membrane trafficking remain incompletely understood. Using a Drosophila model of α-synuclein neurotoxicity with widespread and robust pathology, this study found that human α-synuclein expression impairs autophagic flux in aging adult neurons. Genetic destabilization of the actin cytoskeleton rescues F-actin accumulation, promotes autophagosome clearance, normalizes the autophagolysosomal system, and rescues neurotoxicity in α-synuclein transgenic animals through an Arp2/3 dependent mechanism. Similarly, mitophagosomes accumulate in human α-synuclein-expressing neurons, and reversal of excessive actin stabilization promotes both clearance of these abnormal mitochondria-containing organelles and rescue of mitochondrial dysfunction. These results suggest that Arp2/3 dependent actin cytoskeleton stabilization mediates autophagic and mitophagic dysfunction and implicate failure of autophagosome maturation as a pathological mechanism in Parkinson's disease and related α-synucleinopathies.
Mok, J. W. and Choi, K. W. (2021). Novel function of N-acetyltransferase for microtubule stability and JNK signaling in Drosophila organ development. Proc Natl Acad Sci U S A 118(4). PubMed ID: 33479178
Regulation of microtubule stability is crucial for the maintenance of cell structure and function. This study identified an N-terminal acetyltransferase, Mnat9, that regulates cell signaling and microtubule stability in Drosophila. Loss of Mnat9 causes severe developmental defects in multiple tissues. In the wing imaginal disc, Mnat9 RNAi leads to the ectopic activation of c-Jun N-terminal kinase (JNK) signaling and apoptotic cell death. These defects are suppressed by reducing the level of JNK signaling. Overexpression of Mnat9 can also inhibit JNK signaling. Mnat9 colocalizes with mitotic spindles, and its loss results in various spindle defects during mitosis in the syncytial embryo. Furthermore, overexpression of Mnat9 enhances microtubule stability. Mnat9 is physically associated with microtubules and shows a catalytic activity in acetylating N-terminal peptides of α- and β-tubulin in vitro. Cell death and tissue loss in Mnat9-depleted wing discs are restored by reducing the severing protein Spastin, suggesting that Mnat9 protects microtubules from its severing activity. Remarkably, Mnat9 mutated in the acetyl-CoA binding site is as functional as its wild-type form. This study also found that human NAT9 can rescue Mnat9 RNAi phenotypes in flies, indicating their functional conservation. Taken together, it is proposed that Mnat9 is required for microtubule stability and regulation of JNK signaling to promote cell survival in developing Drosophila organs.
Martin, E., Theis, S., Gay, G., Monier, B., Rouviere, C. and Suzanne, M. (2021). Arp2/3-dependent mechanical control of morphogenetic robustness in an inherently challenging environment. Dev Cell 56(5): 687-701.e687. PubMed ID: 33535069
Epithelial sheets undergo highly reproducible remodeling to shape organs. This stereotyped morphogenesis depends on a well-defined sequence of events leading to the regionalized expression of developmental patterning genes that finally triggers downstream mechanical forces to drive tissue remodeling at a pre-defined position. However, how tissue mechanics controls morphogenetic robustness when challenged by intrinsic perturbations in close proximity has never been addressed. Using Drosophila developing leg, this study shows that a bias in force propagation ensures stereotyped morphogenesis despite the presence of mechanical noise in the environment. Knockdown of the Arp2/3 complex member Arpc5 specifically affects fold directionality while altering neither the developmental nor the force generation patterns. By combining in silico modeling, biophysical tools, and ad hoc genetic tools, these data reveal that junctional myosin II planar polarity favors long-range force channeling and ensures folding robustness, avoiding force scattering and thus isolating the fold domain from surrounding mechanical perturbations.
Nishimura, Y., Shi, S., Zhang, F., Liu, R., Takagi, Y., Bershadsky, A. D., Viasnoff, V. and Sellers, J. R. (2021). The Formin Inhibitor, SMIFH2, Inhibits Members of the Myosin Superfamily. J Cell Sci. PubMed ID: 33589498
The small molecular inhibitor of formin FH2 domains, SMIFH2, is widely used in cell biological studies. It inhibits formin-driven actin polymerization in vitro, but not polymerization of pure actin. It is active against several types of formins from different species. This study found that SMIFH2 inhibits retrograde flow of myosin 2 filaments and contraction of stress fibers. The effect of SMIFH2 was further checked on non-muscle myosin 2A and skeletal muscle myosin 2 in vitro, and SMIFH2 was found to inhibits myosin ATPase activity and ability to translocate actin filaments in the in vitro motility assay. The inhibition of non-muscle myosin 2A in vitro required a higher concentration of SMIFH2 than for the inhibition of retrograde flow and stress fiber contraction in cells. It was also found that SMIFH2 inhibits several other non-muscle myosin types, e.g. mammalian myosin 10, Drosophila myosin 7a and Drosophila myosin 5, more efficient than inhibition of formins. These off-target inhibitions demand additional careful analysis in each case when solely SMIFH2 is used to probe formin functions.
Plochocka, A. Z., Ramirez Moreno, M., Davie, A. M., Bulgakova, N. A. and Chumakova, L. (2021). Robustness of the microtubule network self-organization in epithelia. Elife 10. PubMed ID: 33522481
Robustness of biological systems is crucial for their survival, however, for many systems its origin is an open question. This study analyzed one subcellular level system, the microtubule cytoskeleton. Microtubules self-organize into a network, along which cellular components are delivered to their biologically relevant locations. While the dynamics of individual microtubules is sensitive to the organism's environment and genetics, a similar sensitivity of the overall network would result in pathologies. Large-scale stochastic simulations show that the self-organization of microtubule networks is robust in a wide parameter range in individual cells. This robustness was confirmed in vivo on the tissue-scale using genetic manipulations of Drosophila epithelial cells. Finally, a minimal mathematical model shows that the origin of robustness is the separation of time-scales in microtubule dynamics rates. Altogether, this study demonstrates that the tissue-scale self-organization of a microtubule network depends only on cell geometry and the distribution of the microtubule minus-ends.
Liu, R., Billington, N., Yang, Y., Bond, C., Hong, A., Siththanandan, V., Takagi, Y. and Sellers, J. R. (2021). A binding protein regulates myosin-7a dimerization and actin bundle assembly. Nat Commun 12(1): 563. PubMed ID: 33495456

Myosin-7a, despite being monomeric in isolation, plays roles in organizing actin-based cell protrusions such as filopodia, microvilli and stereocilia, as well as transporting cargoes within them. This study has identified a binding protein for Drosophila myosin-7a termed M7BP and describes how M7BP assembles myosin-7a into a motile complex that enables cargo translocation and actin cytoskeletal remodeling. M7BP binds to the autoinhibitory tail of myosin-7a, extending the molecule and activating its ATPase activity. Single-molecule reconstitution show that M7BP enables robust motility by complexing with myosin-7a as 2:2 translocation dimers in an actin-regulated manner. Meanwhile, M7BP tethers actin, enhancing complex's processivity and driving actin-filament alignment during processive runs. Finally, we show that myosin-7a-M7BP complex assembles actin bundles and filopodia-like protrusions while migrating along them in living cells. Together, these findings provide insights into the mechanisms by which myosin-7a functions in actin protrusions.

Monday, April 26th - Disease Models

Pragati, S. S. (2021). Shaggy functions downstream of dMyc and their concurrent downregulation confers additive rescue against tau toxicity in Drosophila. Biofactors. PubMed ID: 33651466
Neurodegenerative tauopathies such as Alzheimer's and Parkinson's diseases are characterized by hyperphosphorylation of tau protein and their subsequent aggregation in the forms of paired helical filaments and/or neurofibrillary tangles in specific areas of the brain. Despite several attempts, it remains a challenge to develop reliable biomarkers or effective drugs against tauopathies. It is increasingly evident now that due to the involvement of multiple cellular cascades affected by the pathogenic tau molecules, a single genetic modifier or a molecule is unlikely to be efficient enough to provide an inclusive rescue. Hence, multitargets based combinatorial approach(s) have been suggested to provide an efficient rescue against tauopathies. It has been reported that targeted downregulation of dmyc (a Drosophila homolog of human cmyc proto-oncogene) restricts tau etiology by limiting tau hyperphosphorylation and heterochromatin loss. Although, dmyc generates a significant rescue; however, it is not proficient enough to provide a complete alleviation against tauopathies. This study reports that tissue-specific concurrent downregulation of dmyc and gsk3β conveys a near-complete rescue against tau toxicity in Drosophila. It is noted that combinatorial downregulation of dmyc and gsk3β reduces tau hyperphosphorylation, restricts the formation of neurofibrillary tangles, and restores heterochromatin loss to the physiological level. Subsequent investigations revealed that dmyc regulates gsk3β via protein phosphatase 2A (dPP2A) in a dose-dependent manner to regulate tau pathogenesis. It is proposed that dmyc and gsk3β candidates can be utilized in a synergistic manner for the development of an efficient combinatorial therapeutic approach against the devastating human tauopathies.
Odnokoz, O., Nakatsuka, K., Wright, C., Castellanos, J., Klichko, V. I., Kretzschmar, D., Orr, W. C. and Radyuk, S. N. (2021). Mitochondrial Redox Signaling Is Critical to the Normal Functioning of the Neuronal System. Front Cell Dev Biol 9: 613036. PubMed ID: 33585478
Mitochondrial dysfunction often leads to neurodegeneration and is considered one of the main causes of neurological disorders, such as Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS) and other age-related diseases. Mitochondrial dysfunction is tightly linked to oxidative stress and accumulating evidence suggests the association between oxidative stress and neurological disorders. However, there is insufficient knowledge about the role of pro-oxidative shift in cellular redox and impairment of redox-sensitive signaling in the development of neurodegenerative pathological conditions. To gain a more complete understanding of the relationship between mitochondria, redox status, and neurodegenerative disorders, this study investigated the effect of mitochondrial thiol-dependent peroxidases, peroxiredoxins (Prxs), on the physiological characteristics of flies, which change with pathologies such as PD, ALS and during aging. Previous work found that through their ability to sense changes in redox and regulate redox-sensitive signaling, Prxs play a critical role in maintaining global thiol homeostasis, preventing age-related apoptosis and chronic activation of the immune response. It was also found that the phenotype of flies under-expressing Prxs in mitochondria shares many characteristics with the phenotype of Drosophila models of neurological disorders such as ALS, including impaired locomotor activity and compromised redox balance. This study has expanded the study and found that under-expression of mitochondrial Prxs leads to behavioral changes associated with neural function, including locomotor ability, sleep-wake behavior, and temperature-sensitive paralysis. It was also found that under-expression of mitochondrial Prxs with a motor-neuron-specific driver, D42-GAL4, was a determining factor in the development of the phenotype of shortened lifespan and impaired motor activity in flies. The results of the study suggest a causal link between mitochondrial Prx activity and the development of neurological disorders and pre-mature aging.
Nan, Y., Lin, J., Cui, Y., Yao, J., Yang, Y. and Li, Q. (2021). Protective role of vitamin B6 against mitochondria damage in Drosophila models of SCA3. Neurochem Int 144: 104979. PubMed ID: 33535071
Polyglutamine (polyQ)-mediated mitochondria damage is one of the prime causes of polyQ toxicity, which leads to the loss of neurons and the injury of non-neuronal cells. With the discovery of the crucial role of the gut-brain axis and gut microbes in neurological diseases, the relationship between visceral damage and neurological disorders has also received extensive attention. This study successfully simulated the polyQ mitochondrial damage model by expressing 78 or 84 polyglutamine-containing Ataxin3 proteins in Drosophila intestinal enterocytes. In vivo, polyQ expression can reduce mitochondrial membrane potential, mitochondrial DNA damage, abnormal mitochondrial morphology, and loose mitochondrial cristae. Expression profiles evaluated by RNA-seq showed that mitochondrial structural genes and functional genes (oxidative phosphorylation and tricarboxylic acid cycle-related) were significantly down-regulated. More importantly, bioinformatic analyses demonstrated that pathological polyQ expression induced vitamin B6 metabolic pathways abnormality. Active vitamin B6 participates in hundreds of enzymatic reactions and is very important for maintaining mitochondria's activities. In the Spinocerebellar ataxia type 3 Drosophila model, Vitamin B6 supplementation significantly suppressed ECs mitochondria damage in guts and inhibited cellular polyQ aggregates in fat bodies, indicating a promising therapeutic strategy for the treatment of polyQ. Taken together, these results reveal a crucial role for the Vitamin B6-mediated mitochondrial protection in polyQ-induced cellular toxicity, which provides strong evidence for this process as a drug target in polyQ diseases treatment.
Marquilly, C., Busto, G. U., Leger, B. S., Boulanger, A., Giniger, E., Walker, J. A., Fradkin, L. G. and Dura, J. M. (2021). Htt is a repressor of Abl activity required for APP-induced axonal growth. PLoS Genet 17(1): e1009287. PubMed ID: 33465062
Huntington's disease is a progressive autosomal dominant neurodegenerative disorder caused by the expansion of a polyglutamine tract at the N-terminus of a large cytoplasmic protein. The Drosophila huntingtin (htt) gene is widely expressed during all developmental stages from embryos to adults. However, Drosophila htt mutant individuals are viable with no obvious developmental defects. This study asked if such defects could be detected in htt mutants in a background that had been genetically sensitized to reveal cryptic developmental functions. Amyloid precursor protein (APP) is linked to Alzheimer's disease. Appl is the Drosophila APP ortholog and Appl signaling modulates axon outgrowth in the mushroom bodies (MBs), the learning and memory center in the fly, in part by recruiting Abl tyrosine kinase. This study finds that htt mutations suppress axon outgrowth defects of αβ neurons in Appl mutant MB by derepressing the activity of Abl. Abl was shown to be required in MB αβ neurons for their axon outgrowth. Importantly, both Abl overexpression and lack of expression produce similar phenotypes in the MBs, indicating the necessity of tightly regulating Abl activity. Htt was found to behave genetically as a repressor of Abl activity, and consistent with this, in vivo FRET-based measurements reveal a significant increase in Abl kinase activity in the MBs when Htt levels are reduced. Thus, Appl and Htt have essential but opposing roles in MB development, promoting and suppressing Abl kinase activity, respectively, to maintain the appropriate intermediate level necessary for axon growth.
Parkhitko, A. A., Singh, A., Hsieh, S., Hu, Y., Binari, R., Lord, C. J., Hannenhalli, S., Ryan, C. J. and Perrimon, N. (2021). Cross-species identification of PIP5K1-, splicing- and ubiquitin-related pathways as potential targets for RB1-deficient cells. PLoS Genet 17(2): e1009354. PubMed ID: 33591981
The RB1 tumor suppressor is recurrently mutated in a variety of cancers including retinoblastomas, small cell lung cancers, triple-negative breast cancers, prostate cancers, and osteosarcomas. Finding new synthetic lethal (SL) interactions with RB1 could lead to new approaches to treating cancers with inactivated RB1. This study identified 95 SL partners of RB1 based on a Drosophila screen for genetic modifiers of the eye phenotype caused by defects in the RB1 ortholog, Rbf1. 38 mammalian orthologs of Rbf1 modifiers were evaluated as RB1 SL partners in human cancer cell lines with defective RB1 alleles. It was further shown that for many of the RB1 SL genes validated in human cancer cell lines, low activity of the SL gene in human tumors, when concurrent with low levels of RB1 was associated with improved patient survival. Higher order combinatorial gene interactions were investigated by creating a novel Drosophila cancer model with co-occurring Rbf1, Pten and Ras mutations; targeting RB1 SL genes in this background suppressed the dramatic tumor growth and rescued fly survival whilst having minimal effects on wild-type cells. Finally, it was found that drugs targeting the identified RB1 interacting genes/pathways, such as UNC3230, PYR-41, TAK-243, isoginkgetin, madrasin, and celastrol also elicit SL in human cancer cell lines. In summary, this study identified several high confidence, evolutionarily conserved, novel targets for RB1-deficient cells that may be further adapted for the treatment of human cancer.
Nikookar, H., Haddadi, M., Haghi, M. and Masoudi, R. (2021). DNT1 Downregulation and Increased Ethanol Sensitivity in Transgenic Drosophila Models of Alzheimer's Disease. Arch Gerontol Geriatr 94: 104355. PubMed ID: 33550108
Two major pathological hallmarks of Alzheimer's disease (AD) are amyloid plaques and neurofibrillary tangles of hyperphosphorylated tau. Aggregation of amyloid-β (Aβ) is considered as the primary insult in AD. However, failure in treatments based on targeting Aβ without considering the pathologic tau and close correlation between pathological tau and cognitive decline highlighted the crucial role of tau in AD. Loss of synaptic plasticity and cognitive decline, partly due to decrease in Brain Derived Neurotrophic Factor (BDNF), are other hallmarks of AD. Aβ and tau downregulate BDNF at both transcriptional and translational levels. The aim of this research was to study the expression levels of Drosophila Neurotrophin 1 (DNT1), as an orthologue of BDNF, in flies expressing Aβ(42) or tau(R406W). Levels of DNT1 were determined using quantitative real time PCR. Behavioral and Biochemical investigations were also performed in parallel. The results showed that there is a significant decrease in the levels of DNT1 expression in Aβ(42) or tau(R406W) expressing flies. Interestingly, a significant increase was observed in sensitivity to ethanol in both transgenic flies. Rise in Reactive Oxygen Species (ROS) levels was also detected. It is concluded that both Aβ and pathological tau exert their toxic effect on DNT1 expression, ROS production, and response to ethanol, independently. Interestingly, pathological tau showed higher impact on the ROS production compared to Aβ. It seems that Aβ(42) and tau(R406W) transgenic flies are proper models to investigate the interplay between BDNF and oxidative stress, and also to assess the mechanism underlying behavioral response to ethanol.

Friday, April 23rd - Evolution

Johnstun, J. A., Shankar, V., Mokashi, S. S., Sunkara, L. T., Ihearahu, U. E., Lyman, R. L., Mackay, T. F. C. and Anholt, R. R. H. (2021). Functional Diversification, Redundancy and Epistasis among Paralogs of the Drosophila melanogaster Obp50a-d Gene Cluster. Mol Biol Evol. PubMed ID: 33560417
Large multigene families, such as the insect odorant binding proteins (OBPs), are thought to arise through functional diversification after repeated gene duplications. Whereas many OBPs function in chemoreception, members of this family are also expressed in tissues outside chemosensory organs. Paralogs of the Obp50 gene cluster are expressed in metabolic and male reproductive tissues, but their functions and interrelationships remain unknown. This study reports the genetic dissection of four members of the Obp50 cluster, which are in close physical proximity without intervening genes. CRISPR technology was used to excise the entire cluster while introducing a PhiC31 re-integration site to reinsert constructs in which different combinations of the constituent Obp genes were either intact or rendered inactive. Whole transcriptome sequencing was performed and sexually dimorphic changes in transcript abundances ("transcriptional niches") associated with each gene-edited genotype were assessed. Using this approach, it was possible to estimate redundancy, additivity, diversification, and epistasis among Obp50 paralogs. The effects were analyzed of gene editing of this cluster on organismal phenotypes, and a significant skewing was found of sex ratios attributable to Obp50a, and sex-specific effects on starvation stress resistance attributable to Obp50d. Thus, there is functional diversification within the Obp50 cluster with Obp50a contributing to development and Obp50d to stress resistance. The deletion-reinsertion approach applied to the Obp50 cluster provides a general paradigm for the genetic dissection of paralogs of multigene families.
Onal, P., Imaya Gunasinghe, H., Yui Umezawa, K., Zheng, M., Ling, J., Azeez, L., Dalmeus, A., Tazin, T. and Small, S. (2021). Suboptimal Intermediates Underlie Evolution of the Bicoid Homeodomain. Mol Biol Evol. PubMed ID: 33599280
Changes in regulatory networks generate materials for evolution to create phenotypic diversity. For transcription networks, multiple studies have shown that alterations in binding sites of cis-regulatory elements correlate well with the gain or loss of specific features of the body plan. Less is known about alterations in the amino acid sequences of the transcription factors (TFs) that bind these elements. This study examined the evolution of Bicoid (Bcd), a homeodomain (HD) protein that is critical for anterior embryo patterning in Drosophila. The ancestor of Bcd (AncBcd) emerged after a duplication of a Zerknullt (Zen)-like ancestral protein (AncZB) in a suborder of flies. AncBcd diverged from AncZB, gaining novel transcriptional and translational activities. This study focused on the evolution of the HD of AncBcd, which binds to DNA and RNA, and is comprised of four subdomains: an N-terminal arm (NT) and three helices; H1, H2, and Recognition Helix (RH). Using chimeras of subdomains and gene rescue assays in Drosophila, this study showd that robust patterning activity of the Bcd HD (high frequency rescue to adulthood) is achieved only when amino acid substitutions in three separate subdomains (NT, H1, and RH) are combined. Other combinations of subdomains also yield full rescue, but with lower penetrance, suggesting alternative suboptimal activities. The results suggest a multi-step pathway for the evolution of the Bcd HD that involved intermediate HD sequences with suboptimal activities, which constrained and enabled further evolutionary changes. They also demonstrate critical epistatic forces that contribute to the robust function of a DNA-binding domain.
Manat, Y., Lund-Hansen, K. K., Katsianis, G. and Abbott, J. K. (2021). Female-limited X-chromosome evolution effects on male pre- and post-copulatory success. Biol Lett 17(3): 20200915. PubMed ID: 33653095
Intralocus sexual conflict arises when the expression of shared alleles at a single locus generates opposite fitness effects in each sex (i.e. sexually antagonistic alleles), preventing each sex from reaching its sex-specific optimum. Despite its importance to reproductive success, the relative contribution of intralocus sexual conflict to male pre- and post-copulatory success is not well-understood. This study used a female-limited X-chromosome (FLX) evolution experiment in Drosophila melanogaster to limit the inheritance of the X-chromosome to the matriline, eliminating possible counter-selection in males and allowing the X-chromosome to accumulate female-benefit alleles. After more than 100 generations of FLX evolution, the effect of the evolved X-chromosome on male attractiveness and sperm competitiveness was studied. A non-significant increase was found in attractiveness and decrease was found in sperm offence ability in males expressing the evolved X-chromosomes, but a significant increase in their ability to avoid displacement by other males' sperm. This is consistent with a trade-off between these traits, perhaps mediated by differences in body size, causing a small net reduction in overall male fitness in the FLX lines. These results indicate that the X-chromosome in D. melanogaster is subject to selection via intralocus sexual conflict in males.
Lund-Hansen, K. K., Olito, C., Morrow, E. H. and Abbott, J. K. (2021). Sexually antagonistic coevolution between the sex chromosomes of Drosophila melanogaster. Proc Natl Acad Sci U S A 118(8). PubMed ID: 33602805
Antagonistic interactions between the sexes are important drivers of evolutionary divergence. Interlocus sexual conflict is generally described as a conflict between alleles at two interacting loci whose identity and genomic location are arbitrary, but with opposite fitness effects in each sex. This study builds on previous theory by suggesting that when loci under interlocus sexual conflict are located on the sex chromosomes it can lead to cycles of antagonistic coevolution between them and therefore between the sexes. This hypothesis was tested by performing experimental crosses using Drosophila melanogaster where the sex chromosomes was reciprocally exchanged between five allopatric wild-type populations in a round-robin design. Disrupting putatively coevolved sex chromosome pairs resulted in increased male reproductive success in 16 of 20 experimental populations (10 of which were individually significant), but also resulted in lower offspring egg-to-adult viability that affected both male and female fitness. After 25 generations of experimental evolution these sexually antagonistic fitness effects appeared to be resolved. To formalize the hypothesis, population genetic models were developed of antagonistic coevolution using fitness expressions based on the empirical results. The model predictions support the conclusion that antagonistic coevolution between the sex chromosomes is plausible under the fitness effects observed in these experiments. Together, these results lend both empirical and theoretical support to the idea that cycles of antagonistic coevolution can occur between sex chromosomes and illustrate how this process, in combination with autosomal coadaptation, may drive genetic and phenotypic divergence between populations.
Lange, A., Patel, P. H., Heames, B., Damry, A. M., Saenger, T., Jackson, C. J., Findlay, G. D. and Bornberg-Bauer, E. (2021). Structural and functional characterization of a putative de novo gene in Drosophila. Nat Commun 12(1): 1667. PubMed ID: 33712569
Comparative genomic studies have repeatedly shown that new protein-coding genes can emerge de novo from noncoding DNA. Still unknown is how and when the structures of encoded de novo proteins emerge and evolve. Combining biochemical, genetic and evolutionary analyses, this study elucidated the function and structure of goddard, a gene which appears to have evolved de novo at least 50 million years ago within the Drosophila genus. Previous studies found that goddard is required for male fertility. This study shows that Goddard protein localizes to elongating sperm axonemes and that in its absence, elongated spermatids fail to undergo individualization. Combining modelling, NMR and circular dichroism (CD) data, this study showed that Goddard protein contains a large central α-helix, but is otherwise partially disordered. Similar results were found for Goddard's orthologs from divergent fly species and their reconstructed ancestral sequences. Accordingly, Goddard's structure appears to have been maintained with only minor changes over millions of years.
Gray, L. J., Sokolowski, M, B. and Simpson, J. S. (2021). Drosophila as a useful model for understanding the evolutionary physiology of obesity resistance and metabolic thrifte. Fly (Austin) 15(1): 47-59. PubMed ID: 33704003
Evolved metabolic thriftiness in humans is a proposed contributor to the obesity epidemic. Insect models have been shown to evolve both 'metabolic thrift' in response to rearing on high-protein diets that promote leanness, and 'obesity resistance' when reared on fattening high-carbohydrate, low-protein foods. Despite the hypothesis that human obesity is caused by evolved metabolic thrift, genetic contributions to this physiological trait remain elusive. A pilot study was conducted to determine whether thrift and obesity resistance can arise under laboratory based 'quasi-natural selection' in the genetic model organism Drosophila melanogaster. Both these traits were found to evolve within 16 generations. Contrary to predictions from the 'thrifty genotype/phenotype' hypothesis, this study found that when animals from a metabolic thrift inducing high-protein environment are mismatched to fattening high-carbohydrate foods, they did not become 'obese'. Rather, they accumulate less triglyceride than control animals, not more. It is speculated that this may arise through as yet un-quantified parental effects - potentially epigenetic. This study establishes that D. melanogaster could be a useful model for elucidating the role of the trans- and inter-generational effects of diet on the genetics of metabolic traits in higher animals.

Thursday, April 22nd - Adult Physiology

Lourido, F., Quenti, D., Salgado-Canales, D. and Tobar, N. (2021). Domeless receptor loss in fat body tissue reverts insulin resistance induced by a high-sugar diet in Drosophila melanogaster. Sci Rep 11(1): 3263. PubMed ID: 33547367
Insulin resistance is a hallmark of type 2 diabetes resulting from the confluence of several factors, including genetic susceptibility, inflammation, and diet. Under this pathophysiological condition, the dysfunction of the adipose tissue triggered by the excess caloric supply promotes the loss of sensitivity to insulin at the local and peripheral level, a process in which different signaling pathways are involved that are part of the metabolic response to the diet. Besides, the dysregulation of insulin signaling is strongly associated with inflammatory processes in which the JAK/STAT pathway plays a central role. To better understand the role of JAK/STAT signaling in the development of insulin resistance, Drosophila melanogaster was used as a type 2 diabetes model generated by the consumption of a high-sugar diet (HSD). In this model, the effects were studied of inhibiting the expression of the JAK/STAT pathway receptor Domeless, in fat body, on adipose metabolism and glycemic control. The results show that the Domeless receptor loss in fat body cells reverses both hyperglycemia and the increase in the expression of the insulin resistance marker Nlaz, observed in larvae fed a high sugar diet. This effect is consistent with a significant reduction in Dilp2 mRNA expression and an increase in body weight compared to wild-type flies fed high sugar diets. Additionally, the loss of Domeless reduced the accumulation of triglycerides in the fat body cells of larvae fed HSD and also significantly increased the lifespan of adult flies. Taken together, the results show that the loss of Domeless in the fat body reverses at least in part the dysmetabolism induced by a high sugar diet in a Drosophila type 2 diabetes model.
Jorgensen, L. B., Overgaard, J., Hunter-Manseau, F. and Pichaud, N. (2021). Dramatic changes in mitochondrial substrate use at critically high temperatures: a comparative study using Drosophila. J Exp Biol. PubMed ID: 33563650
Ectotherm thermal tolerance is critical to species distribution, but at present the physiological underpinnings of heat tolerance remain poorly understood. Mitochondrial function is perturbed at critically high temperatures in some ectotherms, including insects, suggesting that heat tolerance of these animals is linked to failure of oxidative phosphorylation (OXPHOS) and/or ATP production. To test this hypothesis, mitochondrial oxygen consumption rates were measured in six Drosophila species with different heat tolerance using high-resolution respirometry. Using a substrate-uncoupler-inhibitor titration protocol specific steps of the electron transport system were examined to study how temperatures below, bracketing and above organismal heat limits affected mitochondrial function and substrate oxidation. At benign temperatures (19 and 30°C), complex I-supported respiration (CI-OXPHOS) was the most significant contributor to maximal OXPHOS. At higher temperatures (34, 38, 42 and 46°C), CI-OXPHOS decreased considerably, ultimately to very low levels at 42 and 46°C. The enzymatic catalytic capacity of complex I was intact across all temperatures and accordingly the decreased CI-OXPHOS is unlikely to be caused directly by hyperthermic denaturation/inactivation of complex I. Despite the reduction in CI-OXPHOS, maximal OXPHOS capacities were maintained in all species, through oxidation of alternative substrates; proline, succinate and, particularly, glycerol-3-phosphate, suggesting important mitochondrial flexibility at temperatures exceeding the organismal heat limit. Interestingly, this failure of CI-OXPHOS and compensatory oxidation of alternative substrates occurred at temperatures that tended to correlate with species heat tolerance, such that heat-tolerant species could defend "normal" mitochondrial function at higher temperatures than sensitive species. Future studies should investigate why CI-OXPHOS is perturbed and how this potentially affects ATP production rates.
Liu, X. W., Wu, H. M., Bai, Y., Zeng, Q., Cao, Z. M., Wu, X. S. and Tang, M. (2021). Potassium channel Shaker play a protective role against cardiac aging in Drosophila. Yi Chuan 43(1): 94-99. PubMed ID: 33509778
Potassium channels, which are the most diverse group of the ion channel family, play an important role in the repolarization of cardiomyocytes. Recent studies showed that potassium channels, such as KCNQ and HERG/eag, play an important role in regulating adult heart function through shaping the action potential and maintaining the rhythm of cardiac contraction. The potassium channel protein Shaker is the first voltage-gated potassium channel found in Drosophila to maintain the electrical excitability of neurons and muscle cells, but its role in adult cardiac function is still unclear. In this study, Drosophila was used as a model to study the role of Shaker channel in the maintenance of cardiac function under stress and aging. The incidence of heart failure was observed in shaker mutant after external electrical pacing, which simulates cardiac stress. Additionally, The cardiac-specific driver hand4.2 Gal4 was used to specifically knock down the expression of the potassium channel shaker in Drosophila. The cardiac parameter was analyzed at 1, 3, 5 weeks of age on cardiac specific knockdown of shaker using Drosophila adult cardiac physiological assay. The results showed that the mutation of shaker gene seriously affect the cardiac function under stress, demonstrated by significant increase in heart failure rate under electrical stimulation. In addition, cardiac specific knockdown of shaker increased the incidence of arrhythmias in Drosophila at the age of 5 weeks. Cardiac-specific knockdown of shaker reduces life span. Therefore, the results of this study suggest a vital role of the potassium channel shaker in maintaining normal cardiac function during aging.
Kawecki, T. J., Erkosar, B., Dupuis, C., Hollis, B., Stillwell, R. C. and Kapun, M. (2021). The genomic architecture of adaptation to larval malnutrition points to a trade-off with adult starvation resistance in Drosophila. Mol Biol Evol. PubMed ID: 33677563
Periods of nutrient shortage impose strong selection on animal populations. Experimental studies of genetic adaptation to nutrient shortage largely focus on resistance to acute starvation at adult stage; it is not clear how conclusions drawn from these studies extrapolate to other forms of nutritional stress. The genomic signature of adaptation to chronic juvenile malnutrition was studied in six populations of Drosophila melanogaster evolved for 150 generations on an extremely nutrient-poor larval diet. Comparison with control populations evolved on standard food revealed repeatable genomic differentiation between the two set of population, involving >3,000 candidate SNPs forming >100 independently evolving clusters. The candidate genomic regions were enriched in genes implicated in hormone, carbohydrate, and lipid metabolism, including some with known effects on fitness-related life-history traits. Rather than being close to fixation, a substantial fraction of candidate SNPs segregated at intermediate allele frequencies in all malnutrition-adapted populations. This, together with patterns of among-population variation in allele frequencies and estimates of Tajima's D, suggests that the poor diet results in balancing selection on some genomic regions. Candidate genes for tolerance to larval malnutrition showed a high overlap with genes previously implicated in acute starvation resistance. However, adaptation to larval malnutrition in this study was associated with reduced tolerance to acute adult starvation. Thus, rather than reflecting synergy, the shared genomic architecture appears to mediate an evolutionary trade-off between tolerances to these two forms of nutritional stress.
Oka, M., Suzuki, E., Asada, A., Saito, T., Iijima, K. M. and Ando, K. (2021). Increasing neuronal glucose uptake attenuates brain aging and promotes life span under dietary restriction in Drosophila. iScience 24(1): 101979. PubMed ID: 33490892
Brain neurons play a central role in organismal aging, but there is conflicting evidence about the role of neuronal glucose availability because glucose uptake and metabolism are associated with both aging and extended life span. This study analyzed metabolic changes in the brain neurons of Drosophila during aging. Using a genetically encoded fluorescent adenosine triphosphate (ATP) biosensor, decreased ATP concentration was found in the neuronal somata of aged flies, correlated with decreased glucose content, expression of glucose transporter and glycolytic enzymes and mitochondrial quality. The age-associated reduction in ATP concentration did not occur in brain neurons with suppressed glycolysis or enhanced glucose uptake, suggesting these pathways contribute to ATP reductions. Despite age-associated mitochondrial damage, increasing glucose uptake maintained ATP levels, suppressed locomotor deficits, and extended the life span. Increasing neuronal glucose uptake during dietary restriction resulted in the longest life spans, suggesting an additive effect of enhancing glucose availability during a bioenergetic challenge on aging.
Lovejoy, P. C., Foley, K. E., Conti, M. M., Meadows, S. M., Bishop, C. and Fiumera, A. C. (2021). The genetic basis of susceptibility to low-dose paraquat and variation between the sexes in D. melanogaster. Mol Ecol. PubMed ID: 33710693
Toxicant resistance is a complex trait, affected both by genetics and the environment. Like most complex traits, it can exhibit sexual dimorphism, yet sex is often overlooked as a factor in studies of toxicant resistance. Paraquat, one such toxicant, is a commonly used herbicide and is known to produce mitochondrial oxidative stress, decrease dopaminergic neurons and dopamine (DA) levels, and decrease motor ability. The purpose of this study was to map the genes contributing to low-dose paraquat susceptibility in Drosophila melanogaster, and to determine if susceptibility differs between the sexes. One hundred of the Drosophila Genetic Reference Panel (DGRP) lines were scored for susceptibility via climbing ability and used in a genome wide association study (GWAS). Variation in seventeen genes in females and thirty-five genes in males associated with paraquat susceptibility. Only two candidate genes overlapped between the sexes despite a significant positive correlation between male and female susceptibilities. Many associated polymorphisms had significant interactions with sex, with most having conditionally neutral effects. Conditional neutrality between the sexes likely stems from sex-biased expression which may result from partial resolution of sexual conflict. Candidate genes were verified with RNAi knockdowns, gene expression analyses, and DA quantification. Several of these genes are novel associations with paraquat susceptibility. This research highlights the importance of assessing both sexes when studying toxicant susceptibility.

Wednesday, April 21st - Adult neural development and function

McCurdy, L. Y., Sareen, P., Davoudian, P. A. and Nitabach, M. N. (2021). Dopaminergic mechanism underlying reward-encoding of punishment omission during reversal learning in Drosophila. Nat Commun 12(1): 1115. PubMed ID: 33602917
Animals form and update learned associations between otherwise neutral sensory cues and aversive outcomes (i.e., punishment) to predict and avoid danger in changing environments. When a cue later occurs without punishment, this unexpected omission of aversive outcome is encoded as reward via activation of reward-encoding dopaminergic neurons. How such activation occurs remains unknown. Using real-time in vivo functional imaging, optogenetics, behavioral analysis and synaptic reconstruction from electron microscopy data, this study identified the neural circuit mechanism through which Drosophila reward-encoding dopaminergic neurons are activated when an olfactory cue is unexpectedly no longer paired with electric shock punishment. Reduced activation of punishment-encoding dopaminergic neurons relieves depression of olfactory synaptic inputs to cholinergic neurons. Synaptic excitation by these cholinergic neurons of reward-encoding dopaminergic neurons increases their odor response, thus decreasing aversiveness of the odor. These studies reveal how an excitatory cholinergic relay from punishment- to reward-encoding dopaminergic neurons encodes the absence of punishment as reward, revealing a general circuit motif for updating aversive memories that could be present in mammals.
Melo, N., Capek, M., Arenas, O. M., Afify, A., Yilmaz, A., Potter, C. J., Laminette, P. J., Para, A., Gallio, M. and Stensmyr, M. C. (2021). The irritant receptor TRPA1 mediates the mosquito repellent effect of catnip. Curr Biol. PubMed ID: 33667373
Catnip (Nepeta cataria) is a common garden herb well known for its euphoric and hallucinogenic effects on domestic cats, for its medicinal properties, as well as for its powerful repellent action on insects. Catnip extracts have been proposed as a natural alternative to synthetic insect repellents, such as N,N-diethyl-3-methylbenzamide (DEET), but how catnip triggers aversion in insects is not known. This study shows that, both in Drosophila melanogaster flies and Aedes aegypti mosquitoes, the major mediator of catnip repellency is the widely conserved chemical irritant receptor TRPA1. In vitro, both catnip extract and its active ingredient nepetalactone can directly activate fly and mosquito TRPA1. In vivo, D. melanogaster and Ae. aegypti TRPA1 mutants are no longer repelled by catnip and nepetalactone. Interestingly, the data show that some, but not all, fly and mosquito TRPA1 variants are catnip targets. Moreover, unlike the broad TRPA1 agonist allyl isothiocyanate (AITC) (an active ingredient of tear gas and wasabi), catnip does not activate human TRPA1. These results support the use of catnip and nepetalactone as insect-selective irritants and suggest that, despite TRPA1's broad conservation, insect TRPA1 can be targeted for the development of safe repellents.
Oh, S. M., Jeong, K., Seo, J. T. and Moon, S. J. (2021). Multisensory interactions regulate feeding behavior in Drosophila. Proc Natl Acad Sci U S A 118(7). PubMed ID: 33558226
The integration of two or more distinct sensory cues can help animals make more informed decisions about potential food sources, but little is known about how feeding-related multimodal sensory integration happens at the cellular and molecular levels. This study shows that multimodal sensory integration contributes to a stereotyped feeding behavior in the model organism Drosophila melanogaster Simultaneous olfactory and mechanosensory inputs significantly influence a taste-evoked feeding behavior called the proboscis extension reflex (PER). Olfactory and mechanical information are mediated by antennal Or35a neurons and leg hair plate mechanosensory neurons, respectively. The controlled delivery of three different sensory cues can produce a supra-additive PER via the concurrent stimulation of olfactory, taste, and mechanosensory inputs. It is suggested that the fruit fly is a versatile model system to study multisensory integration related to feeding, which also likely exists in vertebrates.
McLaughlin, C. N., Brbic, M., Xie, Q., Li, T., Horns, F., Kolluru, S. S., Kebschull, J. M., Vacek, D., Xie, A., Li, J., Jones, R. C., Leskovec, J., Quake, S. R., Luo, L. and Li, H. (2021). Single-cell transcriptomes of developing and adult olfactory receptor neurons in Drosophila. Elife 10. PubMed ID: 33555999
Recognition of environmental cues is essential for the survival of all organisms. Transcriptional changes occur to enable the generation and function of the neural circuits underlying sensory perception. To gain insight into these changes, single-cell transcriptomes of Drosophila olfactory- (ORNs), thermo-, and hygro-sensory neurons were generated at an early developmental and adult stage using single-cell and single-nucleus RNA sequencing. It was discovered that ORNs maintain expression of the same olfactory receptors across development. Using receptor expression and computational approaches, transcriptomic clusters corresponding to anatomically and physiologically defined neuron types were matched across multiple developmental stages. It was found that cell-type-specific transcriptomes partly reflected axon trajectory choices in development and sensory modality in adults. Stage-specific genes were uncovered that could regulate the wiring and sensory responses of distinct ORN types. Collectively, these data reveal transcriptomic features of sensory neuron biology and provide a resource for future studies of their development and physiology.
Min, S., Oh, Y., Verma, P., Whitehead, S. C., Yapici, N., Van Vactor, D., Suh, G. S. and Liberles, S. (2021). Control of feeding by Piezo-mediated gut mechanosensation in Drosophila. Elife 10. PubMed ID: 33599608
Across animal species, meals are terminated after ingestion of large food volumes, yet underlying mechanosensory receptors have so far remained elusive. This study identified an essential role for Drosophila Piezo in volume-based control of meal size. A rare population of fly neurons was discovered that express Piezo, innervate the anterior gut and crop (a food reservoir organ), and respond to tissue distension in a Piezo-dependent manner. Activating Piezo neurons decreases appetite, while Piezo knockout and Piezo neuron silencing cause gut bloating and increase both food consumption and body weight. These studies reveal that disrupting gut distension receptors changes feeding patterns and identify a key role for Drosophila Piezo in internal organ mechanosensation.
Nojima, T., Rings, A., Allen, A. M., Otto, N., Verschut, T. A., Billeter, J. C., Neville, M. C. and Goodwin, S. F. (2021). A sex-specific switch between visual and olfactory inputs underlies adaptive sex differences in behavior. Curr Biol. PubMed ID: 33508219
Although males and females largely share the same genome and nervous system, they differ profoundly in reproductive investments and require distinct behavioral, morphological, and physiological adaptations. How can the nervous system, while bound by both developmental and biophysical constraints, produce these sex differences in behavior? This study uncovered a novel dimorphism in Drosophila melanogaster that allows deployment of completely different behavioral repertoires in males and females with minimum changes to circuit architecture. Sexual differentiation of only a small number of higher order neurons in the brain leads to a change in connectivity related to the primary reproductive needs of both sexes-courtship pursuit in males and communal oviposition in females. This study explains how an apparently similar brain generates distinct behavioral repertoires in the two sexes and presents a fundamental principle of neural circuit organization that may be extended to other species.

Tuesday, April 20 - Methods

Loher, P. and Karathanasis, N. (2020). Machine Learning Approaches Identify Genes Containing Spatial Information From Single-Cell Transcriptomics Data. Front Genet 11: 612840. PubMed ID: 33633771
The development of single-cell sequencing technologies has allowed researchers to gain important new knowledge about the expression profile of genes in thousands of individual cells of a model organism or tissue. A common disadvantage of this technology is the loss of the three-dimensional (3-D) structure of the cells. Consequently, the Dialogue on Reverse Engineering Assessment and Methods (DREAM) organized the Single-Cell Transcriptomics Challenge with the aim to address the following two problems: (a) to identify the top 60, 40, and 20 genes of the Drosophila melanogaster embryo that contain the most spatial information and (b) to reconstruct the 3-D arrangement of the embryo using information from those genes. Two independent techniques were developed, leveraging machine learning models from least absolute shrinkage and selection operator (Lasso) and deep neural networks (NNs), which are applied to high-dimensional single-cell sequencing data in order to accurately identify genes that contain spatial information. The first technique, Lasso.TopX, utilizes the Lasso and ranking statistics and allows a user to define a specific number of features they are interested in. The NN approach utilizes weak supervision for linear regression to accommodate for uncertain or probabilistic training labels. It was shown, individually for both techniques, that it is possible to identify important, stable, and a user-defined number of genes containing the most spatial information. The results from both techniques achieve high performance when reconstructing spatial information in D. melanogaster and also generalize to zebrafish (Danio rerio). Furthermore, novel D. melanogaster genes were identified that carry important positional information and were not previously suspected. It was also shown how the indirect use of the full datasets' information can lead to data leakage and generate bias in overestimating the model's performance. Lastly, the applicability of these approaches to other feature selection problems outside the realm of single-cell sequencing is discurssed and the importance of being able to handle probabilistic training labels.
Mishra, P., Yang, S. E., Montgomery, A. B., Reed, A. R., Rodan, A. R. and Rothenfluh, A. (2021). The fly liquid-food electroshock assay (FLEA) suggests opposite roles for neuropeptide F in avoidance of bitterness and shock. BMC Biol 19(1): 31. PubMed ID: 33593351
Proper regulation of feeding is important for an organism's well-being and survival and involves a motivational component directing the search for food. Dissecting the molecular and neural mechanisms of motivated feeding behavior requires assays that allow quantification of both motivation and food intake. Measurements of motivated behavior usually involve assessing physical effort or overcoming an aversive stimulus. Food intake in Drosophila can be determined in a number of ways, including by measuring the time a fly's proboscis interacts with a food source associated with an electrical current in the fly liquid-food interaction counter (FLIC). This study shows that electrical current flowing through flies during this interaction is aversive, and a modified assay is described to measure motivation in Drosophila. Food intake is reduced during the interaction with FLIC when the electrical current is turned on, which provides a confounding variable in studies of motivated behavior. Based on the FLIC, a novel assay, the fly liquid-food electroshock assay (FLEA), was engineered that allows for current adjustments for each feeding well. Using the FLEA, it was shown that both external incentives and internal motivational state can serve as drivers for flies to overcome higher current (electric shock) to obtain superior food. Unlike similar assays in which bitterness is the aversive stimulus for the fly to overcome, it was shown that current perception is not discounted as flies become more food-deprived. Finally, genetically manipulated flies were used to show that neuropeptide F, an orthologue of mammalian NPY previously implicated in regulation of feeding motivation, is required for sensory processing of electrical current. It is concluded that the FLEA is therefore a novel assay to accurately measure incentive motivation in Drosophila. Using the FLEA, it was also shows that neuropeptide F is required for proper perception or processing of an electroshock, a novel function for this neuropeptide involved in the processing of external and internal stimuli.
Mohr, S. E., Tattikota, S. G., Xu, J., Zirin, J., Hu, Y. and Perrimon, N. (2021). Methods and tools for spatial mapping of single-cell RNAseq clusters in Drosophila. Genetics. PubMed ID: 33713129
Single-cell RNA sequencing (scRNAseq) experiments provide a powerful means to identify clusters of cells that share common gene expression signatures. A major challenge in scRNAseq studies is to map the clusters to specific anatomical regions along the body and within tissues. Existing data, such as information obtained from large-scale in situ RNA hybridization studies, cell type specific transcriptomics, gene expression reporters, antibody stainings, and fluorescent tagged proteins, can help to map clusters to anatomy. However, in many cases, additional validation is needed to precisely map the spatial location of cells in clusters. Several approaches are available for spatial resolution in Drosophila, including mining of existing datasets, and use of existing or new tools for direct or indirect detection of RNA, or direct detection of proteins. This study reviews available resources and emerging technologies that will facilitate spatial mapping of scRNAseq clusters at high resolution in Drosophila. Importantly, the need, available approaches, and reagents for multiplexing gene expression detection in situ, as in most cases scRNAseq clusters are defined by the unique coexpression of sets of genes.
Oberhofer, G., Ivy, T. and Hay, B. A. (2021). Split versions of Cleave and Rescue selfish genetic elements for measured self limiting gene drive. PLoS Genet 17(2): e1009385. PubMed ID: 33600432
Gene drive elements promote the spread of linked traits, providing methods for changing the composition or fate of wild populations. Self-sustaining Cleave and Rescue (ClvR) elements include a DNA sequence-modifying enzyme such as Cas9/gRNAs that disrupts endogenous versions of an essential gene, a tightly linked recoded version of the essential gene resistant to cleavage (the Rescue), and a Cargo. ClvR spreads by creating loss-of-function (LOF) conditions in which those without ClvR die because they lack functional copies of the essential gene. Modeling was used to show that when the Rescue-Cargo and one or both components required for LOF allele creation (Cas9 and gRNA) reside at different locations (split ClvR), drive of Rescue-Cargo is self-limiting due to a progressive decrease in Cas9 frequency, and thus opportunities for creation of LOF alleles, as spread occurs. Importantly, drive strength and duration can be extended in a measured manner-which is still self-limiting-by moving the two components close enough to each other that they experience some degree of linkage. With linkage, Cas9 transiently experiences drive by hitchhiking with Rescue-Cargo until linkage disequilibrium between the two disappears, a function of recombination frequency and number of generations, creating a novel point of control. Split ClvR was implemented in Drosophila, with key elements on different chromosomes. Cargo/Rescue/gRNAs spreads to high frequency in a Cas9-dependent manner, while the frequency of Cas9 decreases. These observations show that measured, transient drive, coupled with a loss of future drive potential, can be achieved using the simple toolkit that make up ClvR elements-Cas9 and gRNAs and a Rescue/Cargo.
Mack, J. O. and Zhang, Y. V. (2021). A Rapid Food-Preference Assay in Drosophila. J Vis Exp(168). PubMed ID: 33645577
To select food with nutritional value while avoiding the consumption of harmful agents, animals need a sophisticated and robust taste system to evaluate their food environment. The fruit fly, Drosophila melanogaster, is a genetically tractable model organism that is widely used to decipher the molecular, cellular, and neural underpinnings of food preference. To analyze fly food preference, a robust feeding method is needed. Described here is a two-choice feeding assay, which is rigorous, cost-saving, and fast. The assay is Petri-dish-based and involves the addition of two different foods supplemented with blue or red dye to the two halves of the dish. Then, ~70 prestarved, 2-4-day-old flies are placed in the dish and allowed to choose between blue and red foods in the dark for about 90 min. Examination of the abdomen of each fly is followed by the calculation of the preference index. In contrast to multiwell plates, each Petri dish takes only ~20 s to fill and saves time and effort. This feeding assay can be employed to quickly determine whether flies like or dislike a particular food.
Lazar, A. A., Liu, T., Turkcan, M. K. and Zhou, Y. (2021). Accelerating with FlyBrainLab the discovery of the functional logic of the Drosophila brain in the connectomic era. Elife 10. PubMed ID: 33616035
In recent years, a wealth of Drosophila neuroscience data have become available including cell type, connectome/synaptome datasets for both the larva and adult fly. To facilitate integration across data modalities and to accelerate the understanding of the functional logic of the fly brain, FlyBrainLab, a unique open-source computing platform that integrates 3D exploration and visualization of diverse datasets with interactive exploration of the functional logic of modeled executable brain circuits, was developed. FlyBrainLab's User Interface, Utilities Libraries and Circuit Libraries bring together neuroanatomical, neurogenetic and electrophysiological datasets with computational models of different researchers for validation and comparison within the same platform. Seeking to transcend the limitations of the connectome/synaptome, FlyBrainLab also provides libraries for molecular transduction arising in sensory coding in vision/olfaction. Together with sensory neuron activity data, these libraries serve as entry points for the exploration, analysis, comparison and evaluation of circuit functions of the fruit fly brain.

Monday, April 19 - Behavior

Han, R., Wei, T. M., Tseng, S. C. and Lo, C. C. (2021). Characterizing approach behavior of Drosophila melanogaster in Buridan's paradigm. PLoS One 16(1): e0245990. PubMed ID: 33507934
The Buridan's paradigm is a behavioral task designed for testing visuomotor responses or phototaxis in fruit fly Drosophila melanogaster. In the task, a wing-shortened fruit fly freely moves on a round platform surrounded by a 360° white screen with two vertical black stripes placed at 0° and 180°. A normal fly will tend to approach the stripes one at a time and move back and forth between them. A variety of tasks developed based on the Buridan's paradigm were designed to test other cognitive functions such as visual spatial memory. Although the movement patterns and the behavioral preferences of the flies in the Buridan's or similar tasks have been extensively studies a few decades ago, the protocol and experimental settings are markedly different from what are used today. This study revisited the Buridan's paradigm and systematically investigated the approach behavior of fruit flies under different stimulus settings. While early studies revealed an edge-fixation behavior for a wide stripe in the initial visuomotor responses, no such tendency was discovered in the Buridan's paradigm when observing a longer-term behavior up to minutes, a memory-task relevant time scale. Instead, robust negative photoaxis was observed in which the flies approached the central part of the dark stripes of all sizes. In addition, it was found that stripes of 20°-30° width yielded the best performance of approach. Further, the luminance of the stripes and the background screen were varied; it was discovered that the performance depended on the luminance ratio between the stripes and the screen. This study provided useful information for designing and optimizing the Buridan's paradigm and other behavioral tasks that utilize the approach behavior.
Legros, J., Tang, G., Gautrais, J., Fernandez, M. P. and Trannoy, S. (2020). Long-Term Dietary Restriction Leads to Development of Alternative Fighting Strategies. Front Behav Neurosci 14: 599676. PubMed ID: 33519392
In competition for food, mates and territory, most animal species display aggressive behavior through visual threats and/or physical attacks. Such naturally-complex social behaviors have been shaped by evolution. Environmental pressure, such as the one imposed by dietary regimes, forces animals to adapt to specific conditions and ultimately to develop alternative behavioral strategies. The quality of the food resource during contests influence animals' aggression levels. However, little is known regarding the effects of a long-term dietary restriction-based environmental pressure on the development of alternative fighting strategies. To address this, two lines were employed of the wild-type Drosophila melanogaster Canton-S (CS)which originated from the same population but raised under two distinct diets for years. One diet contained both proteins and sugar, while the second one was sugar-free. Male-male aggression assays were set up using both CS lines; differences were found in aggression levels and the fighting strategies employed to establish dominance relationships. CS males raised on a sugar-containing diet started fights with a physical attack and employed a high number of lunges for establishing dominance but displayed few wing threats throughout the fight. In contrast, the sugar-free-raised males favored wing threats as an initial aggressive demonstration and used fewer lunges to establish dominance, but displayed a higher number of wing threats. This study demonstrates that fruit flies that have been raised under different dietary conditions have adapted their patterns of aggressive behavior and developed distinct fighting strategies: one favoring physical attacks, while the other one favoring visual threats.
Monyak, R. E., Golbari, N. M., Chan, Y. B., Pranevicius, A., Tang, G., Fernandez, M. P. and Kravitz, E. A. (2021). Masculinized Drosophila females adapt their fighting strategies to their opponent. J Exp Biol. PubMed ID: 33568440
Many animal species show aggression to gain mating partners and to protect territories and other resources from competitors. Both male and female fruit flies of the species Drosophila melanogaster exhibit aggression in same-sex pairings, but the strategies used are sexually dimorphic. The biological basis for the differing aggression strategies, and the cues promoting one form of aggression over the other, are being explored. This study describes a line of genetically masculinized females that switch between male and female aggression patterns based on the sexual identity of their opponents. When these masculinized females are paired with more aggressive opponents, they increase the amount of male-like aggression they use, but do not alter the level of female aggression. This suggests that male aggression may be more highly responsive to behavioral cues than female aggression. Although the masculinized females of this line show opponent-dependent changes in aggression and courtship behavior, locomotor activity and sleep are unaffected. Thus, the driver line used may specifically masculinize neurons involved in social behavior. A discussion of possible different roles of male and female aggression in fruit flies is included in this paper. These results can serve as precursors to future experiments aimed at elucidating the circuitry and triggering cues underlying sexually dimorphic aggressive behavior.
Litovchenko, M., Meireles-Filho, A. C. A., Frochaux, M. V., Bevers, R. P. J., Prunotto, A., Anduaga, A. M., Hollis, B., Gardeux, V., Braman, V. S., Russeil, J. M. C., Kadener, S., Dal Peraro, M. and Deplancke, B. (2021). Extensive tissue-specific expression variation and novel regulators underlying circadian behavior. Sci Adv 7(5). PubMed ID: 33514540
Natural genetic variation affects circadian rhythms across the evolutionary tree, but the underlying molecular mechanisms are poorly understood. This study investigated population-level, molecular circadian clock variation by generating >700 tissue-specific transcriptomes of Drosophila melanogaster (w(1118)) and 141 Drosophila Genetic Reference Panel (DGRP) lines. This comprehensive circadian gene expression atlas contains >1700 cycling genes including previously unknown central circadian clock components and tissue-specific regulators. Furthermore, >30% of DGRP lines exhibited aberrant circadian gene expression, revealing abundant genetic variation-mediated, intertissue circadian expression desynchrony. Genetic analysis of one line with the strongest deviating circadian expression uncovered a novel cry mutation that, as shown by protein structural modeling and brain immunohistochemistry, disrupts the light-driven flavin adenine dinucleotide cofactor photoreduction, providing in vivo support for the importance of this conserved photoentrainment mechanism. Together, this study revealed pervasive tissue-specific circadian expression variation with genetic variants acting upon tissue-specific regulatory networks to generate local gene expression oscillations.
Chun, C., Biswas, T. and Bhandawat, V. (2021). Drosophila uses a tripod gait across all walking speeds, and the geometry of the tripod is important for speed control. Elife 10. PubMed ID: 33533718
Changes in walking speed are characterized by changes in both the animal's gait and the mechanics of its interaction with the ground. This study examined these changes in walking Drosophila. he fly's center of mass movement with high spatial resolution and the position of its footprints were measured. Flies predominantly employ a modified tripod gait that only changes marginally with speed. The mechanics of a tripod gait can be approximated with a simple model - angular and radial spring-loaded inverted pendulum (ARSLIP) - which is characterized by two springs of an effective leg that become stiffer as the speed increases. Surprisingly, the change in the stiffness of the spring is mediated by the change in tripod shape rather than a change in stiffness of individual legs. The effect of tripod shape on mechanics can also explain the large variation in kinematics among insects, and ARSLIP can model these variations.
McKinney, R. M., Valdez, R. and Ben-Shahar, Y. (2021). The genetic architecture of larval aggregation behavior in Drosophila. J Neurogenet: 1-16. PubMed ID: 33629904
Many insect species exhibit basal social behaviors such as aggregation, which play important roles in their feeding and mating ecologies. However, the evolutionary, genetic, and physiological mechanisms that regulate insect aggregation remain unknown for most species. This study used natural populations of Drosophila melanogaster to identify the genetic architecture that drives larval aggregation feeding behavior. By using quantitative and reverse genetic approaches, a complex neurogenetic network was identified that plays a role in regulating the decision of larvae to feed in either solitude or as a group. Results from single gene, RNAi-knockdown experiments show that several of the identified genes represent key nodes in the genetic network that determines the level of aggregation while feeding. Furthermore, this study showed that a single non-coding variant in the gene CG14205, a putative acyltransferase, is associated with both decreased mRNA expression and increased aggregate formation, which suggests that it has a specific role in inhibiting aggregation behavior. These results identify, for the first time, the genetic components which interact to regulate naturally occurring levels of aggregation in D. melanogaster larvae.

Friday, April 16th - Adult neural development and function

Leung, A., Cohen, D., van Swinderen, B. and Tsuchiya, N. (2021). Integrated information structure collapses with anesthetic loss of conscious arousal in Drosophila melanogaster. PLoS Comput Biol 17(2): e1008722. PubMed ID: 33635858
The physical basis of consciousness remains one of the most elusive concepts in current science. One influential conjecture is that consciousness is to do with some form of causality, measurable through information. The integrated information theory of consciousness (IIT) proposes that conscious experience, filled with rich and specific content, corresponds directly to a hierarchically organised, irreducible pattern of causal interactions; i.e. an integrated informational structure among elements of a system. This study tested this conjecture in a simple biological system (fruit flies), estimating the information structure of the system during wakefulness and general anesthesia. Consistent with this conjecture, it was found that integrated interactions among populations of neurons during wakefulness collapsed to isolated clusters of interactions during anesthesia. Classification analysis to quantify the accuracy of discrimination between wakeful and anesthetised states, and found that informational structures inferred conscious states with greater accuracy than a scalar summary of the structure, a measure which is generally championed as the main measure of IIT. In stark contrast to a view which assumes feedforward architecture for insect brains, especially fly visual systems, rich information structures were found, which cannot arise from purely feedforward systems, occurred across the fly brain. Further, these information structures collapsed uniformly across the brain during anesthesia. The results speak to the potential utility of the novel concept of an "informational structure" as a measure for level of consciousness, above and beyond simple scalar values.
Klann, M., Schacht, M. I., Benton, M. A. and Stollewerk, A. (2021). Functional analysis of sense organ specification in the Tribolium castaneum larva reveals divergent mechanisms in insects. BMC Biol 19(1): 22. PubMed ID: 33546687
Insects and other arthropods utilise external sensory structures for mechanosensory, olfactory, and gustatory reception. In arthropods other than Drosophila, sense organ subtypes cannot be linked to the same code of gene expression as found for Drosophila. This raises the questions of whether the principles underlying subtype identity in D. melanogaster are representative of other insects. This study provides evidence that such principles cannot be generalised, and suggest that sensory organ diversification followed the recruitment of sensory genes to distinct sensory organ specification mechanism. Sense organ development in a nondipteran insect, the flour beetle Tribolium castaneum, was analyzed by gene expression and RNA interference studies. In contrast to D. melanogaster, T. castaneum sense organs cannot be categorised based on the expression or their requirement for individual or combinations of conserved sense organ transcription factors such as cut and pox neuro, or members of the Achaete-Scute (Tc ASH, Tc asense), Atonal (Tc atonal, Tc cato, Tc amos), and neurogenin families (Tc tap). Rather, the observations support an evolutionary scenario whereby these sensory genes are required for the specification of sense organ precursors and the development and differentiation of sensory cell types in diverse external sensilla which do not fall into specific morphological and functional classes. Based on these findings and past research, an evolutionary scenario is presented suggesting that sense organ subtype identity has evolved by recruitment of a flexible sensory gene network to the different sense organ specification processes. A dominant role of these genes in subtype identity has evolved as a secondary effect of the function of these genes in individual or subsets of sense organs, probably modulated by positional cues.
Kanno, M., Hiramatsu, S., Kondo, S., Tanimoto, H. and Ichinose, T. (2021). Voluntary intake of psychoactive substances is regulated by the dopamine receptor Dop1R1 in Drosophila. Sci Rep 11(1): 3432. PubMed ID: 33564023
Dysregulated motivation to consume psychoactive substances leads to addictive behaviors that often result in serious health consequences. Understanding the neuronal mechanisms that drive drug consumption is crucial for developing new therapeutic strategies. The fruit fly Drosophila melanogaster offers a unique opportunity to approach this problem with a battery of sophisticated neurogenetic tools available, but how they consume these drugs remains largely unknown. This study examined drug self-administration behavior of Drosophila and the underlying neuronal mechanisms. The preference of flies for five different psychoactive substances was measured using a two-choice feeding assay and its long-term changes were monitored. Flies were found to show acute preference for ethanol and methamphetamine, but not for cocaine, caffeine or morphine. Repeated intake of ethanol, but not methamphetamine, increased over time. Preference for methamphetamine and the long-term escalation of ethanol preference required the dopamine receptor Dop1R1 in the mushroom body. The protein level of Dop1R1 increased after repeated intake of ethanol, but not methamphetamine, which correlates with the acquired preference. Genetic overexpression of Dop1R1 enhanced ethanol preference. These results reveal a striking diversity of response to individual drugs in the fly and the role of dopamine signaling and its plastic changes in controlling voluntary intake of drugs.
Karam, C. S., Williams, B. L., Jones, S. K. and Javitch, J. A. (2021). The Role of the Dopamine Transporter in the Effects of Amphetamine on Sleep and Sleep Architecture in Drosophila. Neurochem Res. PubMed ID: 33630236
The dopamine transporter (DAT) mediates the inactivation of released dopamine (DA) through its reuptake, and thereby plays an important homeostatic role in dopaminergic neurotransmission. Amphetamines exert their stimulant effects by targeting DAT and inducing the reverse transport of DA, leading to a dramatic increase of extracellular DA. Animal models have proven critical to investigating the molecular and cellular mechanisms underlying transporter function and its modulation by psychostimulants such as amphetamine. This study established a behavioral model for amphetamine action using adult Drosophila melanogaster. This model was used to characterize the effects of amphetamine on sleep and sleep architecture. The data show that amphetamine induces hyperactivity and disrupts sleep in a DA-dependent manner. Flies that do not express a functional DAT (dDAT null mutants) have been shown to be hyperactive and to exhibit significantly reduced sleep at baseline. The data show that, in contrast to its action in control flies, amphetamine decreases the locomotor activity of dDAT null mutants and restores their sleep by modulating distinct aspects of sleep structure. To begin to explore the circuitry involved in the actions of amphetamine on sleep, the localization of dDAT throughout the fly brain is described, particularly in neuropils known to regulate sleep. Together, these data establish Drosophila as a robust model for studying the regulatory mechanisms that govern DAT function and psychostimulant action.
Mattar, P., Jolicoeur, C., Dang, T., Shah, S., Clark, B. S. and Cayouette, M. (2021). A Casz1-NuRD complex regulates temporal identity transitions in neural progenitors. Sci Rep 11(1): 3858. PubMed ID: 33594190
Neural progenitor cells undergo identity transitions during development to ensure the generation different types of neurons and glia in the correct sequence and proportions. A number of temporal identity factors that control these transitions in progenitor competence have been identified, but the molecular mechanisms underlying their function remain unclear. Here, it was asked how Casz1, the mammalian orthologue of Drosophila castor, regulates competence during retinal development. Casz1 is shown to be required to control the transition between neurogenesis and gliogenesis. Using BioID proteomics, it was revealed that Casz1 interacts with the nucleosome remodeling and deacetylase (NuRD) complex in retinal cells. Finally, it was shown that both the NuRD and the polycomb repressor complexes are required for Casz1 to promote the rod fate and suppress gliogenesis. As additional temporal identity factors have been found to interact with the NuRD complex in other contexts, it is proposed that these factors might act through this common biochemical process to regulate neurogenesis.
Li, H., Lones, L. and DiAntonio, A. (2021). Bidirectional regulation of glial potassium buffering - glioprotection versus neuroprotection. Elife 10. PubMed ID: 33646119
Glia modulate neuronal excitability and seizure sensitivity by maintaining potassium and water homeostasis. A salt inducible kinase 3 (SIK3)-regulated gene expression program controls the glial capacity to buffer K(+) and water in Drosophila, however upstream regulatory mechanisms are unknown. This study identified an octopaminergic circuit linking neuronal activity to glial ion and water buffering. Under basal conditions, octopamine functions through the inhibitory octopaminergic G-protein-coupled receptor (GPCR) OctβR to upregulate glial buffering capacity, while under pathological K(+) stress, octopamine signals through the stimulatory octopaminergic GPCR OAMB1 to downregulate the glial buffering program. Failure to downregulate this program leads to intracellular glia swelling and stress signaling, suggesting that turning down this pathway is glioprotective. In the eag shaker Drosophila seizure model, the SIK3-mediated buffering pathway is inactivated. Reactivation of the glial buffering program dramatically suppresses neuronal hyperactivity, seizures, and shortened life span in this mutant. These findings highlight the therapeutic potential of a glial-centric therapeutic strategy for diseases of hyperexcitability.

Thursday, April 16th - Chromatin & Recombination

Liao, Y., Zhang, X., Chakraborty, M. and Emerson, J. J. (2021). Topologically associating domains and their role in the evolution of genome structure and function in Drosophila. Genome Res 31(3): 397-410. PubMed ID: 33563719
Topologically associating domains (TADs) were recently identified as fundamental units of three-dimensional eukaryotic genomic organization, although knowledge of the influence of TADs on genome evolution remains preliminary. To study the molecular evolution of TADs in Drosophila species, a new reference-grade genome assembly and accompanying high-resolution TAD map for D. pseudoobscura was constructed. Comparison of D. pseudoobscura and D. melanogaster, which are separated by ∼49 million years of divergence, showed that ∼30%-40% of their genomes retain conserved TADs. Comparative genomic analysis of 17 Drosophila species revealed that chromosomal rearrangement breakpoints are enriched at TAD boundaries but depleted within TADs. Additionally, genes within conserved TADs show lower expression divergence than those located in nonconserved TADs. Furthermore, it was found that a substantial proportion of long genes (>50 kbp) in D. melanogaster (42%) and D. pseudoobscura (26%) constitute their own TADs, implying transcript structure may be one of the deterministic factors for TAD formation. By using structural variants (SVs) identified from 14 D. melanogaster strains, its three closest sibling species from the D. simulans species complex, and two obscura clade species, evidence was uncovered of selection acting on SVs at TAD boundaries, but with the nature of selection differing between SV types. Deletions are depleted at TAD boundaries in both divergent and polymorphic SVs, suggesting purifying selection, whereas divergent tandem duplications are enriched at TAD boundaries relative to polymorphism, suggesting they are adaptive. These findings highlight how important TADs are in shaping the acquisition and retention of structural mutations that fundamentally alter genome organization.
Carvajal-Garcia, J., Crown, K. N., Ramsden, D. A. and Sekelsky, J. (2021). DNA polymerase theta suppresses mitotic crossing over. PLoS Genet 17(3): e1009267. PubMed ID: 33750946
Polymerase theta-mediated end joining (TMEJ) is a chromosome break repair pathway that is able to rescue the lethality associated with the loss of proteins involved in early steps in homologous recombination (e.g., BRCA1/2). This is due to the ability of polymerase theta (Pol theta) to use resected, 3' single stranded DNA tails to repair chromosome breaks. These resected DNA tails are also the starting substrate for homologous recombination. However, it remains unknown if TMEJ can compensate for the loss of proteins involved in more downstream steps during homologous recombination. This study shows that the Holliday junction resolvases SLX4 and GEN1 are required for viability in the absence of Pol theta in Drosophila melanogaster, and lack of all three proteins results in high levels of apoptosis. Flies deficient in Pol theta and SLX4 are extremely sensitive to DNA damaging agents, and mammalian cells require either Pol theta or SLX4 to survive. The current results suggest that TMEJ and Holliday junction formation/resolution share a common DNA substrate, likely a homologous recombination intermediate, that when left unrepaired leads to cell death. One major consequence of Holliday junction resolution by SLX4 and GEN1 is cancer-causing loss of heterozygosity due to mitotic crossing over. Mitotic crossovers were measured in flies after a Cas9-induced chromosome break; this mutagenic form of repair is increased in the absence of Pol theta. This demonstrates that TMEJ can function upstream of the Holiday junction resolvases to protect cells from loss of heterozygosity. This work argues that Pol theta can thus compensate for the loss of the Holliday junction resolvases by using homologous recombination intermediates, suppressing mitotic crossing over and preserving the genomic stability of cells.
Kaushal, A., Mohana, G., Dorier, J., Ozdemir, I., Omer, A., Cousin, P., Semenova, A., Taschner, M., Dergai, O., Marzetta, F., Iseli, C., Eliaz, Y., Weisz, D., Shamim, M. S., Guex, N., Lieberman Aiden, E. and Gambetta, M. C. (2021). CTCF loss has limited effects on global genome architecture in Drosophila despite critical regulatory functions. Nat Commun 12(1): 1011. PubMed ID: 33579945
Vertebrate genomes are partitioned into contact domains defined by enhanced internal contact frequency and formed by two principal mechanisms: compartmentalization of transcriptionally active and inactive domains, and stalling of chromosomal loop-extruding cohesin by CTCF bound at domain boundaries. While Drosophila has widespread contact domains and CTCF, it is currently unclear whether CTCF-dependent domains exist in flies. CTCF was genetically ablate in Drosophila, and mpacts on genome folding and transcriptional regulation were examined in the central nervous system. CTCF was found to be required to form a small fraction of all domain boundaries, while critically controlling expression patterns of certain genes and supporting nervous system function. It was also found that CTCF recruits the pervasive boundary-associated factor Cp190 to CTCF-occupied boundaries and co-regulates a subset of genes near boundaries together with Cp190. These results highlight a profound difference in CTCF-requirement for genome folding in flies and vertebrates, in which a large fraction of boundaries are CTCF-dependent and suggest that CTCF has played mutable roles in genome architecture and direct gene expression control during metazoan evolution.
Kwon, S. Y., Jang, B. and Badenhorst, P. (2021). The ISWI chromatin remodelling factor NURF is not required for mitotic male X chromosome organisation. MicroPubl Biol 2021. PubMed ID: 33537560
The nucleosome remodelling factor (NURF) is an ISWI-class ATP-dependent chromatin remodeling enzyme required both for gene expression and higher order chromatin organisation. NURF binds to histone modifications that decorate the Drosophila polytene male X chromosome and is required to maintain correct organisation of this chromosome. NURF mutants exhibit distorted and decondensed polytene male X chromosomes dependent on the presence of the male-specific lethal (MSL) complex. This study tested whether mitotic chromosomes similarly require NURF to maintain correct morphology. Surprisingly, although the MSL complex remains associated with mitotic male X chromosomes, NURF is not required to maintain morphology. While the ISWI subunit of NURF is known to remain associated with mitotic chromosomes, this study shows that the NURF specificity subunit Nurf301/BPTF dissociates from chromatin during both Drosophila and human mitosis, further illuminating that NURF is dispensable for mitotic chromosome organisation.
Fursova, N. A., Mazina, M. Y., Nikolenko, J. V., Vorobyova, N. E. and Krasnov, A. N. (2020). Drosophila Zinc Finger Protein CG9890 Is Colocalized with Chromatin Modifying and Remodeling Complexes on Gene Promoters and Involved in Transcription Regulation. Acta Naturae 12(4): 114-119. PubMed ID: 33456983
In this work, a genome-wide study of the zinc finger protein CG9890 was conducted and showed that it is localized mostly on the promoters of active genes. The CG9890 binding sites are low-nucleosome-density regions and are colocalized with the chromatin modifying and remodeling complexes SAGA and dSWI/SNF, as well as with the ORC replication complex. The CG9890 protein was shown to be involved in the regulation of the expression of some genes on the promoters of which it is located, with the ecdysone cascade genes accounting for a significant percentage of these genes. Thus, the CG9890 protein is a new member of the transcriptional network which is localized on active promoters, interacts with the main transcription and replication complexes, and is involved in the regulation of both basal and inducible transcription.
Gong, N. N., Dilley, L. C., Williams, C. E., Moscato, E. H., Szuperak, M., Wang, Q., Jensen, M., Girirajan, S., Tan, T. Y., Deardorff, M. A., Li, D., Song, Y. and Kayser, M. S. (2021). The chromatin remodeler ISWI acts during Drosophila development to regulate adult sleep. Sci Adv 7(8). PubMed ID: 33597246
Sleep disruptions are among the most commonly reported symptoms across neurodevelopmental disorders (NDDs), but mechanisms linking brain development to normal sleep are largely unknown. From a Drosophila screen of human NDD-associated risk genes, the chromatin remodeler Imitation SWItch/SNF (ISWI) was identified as being required for adult fly sleep. Loss of ISWI also results in disrupted circadian rhythms, memory, and social behavior, but ISWI acts in different cells and during distinct developmental times to affect each of these adult behaviors. Specifically, ISWI expression in type I neuroblasts is required for both adult sleep and formation of a learning-associated brain region. Expression in flies of the human ISWI homologs SMARCA1 and SMARCA5 differentially rescues adult phenotypes, while de novo SMARCA5 patient variants fail to rescue sleep. It is proposed that sleep deficits are a primary phenotype of early developmental origin in NDDs and point toward chromatin remodeling machinery as critical for sleep circuit formation.

Wednesday, April 14 - RNA and Transposons

Kolasa, A. M., Bhogal, J. K. and DiAngelo, J. R. (2021). The heterogeneous nuclear ribonucleoprotein (hnRNP) glorund functions in the Drosophila fat body to regulate lipid storage and transport. Biochem Biophys Rep 25: 100919. PubMed ID: 33537463
The availability of excess nutrients in Western diets has led to the overaccumulation of these nutrients as triglycerides, a condition known as obesity. The full complement of genes important for regulating triglyceride storage is not completely understood. Genome-wide RNAi screens in Drosophila cells have identified genes involved in mRNA splicing as important lipid storage regulators. Previous work showed that a group of splicing factors called heterogeneous nuclear ribonucleoproteins (hnRNPs) regulate lipid metabolism in the fly fat body; however, the identities of all the hnRNPs that function to control triglyceride storage are not known. This study used the GAL4/UAS system to induce RNAi to the hnRNP glorund (glo) in the Drosophila fat body to assess whether this hnRNP has any metabolic functions. Decreasing glo levels resulted in less triglycerides being stored throughout the fly. Interestingly, decreasing fat body glo expression resulted in increased triglyceride storage in the fat body, but blunted triglyceride storage in non-fat body tissues, suggesting a defect in lipid transport. Consistent with this hypothesis, the expression of apolipophorin (apolpp), microsomal triglyceride transfer protein (mtp), and apolipoprotein lipid transfer particle (apoltp), apolipoprotein genes important for lipid transport through the fly hemolymph, was decreased in glo-RNAi flies, suggesting that glo regulates the transport of lipids from the fly fat body to surrounding tissues. Together, these results indicate that glorund plays a role in controlling lipid transport and storage and provide additional evidence of the link between gene expression and the regulation of lipid metabolism.
Fabian, D. K., Melike Donertaş, H., Fuentealba, M., Partridge, L. and Thornton, J. M. (2021). Transposable element landscape in Drosophila populations selected for longevity. Genome Biol Evol. PubMed ID: 33595657
Transposable elements (TEs) inflict numerous negative effects on health and fitness as they replicate by integrating into new regions of the host genome. Even though organisms employ powerful mechanisms to demobilize TEs, transposons gradually lose repression during aging. The rising TE activity causes genomic instability and was implicated in age-dependent neurodegenerative diseases, inflammation and the determination of lifespan. It is therefore conceivable that long-lived individuals have improved TE silencing mechanisms resulting in reduced TE expression relative to their shorter-lived counterparts and fewer genomic insertions. This study tested this hypothesis by performing the first genome-wide analysis of TE insertions and expression in populations of Drosophila melanogaster selected for longevity through late-life reproduction for 50-170 generations from four independent studies. Contrary to expectation, TE families were generally more abundant in long-lived populations compared to non-selected controls. Although simulations showed that this was not expected under neutrality, little evidence was found for selection driving TE abundance differences. Additional RNA-seq analysis revealed a tendency for reducing TE expression in selected populations, which might be more important for lifespan than regulating genomic insertions. Limited evidence was found of parallel selection on genes related to TE regulation and transposition. However, telomeric TEs were genomically and transcriptionally more abundant in long-lived flies, suggesting improved telomere maintenance as a promising TE-mediated mechanism for prolonging lifespan. The results provide a novel viewpoint indicating that reproduction at old age increases the opportunity of TEs to be passed on to the next generation with little impact on longevity.
Hudson, A. M., Szabo, N. L., Loughran, G., Wills, N. M., Atkins, J. F. and Cooley, L. (2021). Tissue-specific dynamic codon redefinition in Drosophila. Proc Natl Acad Sci U S A 118(5). PubMed ID: 33500350
Translational stop codon readthrough occurs in organisms ranging from viruses to mammals and is especially prevalent in decoding Drosophila and viral mRNAs. Recoding of UGA, UAG, or UAA to specify an amino acid allows a proportion of the protein encoded by a single gene to be C-terminally extended. The extended product from Drosophila kelch mRNA is 160 kDa, whereas unextended Kelch protein, a subunit of a Cullin3-RING ubiquitin ligase, is 76 kDa. Previously tissue-specific regulation of readthrough of the first kelch stop codon was reported. This study characterizes major efficiency differences in a variety of cell types. Immunoblotting revealed low levels of readthrough in malpighian tubules, ovary, and testis but abundant readthrough product in lysates of larval and adult central nervous system (CNS) tissue. Reporters of readthrough demonstrated greater than 30% readthrough in adult brains, and imaging in larval and adult brains showed that readthrough occurred in neurons but not glia. The extent of readthrough stimulatory sequences flanking the readthrough stop codon was assessed in transgenic Drosophila and in human tissue culture cells where inefficient readthrough occurs. A 99-nucleotide sequence with potential to form an mRNA stem-loop 3' of the readthrough stop codon stimulated readthrough efficiency. However, even with just six nucleotides of kelch mRNA sequence 3' of the stop codon, readthrough efficiency only dropped to 6% in adult neurons. Finally, it was shown that high-efficiency readthrough in the Drosophila CNS is common; for many neuronal proteins, C-terminal extended forms of individual proteins are likely relatively abundant.
Hervas, R., Del Carmen Fernandez-Ramirez, M., Galera-Prat, A., Suzuki, M., Nagai, Y., Bruix, M., Menendez, M., Laurents, D. V. and Carrion-Vazquez, M. (2021). Divergent CPEB prion-like domains reveal different assembly mechanisms for a generic amyloid-like fold. BMC Biol 19(1): 43. PubMed ID: 33706787
Amyloids are ordered, insoluble protein aggregates, characterized by a cross-β sheet quaternary structure in which molecules in a β-strand conformation are stacked along the filament axis via intermolecular interactions. While amyloids are typically associated with pathological conditions, functional amyloids have also been identified and are present in a wide variety of organisms ranging from bacteria to humans. The cytoplasmic polyadenylation element-binding (CPEB) prion-like protein is an mRNA-binding translation regulator, whose neuronal isoforms undergo activity-dependent aggregation, a process that has emerged as a plausible biochemical substrate for memory maintenance. CPEB aggregation is driven by prion-like domains (PLD) that are divergent in sequence across species, and it remains unknown whether such divergent PLDs follow a similar aggregating assembly pathway. This study describes the amyloid-like features of the neuronal Aplysia CPEB (ApCPEB) PLD and compares them to those of the Drosophila ortholog, Orb2 PLD. Using in vitro single-molecule and bulk biophysical methods, this study found transient oligomers and mature amyloid-like filaments that suggest similarities in the late stages of the assembly pathway for both ApCPEB and Orb2 PLDs. However, while prior to aggregation the Orb2 PLD monomer remains mainly as a random coil in solution, ApCPEB PLD adopts a diversity of conformations comprising α-helical structures that evolve to coiled-coil species, indicating structural differences at the beginning of their amyloid assembly pathways. These results indicate that divergent PLDs of CPEB proteins from different species retain the ability to form a generic amyloid-like fold through different assembly mechanisms.
Glukhova, A. A., Kurshakova, M. M., Nabirochkina, E. N., Georgieva, S. G. and Kopytova, D. V. (2021). PCID2, a subunit of the Drosophila TREX-2 nuclear export complex, is essential for both mRNA nuclear export and its subsequent cytoplasmic trafficking. RNA Biol: 1-12. PubMed ID: 33602059
The TREX-2 complex is essential for the general nuclear mRNA export in eukaryotes. TREX-2 interacts with the nuclear pore and transcriptional apparatus and links transcription to the mRNA export. However, it remains poorly understood how the TREX-2-dependent nuclear export is connected to the subsequent stages of mRNA trafficking. This study shows that the PCID2 subunit of Drosophila TREX-2 is present in the cytoplasm of the cell. The cytoplasmic PCID2 directly interacts with the NudC protein and this interaction maintains its stability in the cytoplasm. Moreover, PCID2 is associated with the cytoplasmic mRNA and microtubules. The PCID2 knockdown blocks nuclear export of mRNA and also affects the general mRNA transport into the cytoplasm. These data suggest that PCID2 could be the link between the nuclear TREX-2-dependent export and the subsequent cytoplasmic trafficking of mRNA.
Liu, N., Neuenkirchen, N., Zhong, M. and Lin, H. (2021). Genome-wide mapping of Piwi association with specific loci in Drosophila ovaries. G3 (Bethesda) 11(2). PubMed ID: 33609367
Small noncoding RNA pathways have been implicated in diverse mechanisms of gene regulation. In Drosophila ovaries, Piwi binds to Piwi-interacting RNAs (piRNAs) of mostly 24-28 nucleotides (nt) and plays an important role in germline stem cell maintenance, transposon repression, and epigenetic regulation. To understand the mechanism underlying these functions, this paper reports the application of the DamID-seq method to identify genome-wide binding sites of Piwi in Drosophila ovaries. Piwi localizes to at least 4535 euchromatic regions that are enriched with piRNA target sites. Surprisingly, the density of Piwi binding to euchromatin is much higher than in heterochromatin. Disrupting the piRNA binding of Piwi results in an overall change of the genomic binding profile, which indicates the role of piRNAs in directing Piwi to specific genomic sites. Most Piwi binding sites were either within or in the vicinity of protein-coding genes, particularly enriched near the transcriptional start and termination sites. The methylation signal near the transcriptional termination sites is significantly reduced when Piwi was mutated to become defective in piRNA binding. These observations indicate that Piwi might directly regulate the expression of many protein-coding genes, especially through regulating the 3' ends of targeted transcripts.

Tuesday, April 13th - Cytoskeleton and Junctions

Hildebrand, J. D., Leventry, A. D., Aideyman, O. P., Majewski, J. C., Haddad, J. A., Bisi, D. C. and Kaufmann, N. (2021). A modifier screen identifies regulators of cytoskeletal architecture as mediators of Shroom-dependent changes in tissue morphology. Biol Open 10(2). PubMed ID: 33504488
Regulation of cell architecture is critical in the formation of tissues during animal development. The mechanisms that control cell shape must be both dynamic and stable in order to establish and maintain the correct cellular organization. Previous work has identified Shroom family proteins as essential regulators of cell morphology during vertebrate development. Shroom proteins regulate cell architecture by directing the subcellular distribution and activation of Rho-kinase, which results in the localized activation of non-muscle myosin II. Because the Shroom-Rock-myosin II module is conserved in most animal model systems, Drosophila melanogaster was used to further investigate the pathways and components that are required for Shroom to define cell shape and tissue architecture. Using a phenotype-based heterozygous F1 genetic screen for modifiers of Shroom activity, several cytoskeletal and signaling protein were identified that may cooperate with Shroom. Two of these proteins, Enabled and Short stop, are required for ShroomA-induced changes in tissue morphology and are apically enriched in response to Shroom expression. While the recruitment of Ena is necessary, it is not sufficient to redefine cell morphology. Additionally, this requirement for Ena appears to be context dependent, as a variant of Shroom that is apically localized, binds to Rock, but lacks the Ena binding site, is still capable of inducing changes in tissue architecture. These data point to important cellular pathways that may regulate contractility or facilitate Shroom-mediated changes in cell and tissue morphology.
Chandran, R., Kale, G., Philippe, J. M., Lecuit, T. and Mayor, S. (2021). Distinct actin-dependent nanoscale assemblies underlie the dynamic and hierarchical organization of E-cadherin. Curr Biol. PubMed ID: 33607036
Cadherins are transmembrane adhesion proteins required for the formation of cohesive tissues. Intracellular interactions of E-cadherin with the Catenin family proteins, α- and β-catenin, facilitate connections with the cortical actomyosin network. This is necessary for maintaining the integrity of cell-cell adhesion in epithelial tissues. The supra-molecular architecture of E-cadherin is an important feature of its adhesion function; cis and trans interactions of E-cadherin are deployed to form clusters, both in cis and trans. Studies in Drosophila embryo have also shown that Drosophila E-cadherin (dE-cad) is organized as finite-sized dynamic clusters that localize with actin patches at cell-cell junctions, in continuous exchange with the extra-junctional pool of dE-cad surrounding the clusters. This study used the ectopic expression of dE-cad in larval hemocytes, which lack endogenous dE-cad to recapitulate functional cell-cell junctions in a convenient model system. While dE-cad at cell-cell junctions in hemocytes exhibits a clustered trans-paired organization similar to that reported previously in embryonic epithelial tissue, extra-junctional dE-cad is also organized as relatively immobile nanoclusters as well as more loosely packed diffusive oligomers. Oligomers are promoted by cis interactions of the ectodomain, and their growth is counteracted by the activity of cortical actomyosin. Oligomers in turn promote assembly of dense nanoclusters that require cortical actomyosin activity. Thus, cortical actin activity remodels oligomers and generates nanoclusters. The requirement for dynamic actin in the organization of dE-cad at the nanoscale may provide a mechanism to dynamically tune junctional strength.
Izumi, Y., Furuse, K. and Furuse, M. (2021). A novel membrane protein Hoka regulates septate junction organization and stem cell homeostasis in the Drosophila gut. J Cell Sci. PubMed ID: 33589496
Smooth septate junctions (sSJs) regulate the paracellular transport in the intestinal tract in arthropods. In Drosophila, the organization and physiological function of sSJs are regulated by at least three sSJ-specific membrane proteins: Ssk, Mesh, and Tsp2A. This study reports a novel sSJ membrane protein Hoka, which has a single membrane-spanning segment with a short extracellular region, and a cytoplasmic region with the Tyr-Thr-Pro-Ala motifs. The larval midgut in hoka-mutants shows a defect in sSJ structure. Hoka forms a complex with Ssk, Mesh, and Tsp2A and is required for the correct localization of these proteins to sSJs. Knockdown of hoka in the adult midgut leads to intestinal barrier dysfunction, and stem cell overproliferation. In hoka-knockdown midguts, aPKC is up-regulated in the cytoplasm and the apical membrane of epithelial cells. The depletion of aPKC and yki in hoka-knockdown midguts results in reduced stem cell overproliferation. These findings indicate that Hoka cooperates with the sSJ-proteins Ssk, Mesh, and Tsp2A to organize sSJs, and is required for maintaining intestinal stem cell homeostasis through the regulation of aPKC and Yki activities in the Drosophila midgut.
Li, B., Li, S. and Yan, Z. (2021). Axonemal Dynein DNAH5 is Required for Sound Sensation in Drosophila Larvae. Neurosci Bull. PubMed ID: 33570705
Chordotonal neurons are responsible for sound sensation in Drosophila. However, little is known about how they respond to sound with high sensitivity. Using genetic labeling, it was found that one of the Drosophila axonemal dynein heavy chains, CG9492 (DNAH5), was specifically expressed in larval chordotonal neurons and showed a distribution restricted to proximal cilia. While DNAH5 mutation did not affect the cilium morphology or the trafficking of Inactive, a candidate auditory transduction channel, larvae with DNAH5 mutation had reduced startle responses to sound at low and medium intensities. Calcium imaging confirmed that DNAH5 functioned autonomously in chordotonal neurons for larval sound sensation. Furthermore, disrupting DNAH5 resulted in a decrease of spike firing responses to low-level sound in chordotonal neurons. Intriguingly, DNAH5 mutant larvae displayed an altered frequency tuning curve of the auditory organs. All together, these findings support a critical role of DNAH5 in tuning the frequency selectivity and the sound sensitivity of larval auditory neurons.
Bajusz, C., Kristo, I., Abonyi, C., Venit, T., Vedelek, V., Lukacsovich, T., Farkas, A., Borkuti, P., Kovacs, Z., Bajusz, I., Marton, A., Vizler, C., Lipinszki, Z., Sinka, R., Percipalle, P. and Vilmos, P. (2021). The nuclear activity of the actin-binding Moesin protein is necessary for gene expression in Drosophila. FEBS J. PubMed ID: 33606336
Ezrin-Radixin-Moesin (ERM) proteins play an essential role in the cytoplasm by cross-linking actin filaments with plasma membrane proteins. Research has identified the nuclear localization of ERMs, as well as the involvement of a single Drosophila ERM protein, Moesin, in nuclear mRNA exports. However, the question of how important the nuclear activity of ERM proteins are for the life of an organism has so far not been explored. This study presents the first attempt to reveal the in vivo relevance of nuclear localization of Moesin in Drosophila. With the help of a nuclear export signal, the amount of Moesin in the nuclei of the animals was decreased. Furthermore, various developmental defects were observed, demonstrating the importance of ERM function in the nucleus for the first time. Transcriptome analysis of the mutant flies revealed that the lack of nuclear Moesin function leads to expression changes in nearly 700 genes, among them heat-shock genes. This result together with additional findings revealed that in Drosophila the expression of protein chaperones requires the nuclear functions of Moesin.
Bischoff, M. C., Lieb, S., Renkawitz-Pohl, R. and Bogdan, S. (2021). Filopodia-based contact stimulation of cell migration drives tissue morphogenesis. Nat Commun 12(1): 791. PubMed ID: 33542237
Cells migrate collectively to form tissues and organs during morphogenesis. Contact inhibition of locomotion (CIL) drives collective migration by inhibiting lamellipodial protrusions at cell-cell contacts and promoting polarization at the leading edge. This study reports a CIL-related collective cell behavior of myotubes that lack lamellipodial protrusions, but instead use filopodia to move as a cohesive cluster in a formin-dependent manner. Genetic, pharmacological and mechanical perturbation analyses were performed to reveal the essential roles of Rac2, Cdc42 and Rho1 in myotube migration. These factors differentially control protrusion dynamics and cell-matrix adhesion formation. Active Rho1 GTPase localizes at retracting free edge filopodia and Rok-dependent actomyosin contractility does not mediate a contraction of protrusions at cell-cell contacts, but likely plays an important role in the constriction of supracellular actin cables. Based on these findings, it is proposed that contact-dependent asymmetry of cell-matrix adhesion drives directional movement, whereas contractile actin cables contribute to the integrity of the migrating cell cluster.

Monday, April 12 - Disease Models

Lee, K. M., Talikoti, A., Shelton, K. and Grotewiel, M. (2021). Tyramine synthesis, vesicular packaging, and the SNARE complex function coordinately in astrocytes to regulate Drosophila alcohol sedation. Addict Biol: e13019. PubMed ID: 33538092
Identifying mechanisms underlying alcohol-related behaviors could provide important insights regarding the etiology of alcohol use disorder. To date, most genetic studies on alcohol-related behavior in model organisms have focused on neurons, leaving the causal roles of glial mechanisms less comprehensively investigated. This paper reports studies on the role of Tyrosine decarboxylase 2 (Tdc2), which converts tyrosine to the catecholamine tyramine, in glial cells in Drosophila alcohol sedation. Using genetic approaches that drove transgene expression constitutively in all glia, constitutively in astrocytes and conditionally in glia during adulthood, this study found that knockdown and overexpression of Tdc2, respectively, increased and decreased the sensitivity to alcohol sedation in flies. Manipulation of the genes tyramine β-hydroxylase and tyrosine hydroxylase, which respectively synthesize octopamine and dopamine from tyramine and tyrosine, had no discernable effect on alcohol sedation, suggesting that Tdc2 affects alcohol sedation by regulating tyramine production. It was also found that knockdown of the vesicular monoamine transporter (VMAT) and disruption of the SNARE complex in all glia or selectively in astrocytes increased sensitivity to alcohol sedation and that both VMAT and the SNARE complex functioned downstream of Tdc2. These studies support a model in which the synthesis of tyramine and vesicle-mediated release of tyramine from adult astrocytes regulates alcohol sedation in Drosophila. Considering that tyramine is functionally orthologous to norepinephrine in mammals, these results raise the possibility that gliotransmitter synthesis release could be a conserved mechanism influencing behavioral responses to alcohol as well as alcohol use disorder.
Jewett, K. A., Thomas, R. E., Phan, C. Q., Lin, B., Milstein, G., Yu, S., Bettcher, L. F., Neto, F. C., Djukovic, D., Raftery, D., Pallanck, L. J. and Davis, M. Y. (2021). Glucocerebrosidase reduces the spread of protein aggregation in a Drosophila melanogaster model of neurodegeneration by regulating proteins trafficked by extracellular vesicles. PLoS Genet 17(2): e1008859. PubMed ID: 33539341
Abnormal protein aggregation within neurons is a key pathologic feature of Parkinson's disease (PD). The spread of brain protein aggregates is associated with clinical disease progression, but how this occurs remains unclear. Mutations in glucosidase, beta acid 1 (GBA), which encodes glucocerebrosidase (GCase), are the most penetrant common genetic risk factor for PD and dementia with Lewy bodies and associate with faster disease progression. To explore how GBA mutations influence pathogenesis, a Drosophila model of GBA deficiency (Gba1b) was created that manifests neurodegeneration and accelerated protein aggregation. Proteomic analysis of Gba1b mutants revealed dysregulation of proteins involved in extracellular vesicle (EV) biology, and altered protein composition of EVs was found from Gba1b mutants. Accordingly, it was hypothesized that GBA may influence pathogenic protein aggregate spread via EVs. It was found that accumulation of ubiquitinated proteins and Ref(2)P, Drosophila homologue of mammalian p62, were reduced in muscle and brain tissue of Gba1b flies by ectopic expression of wildtype GCase in muscle. Neuronal GCase expression also rescued protein aggregation both cell-autonomously in brain and non-cell-autonomously in muscle. Muscle-specific GBA expression reduced the elevated levels of EV-intrinsic proteins and Ref(2)P found in EVs from Gba1b flies. Perturbing EV biogenesis through neutral sphingomyelinase (nSMase), an enzyme important for EV release and ceramide metabolism, enhanced protein aggregation when knocked down in muscle, but did not modify Gba1b mutant protein aggregation when knocked down in neurons. Lipidomic analysis of nSMase knockdown on ceramide and glucosylceramide levels suggested that Gba1b mutant protein aggregation may depend on relative depletion of specific ceramide species often enriched in EVs. Finally, ectopically expressed GCase was identified within isolated EVs. Together, these findings suggest that GCase deficiency promotes accelerated protein aggregate spread between cells and tissues via dysregulated EVs, and EV-mediated trafficking of GCase may partially account for the reduction in aggregate spread.
Kim, Y. W., Al-Ramahi, I., Koire, A., Wilson, S. J., Konecki, D. M., Mota, S., Soleimani, S., Botas, J. and Lichtarge, O. (2020). Harnessing the paradoxical phenotypes of APOE epsilon2 and APOE epsilon4 to identify genetic modifiers in Alzheimer's disease. Alzheimers Dement. PubMed ID: 33576571
The strongest genetic risk factor for idiopathic late-onset Alzheimer's disease (LOAD) is apolipoprotein E (APOE) ε4, while the APOE ε2 allele is protective. However, there are paradoxical APOE ε4 carriers who remain disease-free and APOE ε2 carriers with LOAD. Exomes of healthy APOE ε4 carriers and APOE ε2 Alzheimer's disease (AD) patients were compared, prioritizing coding variants based on their predicted functional impact; 216 genes were identified with differential mutational load between these two populations. These candidate genes were significantly dysregulated in LOAD brains, and many modulated tau- or β42-induced neurodegeneration in Drosophila. Variants in these genes were associated with AD risk, even in APOE ε3 homozygotes, showing robust predictive power for risk stratification. Network analyses revealed involvement of candidate genes in brain cell type-specific pathways including synaptic biology, dendritic spine pruning and inflammation. These potential modifiers of LOAD may constitute novel biomarkers, provide potential therapeutic intervention avenues, and support applying this approach as larger whole exome sequencing cohorts become available.
Lee, B., Shin, C., Shin, M., Choi, B., Yuan, C. and Cho, K. S. (2021). The linear ubiquitin E3 ligase-Relish pathway is involved in the regulation of proteostasis in Drosophila muscle during aging. Biochem Biophys Res Commun 550: 184-190. PubMed ID: 33706102
Linear ubiquitination is an atypic ubiquitination process that directly connects the N- and C-termini of ubiquitin and is catalyzed by HOIL-1-interacting protein (HOIP). It is involved in the immune response or apoptosis by activating the nuclear factor-κB pathway and is associated with polyglucosan body myopathy 1, an autosomal recessive disorder with progressive muscle weakness and cardiomyopathy. However, little is currently known regarding the function of linear ubiquitination in muscles. This study investigated the role of linear ubiquitin E3 ligase (LUBEL), a Drosophila HOIP ortholog, in the development and aging of muscles. The muscles of the flies with down-regulation of LUBEL or its downstream factors, kenny and Relish, developed normally, and there were no obvious abnormalities in function in young flies. However, the locomotor activity of the LUBEL RNAi flies was reduced compared to age-matched control, while LUBEL RNAi did not affect the increased mitochondrial fusion or myofiber disorganization during aging. Interestingly, the accumulation of polyubiquitinated protein aggregation during aging decreased in muscles by silencing LUBEL, kenny, or Relish. Meanwhile, the levels of autophagy and global translation, which are implicated in the maintenance of proteostasis, did not change due to LUBEL down-regulation. In conclusion, a new role of linear ubiquitination is proposed in proteostasis in the muscle aging.
Kim, K., Cha, S. J., Choi, H. J., Kang, J. S. and Lee, E. Y. (2021). Dysfunction of Mitochondrial Dynamics in Drosophila Model of Diabetic Nephropathy. Life (Basel) 11(1). PubMed ID: 33477666
Although mitochondrial dysfunction is associated with the development and progression of diabetic nephropathy (DN), its mechanisms are poorly understood, and it remains debatable whether mitochondrial morphological change is a cause of DN. In this study, a Drosophila DN model was established by treating a chronic high-sucrose diet that exhibits similar phenotypes in animals. Results showed that flies fed a chronic high-sucrose diet exhibited a reduction in lifespan, as well as increased lipid droplets in fat body tissue. Furthermore, the chronic high-sucrose diet effectively induced the morphological abnormalities of nephrocytes in Drosophila. High-sucrose diet induced mitochondria fusion in nephrocytes by increasing Opa1 and Marf expression. These findings establish Drosophila as a useful model for studying novel regulators and molecular mechanisms for imbalanced mitochondrial dynamics in the pathogenesis of DN. Furthermore, understanding the pathology of mitochondrial dysfunction regarding morphological changes in DN would facilitate the development of novel therapeutics.
Kim, J., Kim, S., Nahm, M., Li, T. N., Lin, H. C., Kim, Y. D., Lee, J., Yao, C. K. and Lee, S. (2021). ALS2 regulates endosomal trafficking, postsynaptic development, and neuronal survival. J Cell Biol 220(5). PubMed ID: 33683284
Mutations in the human ALS2 gene cause recessive juvenile-onset amyotrophic lateral sclerosis and related motor neuron diseases. Although the ALS2 protein has been identified as a guanine-nucleotide exchange factor for the small GTPase Rab5, its physiological roles remain largely unknown. This study demonstrates that the Drosophila homologue of ALS2 (dALS2) promotes postsynaptic development by activating the Frizzled nuclear import (FNI) pathway. dALS2 loss causes structural defects in the postsynaptic subsynaptic reticulum (SSR), recapitulating the phenotypes observed in FNI pathway mutants. Consistently, these developmental phenotypes are rescued by postsynaptic expression of the signaling-competent C-terminal fragment of Drosophila Frizzled-2 (dFz2). It was further demonstrated that dALS2 directs early to late endosome trafficking and that the dFz2 C terminus is cleaved in late endosomes. Finally, dALS2 loss causes age-dependent progressive defects resembling ALS, including locomotor impairment and brain neurodegeneration, independently of the FNI pathway. These findings establish novel regulatory roles for dALS2 in endosomal trafficking, synaptic development, and neuronal survival.

Friday, April 9th - Methods

Kaur, P., Kibat, C., Teo, E., Gruber, J., Mathuru, A. and Tolwinski, A. N. S. (2020). Use of Optogenetic Amyloid-beta to Monitor Protein Aggregation in Drosophila melanogaster, Danio rerio and Caenorhabditis elegans. Bio Protoc 10(23): e3856. PubMed ID: 33659494
Alzheimer's Disease (AD) has long been associated with accumulation of extracellular amyloid plaques (Aβ) originating from the Amyloid Precursor Protein. Plaques have, however, been discovered in healthy individuals and not all AD brains show plaques, suggesting that extracellular Aβ aggregates may play a smaller role than anticipated. One limitation to studying Aβ peptide in vivo during disease progression is the inability to induce aggregation in a controlled manner. This study developed an optogenetic method to induce Aβ aggregation, and its biological influence was tested in three model organisms-D. melanogaster, C. elegans and D. rerio. A fluorescently labeled, optogenetic Aβ peptide was generated that oligomerizes rapidly in vivo in the presence of blue light in all organisms. This paper details the procedures for expressing this fusion protein in animal models, investigating the effects on the nervous system using time lapse light-sheet microscopy, and metabolic assays were perfomred to measure changes due to intracellular Aβ aggregation. This method, employing optogenetics to study the pathology of AD, allows spatial and temporal control in vivo that cannot be achieved by any other method at present.
Kobler, O., Weiglein, A., Hartung, K., Chen, Y. C., Gerber, B. and Thomas, U. (2021). A quick and versatile protocol for the 3D visualization of transgene expression across the whole body of larval Drosophila. J Neurogenet: 1-14. PubMed ID: 33688796
This paper reports a fast, robust and user-friendly procedure for the whole-body multi-fluorescence imaging of Drosophila larvae; the protocol has been optimized specifically for larvae by systematically tackling the pitfalls associated with clearing this small but cuticularized organism. Tests on various fluorescent proteins reveal that the recently introduced monomeric infrared fluorescent protein (mIFP) is particularly suitable for this approach. This approach combines a success rate high enough to allow for small-scale screening approaches and a resolution sufficient for cellular-level analyses with light sheet and confocal microscopy. Given that publications and database documentations typically specify expression patterns of transgenic driver lines only within a given organ system of interest, the present procedure should be versatile enough to extend such documentation systematically to the whole body. As examples, the expression patterns of transgenic driver lines covering the majority of neurons, or subsets of chemosensory, central brain or motor neurons, are documented in the context of whole larval body volumes (using nsyb-Gal4, IR76b-Gal4, APL-Gal4 and mushroom body Kenyon cells, or OK371-Gal4, respectively). Notably, the presented protocol allows for triple-color fluorescence imaging with near-infrared, red and yellow fluorescent proteins.
Chowdhury, B., Wang, M., Gnerer, J. P. and Dierick, H. A. (2021). The Divider Assay is a high-throughput pipeline for aggression analysis in Drosophila. Commun Biol 4(1): 85. PubMed ID: 33469118
Aggression is a complex social behavior that remains poorly understood. Drosophila has become a powerful model system to study the underlying biology of aggression but lack of high throughput screening and analysis continues to be a barrier for comprehensive mutant and circuit discovery. This paper describes the Divider Assay, a simplified experimental procedure to make aggression analysis in Drosophila fast and accurate. In contrast to existing methods, aggression was analyzed over long time intervals and in complete darkness. While aggression is reduced in the dark, flies are capable of intense fighting without seeing their opponent. Twenty-four-hour behavioral analysis showed a peak in fighting during the middle of the day, a drastic drop at night, followed by re-engagement with a further increase in aggression in anticipation of the next day. This pipeline is easy to implement and will facilitate high throughput screening for mechanistic dissection of aggression.
Kandul, N. P., Liu, J., Bennett, J. B., Marshall, J. M. and Akbari, O. S. (2021). A confinable home and rescue gene drive for population modification. Elife 10. PubMed ID: 33666174
Homing based gene drives, engineered using CRISPR/Cas9, have been proposed to spread desirable genes throughout populations. However, invasion of such drives can be hindered by the accumulation of resistant alleles. To limit this obstacle, a confinable population modification Home-and-Rescue (HomeR) drive was engineered in Drosophila targeting an essential gene. In these experiments, resistant alleles that disrupt the target gene function were recessive lethal, and therefore disadvantaged. HomeR can achieve an increase in frequency in population cage experiments, but fitness costs due to the Cas9 insertion limit drive efficacy. Finally, mathematical modeling was conducte comparing HomeR to contemporary gene drive architectures for population modification over wide ranges of fitness costs, transmission rates, and release regimens. HomeR could potentially be adapted to other species, as a means for safe, confinable, modification of wild populations.
Landskron, L., Bonnay, F., Burkard, T. R. and Knoblich, J. A. (2020). DigiTAG-a RNA Sequencing Approach to Analyze Transcriptomes of Rare Cell Populations in Drosophila melanogaster. Bio Protoc 10(21): e3809. PubMed ID: 33659463
Cell-type specific transcriptional programs underlie the development and maintenance of organs. Not only distinct cell types within a tissue, even cells with supposedly identical cell fates show a high degree of transcriptional heterogeneity. Inevitable, low cell numbers are a major hurdle to study transcriptomes of pure cell populations. This study described DigiTAG, a high-throughput method that combines transposase fragmentation and molecular barcoding to retrieve high quality transcriptome data of rare cell types in Drosophila melanogaster. The protocol showcases how DigiTAG can be used to analyse the transcriptome of rare neural stem cells (type II neuroblasts) of Drosophila larval brains, but can also be utilized for other cell types or model systems.
Campos, T. L., Korhonen, P. K., Hofmann, A., Gasser, R. B. and Young, N. D. (2020). Combined use of feature engineering and machine-learning to predict essential genes in Drosophila melanogaster. NAR Genom Bioinform 2(3): lqaa051. PubMed ID: 33575603
Characterizing genes that are critical for the survival of an organism (i.e. essential) is important to gain a deep understanding of the fundamental cellular and molecular mechanisms that sustain life. Functional genomic investigations of the vinegar fly, Drosophila melanogaster, have unravelled the functions of numerous genes of this model species, but results from phenomic experiments can sometimes be ambiguous. Moreover, the features underlying gene essentiality are poorly understood, posing challenges for computational prediction. This study harnessed comprehensive genomic-phenomic datasets publicly available for D. melanogaster and a machine-learning-based workflow to predict essential genes of this fly. Strong predictors were discovered of such genes, paving the way for computational predictions of essentiality in less-studied arthropod pests and vectors of infectious diseases.

Thursday, April 8th - Gonads

Garcez, M., Branco-Santos, J., Gracio, P. C. and Homem, C. C. F. (2020). Mitochondrial Dynamics in the Drosophila Ovary Regulates Germ Stem Cell Number, Cell Fate, and Female Fertility. Front Cell Dev Biol 8: 596819. PubMed ID: 33585443
The fate and proliferative capacity of stem cells have been shown to strongly depend on their metabolic state. Mitochondria are the powerhouses of the cell being responsible for energy production via oxidative phosphorylation (OxPhos) as well as for several other metabolic pathways. Mitochondrial activity strongly depends on their structural organization, with their size and shape being regulated by mitochondrial fusion and fission, a process known as mitochondrial dynamics. However, the significance of mitochondrial dynamics in the regulation of stem cell metabolism and fate remains elusive. This study characterized the role of mitochondria morphology in female germ stem cells (GSCs) and in their more differentiated lineage. Mitochondria are particularly important in the female GSC lineage. Not only do they provide these cells with their energy requirements to generate the oocyte but they are also the only mitochondria pool to be inherited by the offspring. The undifferentiated GSCs predominantly have fissed mitochondria, whereas more differentiated germ cells have more fused mitochondria. By reducing the levels of mitochondrial dynamics regulators, it was shown that both fused and fissed mitochondria are required for the maintenance of a stable GSC pool. Surprisingly, it was found that disrupting mitochondrial dynamics in the germline also strongly affects nurse cells morphology, impairing egg chamber development and female fertility. Interestingly, reducing the levels of key enzymes in the Tricarboxylic Acid Cycle (TCA), known to cause OxPhos reduction, also affects GSC number. This defect in GSC self-renewal capacity indicates that at least basal levels of TCA/OxPhos are required in GSCs. These findings show that mitochondrial dynamics is essential for female GSC maintenance and female fertility, and that mitochondria fusion and fission events are dynamically regulated during GSC differentiation, possibly to modulate their metabolic profile.
Dorogova, N. V., Khruscheva, A. S., Galimova, I. A., Oshchepkov, D. Y., Maslov, D. E., Shvedkina, E. D., Akhmetova, K. A. and Fedorova, S. A. (2020). Migration of primordial germline cells is negatively regulated by surrounding somatic cells during early embryogenesis in Drosophila melanogaster. Vavilovskii Zhurnal Genet Selektsii 24(5): 525-532. PubMed ID: 33659837
The initiation and maintenance of the cell movement state requires the activation of many factors involved in the regulation of transcription, signal transduction, adhesive interactions, modulation of membranes and the cytoskeleton. However, cell movement depends on the status of both migrating and surrounding cells, interacting with each other during movement. The surrounding cells or cell matrix not only form a substrate for movement, but can also participate in the spatio-temporal regulation of the migration. To determine the role of the cell environment in the regulation of individual cell migration, the migration of primordial germline cells (PGC) was studied during early embryogenesis in Drosophila melanogaster. Normally, PGC are formed at the 3rd stage of embryogenesis at the posterior pole of the embryo. During gastrulation (stages 6-7), PGC as a consolidated cell group passively transfers into the midgut primordium. Further, PGC are individualized, acquire an amoeboid form, and actively move through the midgut epithelium and migrate to the 5-6 abdominal segment of the embryo, where they form paired embryonic gonads. A screen was performed for genes expressed in the epithelium surrounding PGC during early embryogenesis and affecting their migration. The myc, Hph, stat92E, Tre-1, and hop genes, whose RNA interference leads to premature active PGC migration at stages 4-7 of embryogenesis, were identified. These genes can be divided into two groups: 1) modulators of JAK/STAT pathway activity inducing PGC migration (stat92E, Tre-1, hop), and 2) myc and Hph involved in epithelial morphogenesis and polarization, i. e. modifying the permeability of the epithelial barrier. Since a depletion of each of these gene products resulted in premature PGC migration, it can be concluded that, normally, the somatic environment negatively regulates PGC migration during early Drosophila embryogenesis.
Imran Alsous, J., Romeo, N., Jackson, J. A., Mason, F. M., Dunkel, J. and Martin, A. C. (2021). Dynamics of hydraulic and contractile wave-mediated fluid transport during Drosophila oogenesis. Proc Natl Acad Sci U S A 118(10). PubMed ID: 33658367
From insects to mice, oocytes develop within cysts alongside nurse-like sister germ cells. Prior to fertilization, the nurse cells' cytoplasmic contents are transported into the oocyte, which grows as its sister cells regress and die. Although critical for fertility, the biological and physical mechanisms underlying this transport process are poorly understood. This study combined live imaging of germline cysts, genetic perturbations, and mathematical modeling to investigate the dynamics and mechanisms that enable directional and complete cytoplasmic transport in Drosophila melanogaster egg chambers. During "nurse cell (NC) dumping" most cytoplasm was discovered to be transported into the oocyte independently of changes in myosin-II contractility, with dynamics instead explained by an effective Young-Laplace law, suggesting hydraulic transport induced by baseline cell-surface tension. A minimal flow-network model inspired by the famous two-balloon experiment and motivated by genetic analysis of a myosin mutant correctly predicts the directionality, intercellular pattern, and time scale of transport. Long thought to trigger transport through "squeezing," changes in actomyosin contractility are required only once NC volume has become comparable to nuclear volume, in the form of surface contractile waves that drive NC dumping to completion. This work thus demonstrates how biological and physical mechanisms cooperate to enable a critical developmental process that, until now, was thought to be mainly biochemically regulated.
Lebo, D. P. V., Chirn, A., Taylor, J. D., Levan, A., Doerre Torres, V., Agreda, E., Serizier, S. B., Lord, A. K., Jenkins, V. K. and McCall, K. (2021). An RNAi screen of the kinome in epithelial follicle cells of the Drosophila melanogaster ovary reveals genes required for proper germline death and clearance. G3 (Bethesda) 11(2). PubMed ID: 33693600
Programmed cell death and cell corpse clearance are an essential part of organismal health and development. Cell corpses are often cleared away by professional phagocytes such as macrophages. However, in certain tissues, neighboring cells known as nonprofessional phagocytes can also carry out clearance functions. This study used the Drosophila melanogaster ovary to identify novel genes required for clearance by nonprofessional phagocytes. In the Drosophila ovary, germline cells can die at multiple time points. As death proceeds, the epithelial follicle cells act as phagocytes to facilitate the clearance of these cells. An unbiased kinase screen was performe to identify novel proteins and pathways involved in cell clearance during two death events. Of 224 genes examined, 18 demonstrated severe phenotypes during developmental death and clearance while 12 demonstrated severe phenotypes during starvation-induced cell death and clearance, representing a number of pathways not previously implicated in phagocytosis. Interestingly, it was found that several genes not only affected the clearance process in the phagocytes, but also non-autonomously affected the process by which germline cells died. This kinase screen has revealed new avenues for further exploration and investigation.
Doherty, C. A., Diegmiller, R., Kapasiawala, M., Gavis, E. R. and Shvartsman, S. Y. (2021). Coupled oscillators coordinate collective germline growth. Dev Cell. PubMed ID: 33689691
Developing oocytes need large supplies of macromolecules and organelles. A conserved strategy for accumulating these products is to pool resources of oocyte-associated germline nurse cells. In Drosophila, these cells grow more than 100-fold to boost their biosynthetic capacity. No previously known mechanism explains how nurse cells coordinate growth collectively. This study reports a cell cycle-regulating mechanism that depends on bidirectional communication between the oocyte and nurse cells, revealing the oocyte as a critical regulator of germline cyst growth. Transcripts encoding the cyclin-dependent kinase inhibitor, Dacapo, are synthesized by the nurse cells and actively localized to the oocyte. Retrograde movement of the oocyte-synthesized Dacapo protein to the nurse cells generates a network of coupled oscillators that controls the cell cycle of the nurse cells to regulate cyst growth. It is proposed that bidirectional nurse cell-oocyte communication establishes a growth-sensing feedback mechanism that regulates the quantity of maternal resources loaded into the oocyte.
Grewal, G., Patlar, B. and Civetta, A. (2021). Expression of Mst89B and CG31287 is Needed for Effective Sperm Storage and Egg Fertilization in Drosophila. Cells 10(2). PubMed ID: 33535499
In Drosophila, male reproductive fitness can be affected by any number of processes, ranging from development of gametes, transfer to and storage of mature sperm within the female sperm storage organs, and utilization of sperm for fertilization. Previous work has identified the 89B cytogenetic map position of D. melanogaster as a hub for genes that effect male paternity success when disturbed. This study used RNA interference to test 11 genes that are highly expressed in the testes and located within the 89B region for their role in sperm competition and male fecundity when their expression is perturbed. Testes-specific knockdown (KD) of bor and CSN5 resulted in complete sterility, whereas KD of CG31287, Manf and Mst89B, showed a breakdown in sperm competitive success when second to mate and reduced fecundity in single matings. The low fecundity of Manf KD is explained by a significant reduction in the amount of mature sperm produced. KD of Mst89B and CG31287 does not affect sperm production, sperm transfer into the female bursa or storage within 30 min after mating. Instead, a significant reduction of sperm in female storage is observed 24 h after mating. Egg hatchability 24 h after mating is also drastically reduced for females mated to Mst89B or CG31287 KD males, and this reduction parallels the decrease in fecundity. Normal germ-line expression of Mst89B and CG31287 is needed for effective sperm usage and egg fertilization.

Wednesday, April 7th - Cell cycle

Wesley, E. R., Hawley, R. S. and Billmyre, K. K. (2020). Genetic background impacts the timing of synaptonemal complex breakdown in Drosophila melanogaster. Chromosoma 129(3-4): 243-254. PubMed ID: 33068154
Experiments performed in different genetic backgrounds occasionally exhibit failure in experimental reproducibility. This is a serious issue in Drosophila where there are no standard control stocks. This study illustrates the importance of controlling genetic background by showing that the timing of a major meiotic event, the breakdown of the synaptonemal complex (SC), varies in different genetic backgrounds. SC breakdown was assessed in three different control stocks and found that in one control stock, y w; sv(spa-pol), the SC broke down earlier than in Oregon-R and w(1118) stocks. SC breakdown was further examined in these three control backgrounds with flies heterozygous for a null mutation in c(3)G, which encodes a key structural component of the SC. Flies heterozygous for c(3)G displayed differences in the timing of SC breakdown in different control backgrounds, providing evidence of a sensitizing effect of this mutation. These observations suggest that SC maintenance is associated with the dosage of c(3)G in some backgrounds. Lastly, chromosome segregation was not affected by premature SC breakdown in mid-prophase, consistent with previous findings that chromosome segregation is not dependent on full-length SC in mid-prophase. Thus, genetic background is an important variable to consider with respect to SC behavior during Drosophila meiosis.
Duan, T., Cupp, R. and Geyer, P. K. (2021). Drosophila female germline stem cells undergo mitosis without nuclear breakdown. Curr Biol. PubMed ID: 33548191
Stem cell homeostasis requires nuclear lamina (NL) integrity. In Drosophila germ cells, compromised NL integrity activates the ataxia telangiectasia and Rad3-related (ATR) and checkpoint kinase 2 (Chk2) checkpoint kinases, blocking germ cell differentiation and causing germline stem cell (GSC) loss. Checkpoint activation occurs upon loss of either the NL protein emerin or its partner barrier-to-autointegration factor, two proteins required for nuclear reassembly at the end of mitosis. This study examined how mitosis contributes to NL structural defects linked to checkpoint activation. These analyses led to the unexpected discovery that wild-type female GSCs utilize a non-canonical mode of mitosis, one that retains a permeable but intact nuclear envelope and NL. The interphase NL is remodeled during mitosis for insertion of centrosomes that nucleate the mitotic spindle within the confines of the nucleus. Depletion or loss of NL components causes mitotic defects, including compromised chromosome segregation associated with altered centrosome positioning and structure. Further, in emerin mutant GSCs, centrosomes remain embedded in the interphase NL. Notably, these embedded centrosomes carry large amounts of pericentriolar material and nucleate astral microtubules, revealing a role for emerin in the regulation of centrosome structure. Epistasis studies demonstrate that defects in centrosome structure are upstream of checkpoint activation, suggesting that these centrosome defects might trigger checkpoint activation and GSC loss. Connections between NL proteins and centrosome function have implications for mechanisms associated with NL dysfunction in other stem cell populations, including NL-associated diseases, such as laminopathies.
Falahati, H., Hur, W., Di Talia, S. and Wieschaus, E. (2021). Temperature-Induced uncoupling of cell cycle regulators. Dev Biol 470: 147-153. PubMed ID: 33278404
The early stages of development involve complex sequences of morphological changes that are both reproducible from embryo to embryo and often robust to environmental variability. To investigate the relationship between reproducibility and robustness this study examined cell cycle progression in early Drosophila embryos at different temperatures. The experiments show that while the subdivision of cell cycle steps is conserved across a wide range of temperatures (5-35 °C), the relative duration of individual steps varies with temperature. The transition into prometaphase is delayed at lower temperatures relative to other cell cycle events, arguing that it has a different mechanism of regulation. Using an in vivo biosensor, the ratio of activities were quantified of the major mitotic kinase, Cdk1 and one of the major mitotic phosphatases PP1. Comparing activation profile with cell cycle transition times at different temperatures indicates that in early fly embryos activation of Cdk1 drives entry into prometaphase but is not required for earlier cell cycle events. In fact, chromosome condensation can still occur when Cdk1 activity is inhibited pharmacologically. These results demonstrate that different kinases are rate-limiting for different steps of mitosis, arguing that robust inter-regulation may be needed for rapid and ordered mitosis.
Kim, M., Delos Santos, K. and Moon, N. S. (2021). Proper CycE-Cdk2 activity in endocycling tissues requires regulation of the cyclin-dependent kinase inhibitor Dacapo by dE2F1b in Drosophila. Genetics 217(1): 1-15. PubMed ID: 33683365
Polyploidy is an integral part of development and is associated with cellular stress, aging, and pathological conditions. The endocycle, comprised of successive rounds of G and S phases without mitosis, is widely employed to produce polyploid cells in plants and animals. In Drosophila, maintenance of the endocycle is dependent on E2F-governed oscillations of Cyclin E (CycE)-Cdk2 activity, which is known to be largely regulated at the level of transcription. This study reports an additional level of E2F-dependent control of CycE-Cdk2 activity during the endocycle. Genetic experiments revealed that an alternative isoform of Drosophila de2f1, dE2F1b, regulates the expression of the p27CIP/KIP-like Cdk inhibitor Dacapo (Dap). Evidence is provided showing that dE2F1b-dependent Dap expression in endocycling tissues is necessary for setting proper CycE-Cdk2 activity. Furthermore, this study demonstrated that dE2F1b is required for proliferating cell nuclear antigen expression that establishes a negative feedback loop in S phase. Overall, this study reveals previously unappreciated E2F-dependent regulatory networks that are critical for the periodic transition between G and S phases during the endocycle.
Barbosa, P., Zhaunova, L., Debilio, S., Steccanella, V., Kelly, V., Ly, T. and Ohkura, H. (2021). SCF-Fbxo42 promotes synaptonemal complex assembly by downregulating PP2A-B56. J Cell Biol 220(2). PubMed ID: 33382409
Meiosis creates genetic diversity by recombination and segregation of chromosomes. The synaptonemal complex assembles during meiotic prophase I and assists faithful exchanges between homologous chromosomes, but how its assembly/disassembly is regulated remains to be understood. This study reports how two major posttranslational modifications, phosphorylation and ubiquitination, cooperate to promote synaptonemal complex assembly. The ubiquitin ligase complex SCF is important for assembly and maintenance of the synaptonemal complex in Drosophila female meiosis. This function of SCF is mediated by two substrate-recognizing F-box proteins, Slmb/βTrcp and Fbxo42. SCF-Fbxo42 down-regulates the phosphatase subunit PP2A-B56, which is important for synaptonemal complex assembly and maintenance.
Willms, R. J., Zeng, J. and Campbell, S. D. (2020). Myt1 Kinase Couples Mitotic Cell Cycle Exit with Differentiation in Drosophila. Cell Rep 33(7): 108400. PubMed ID: 33207203
The Drosophila midgut is an excellent system for characterizing cell cycle regulation in the context of tissue homeostasis. Two major progenitor cell types populate the midgut: mitotic intestinal stem cells and their post-mitotic daughters, enteroblasts. Although regulatory networks that control stem cell proliferation are well characterized, how enteroblast mitotic-cell-cycle exit is coordinated with endocycle entry and enterocyte specification remains poorly defined. Myt1 is a conserved Cdk1 inhibitory kinase that regulates mitotic timing during animal development. This study used myt1-null mutants and cell-specific RNA interference to investigate Myt1 function in stem cells and enteroblast progenitors. Myt1 depletion alters cell cycle kinetics and promotes ectopic stem cell and enteroblast mitoses at the expense of enteroblast-enterocyte differentiation. These aberrant enteroblast mitoses rely upon cyclin A, implicating Myt1 inhibition of cyclin A/Cdk1 as a mechanism for the coupling mitotic exit with differentiation in enteroblasts.

Tuesday April 6th - Adult and larval Physiology

Devilliers, M., Garrido, D., Poidevin, M., Rubin, T., Le Rouzic, A. and Montagne, J. (2021). Differential metabolic sensitivity of insulin-like-response- and TORC1-dependent overgrowth in Drosophila fat cells. Genetics 217(1): 1-12. PubMed ID: 33683355
Glycolysis and fatty acid (FA) synthesis directs the production of energy-carrying molecules and building blocks necessary to support cell growth, although the absolute requirement of these metabolic pathways must be deeply investigated. This study used Drosophila genetics and focused on the TOR (Target of Rapamycin) signaling network that controls cell growth and homeostasis. In mammals, mTOR (mechanistic-TOR) is present in two distinct complexes, mTORC1 and mTORC2; the former directly responds to amino acids and energy levels, whereas the latter sustains insulin-like-peptide (Ilp) response. The TORC1 and Ilp signaling branches can be independently modulated in most Drosophila tissues. This study shows that TORC1 and Ilp-dependent overgrowth can operate independently in fat cells and that ubiquitous over-activation of TORC1 or Ilp signaling affects basal metabolism, supporting the use of Drosophila as a powerful model to study the link between growth and metabolism. Cell-autonomous restriction of glycolysis or FA synthesis in fat cells was shown to retrain overgrowth dependent on Ilp signaling but not TORC1 signaling. Additionally, the mutation of FASN (Fatty acid synthase) results in a drop in TORC1 but not Ilp signaling, whereas, at the cell-autonomous level, this mutation affects none of these signals in fat cells. These findings thus reveal differential metabolic sensitivity of TORC1- and Ilp-dependent growth and suggest that cell-autonomous metabolic defects might elicit local compensatory pathways. Conversely, enzyme knockdown in the whole organism results in animal death. Importantly, this study weakens the use of single inhibitors to fight mTOR-related diseases and strengthens the use of drug combination and selective tissue-targeting.
Jarvis, K. J., Bell, K. M., Loya, A. K., Swank, D. M. and Walcott, S. (2021). Force-velocity and tension transient measurements from Drosophila jump muscle reveal the necessity of both weakly-bound cross-bridges and series elasticity in models of muscle contraction. Arch Biochem Biophys 701: 108809. PubMed ID: 33610561
Muscle contraction is a fundamental biological process where molecular interactions between the myosin molecular motor and actin filaments result in contraction of a whole muscle, a process spanning size scales differing in eight orders of magnitude. It was hypothesized that a model based on molecular measurements must be modified to include a weakly-bound interaction between myosin and actin in order to fit measurements at the muscle fiber or whole muscle scales. This hypothesis is further supported by the model's need for a weakly-bound state in order to qualitatively reproduce the force response that occurs when a muscle fiber is rapidly stretched a small distance. This hypothesis was tested by measuring steady-state force as a function of shortening velocity, and the force transient caused by a rapid length step in Drosophila jump muscle fibers. Then, by performing global parameter optimization, the predictions of two mathematical models, one lacking a weakly-bound state and one with a weakly-bound state, were quantitatively compared to these measurements. Both models could reproduce the force-velocity measurements, but only the model with a weakly-bound state could reproduce the force transient measurements. However, neither model could concurrently fit both measurements. It was found that only a model that includes weakly-bound cross-bridges with force-dependent detachment and an elastic element in series with the cross-bridges is able to fit both the measurements. This result suggests that the force response after stretch is not a reflection of distinct steps in the cross-bridge cycle, but rather arises from the interaction of cross-bridges with a series elastic element. Additionally, the model suggests that the curvature of the force-velocity relationship arises from a combination of the force-dependence of weakly- and strongly-bound cross-bridges. Overall, this work presents a minimal cross-bridge model that has predictive power at the fiber level.
Hughson, B. N., Shimell, M. and O'Connor, M. B. (2021). AKH Signaling in D. melanogaster Alters Larval Development in a Nutrient-Dependent Manner That Influences Adult Metabolism. Front Physiol 12: 619219. PubMed ID: 33708137
Metabolism, growth, and development are intrinsically linked, and their coordination is dependent upon inter-organ communication mediated by anabolic, catabolic, and steroid hormones. In Drosophila melanogaster, the corpora cardiaca (CC) influences metabolic homeostasis through adipokinetic hormone (AKH) signaling. AKH has glucagon-like properties and is evolutionarily conserved in mammals as the gonadotropin-releasing hormone, but its role in insect development is unknown. This study reports that AKH signaling alters larval development in a nutrient stress-dependent manner. This activity is regulated by the locus dg2 (foraging), which encodes a cGMP-dependent protein kinase (PKG). CC-specific downregulation of dg2 expression delayed the developmental transition from larval to pupal life, and altered adult metabolism and behavior. These developmental effects were AKH-dependent, and were observed only in flies that experienced low nutrient stress during larval development. Calcium-mediated vesicle exocytosis regulates ecdysteroid secretion from the prothoracic gland (PG), and this study found that AKH signaling increased cytosolic free calcium levels in the PG. A novel pathway was identified through which PKG acts in the CC to communicate metabolic information to the PG via AKH signaling. AKH signaling provides a means whereby larval nutrient stress can alter developmental trajectories into adulthood.
Everman, E. R., Cloud-Richardson, K. M. and Macdonald, S. J. (2021). Characterizing the genetic basis of copper toxicity in Drosophila reveals a complex pattern of allelic, regulatory, and behavioral variation. Genetics 217(1): 1-20. PubMed ID: 33683361
A range of heavy metals are required for normal cell function and homeostasis. However, the anthropogenic release of metal compounds into soil and water sources presents a pervasive health threat. Copper is one of many heavy metals that negatively impacts diverse organisms at a global scale. Using a combination of quantitative trait locus (QTL) mapping and RNA sequencing in the Drosophila Synthetic Population Resource, this study demonstrates that resistance to the toxic effects of ingested copper in D. melanogaster is genetically complex and influenced by allelic and expression variation at multiple loci. QTL mapping identified several QTL that account for a substantial fraction of heritability. Additionally, it was found that copper resistance is impacted by variation in behavioral avoidance of copper and may be subject to life-stage specific regulation. Gene expression analysis further demonstrated that resistant and sensitive strains are characterized by unique expression patterns. Several of the candidate genes identified via QTL mapping and RNAseq have known copper-specific functions (e.g., Ccs, Sod3, CG11825), and others are involved in the regulation of other heavy metals (e.g., Catsup, whd). Several of these candidate genes were validated with RNAi suggesting they contribute to variation in adult copper resistance. This study illuminates the interconnected roles that allelic and expression variation, organism life stage, and behavior play in copper resistance, allowing a deeper understanding of the diverse mechanisms through which metal pollution can negatively impact organisms.
Galenza, A. and Foley, E. (2021). A glucose-supplemented diet enhances gut barrier integrity in Drosophila. Biol Open 10(3). PubMed ID: 33579694
Dietary intervention has received considerable attention as an approach to extend lifespan and improve aging. However, questions remain regarding optimal dietary regimes and underlying mechanisms of lifespan extension. This study asked how an increase of glucose in a chemically defined diet extends the lifespan of adult Drosophila melanogaster. Glucose-dependent lifespan extension was shown to not be result of diminished caloric intake, or changes to systemic insulin activity, two commonly studied mechanisms of lifespan extension. Instead, this study found that flies raised on glucose-supplemented food increased the expression of cell-adhesion genes, delaying age-dependent loss of intestinal barrier integrity. Furthermore, it was shown that chemical disruption of the gut barrier negated the lifespan extension associated with glucose treatment, suggesting that glucose-supplemented food prolongs adult viability by enhancing the intestinal barrier. It is believed these data contribute to understanding intestinal homeostasis, and may assist efforts to develop preventative measures that limit effects of aging on health.
El-Merhie, N., Kruger, A., Uliczka, K., Papenmeier, S., Roeder, T., Rabe, K. F., Wagner, C., Angstmann, H. and Krauss-Etschmann, S. (2021). Sex dependent effect of maternal e-nicotine on F1 Drosophila development and airways. Sci Rep 11(1): 4441. PubMed ID: 33627715
E-cigarettes are heavily advertised as healthier alternative to common tobacco cigarettes, leading more and more women to switch from regular cigarettes to ENDS (electronic nicotine delivery system) during pregnancy. While the noxious consequences of tobacco smoking during pregnancy on the offspring health are well-described, information on the long-term consequences due to maternal use of e-cigarettes do not exist so far. Therefore, this study aimed to investigate how maternal e-nicotine influences offspring development from earliest life until adulthood. To this end, virgin female Drosophila melanogaster flies were exposed to nicotine vapor (8 μg nicotine) once per hour for a total of eight times. Following the last exposure, e-nicotine or sham exposed females were mated with non-exposed males. The F1-generation was then analyzed for viability, growth and airway structure. Maternal exposure to e-nicotine not only leads to reduced maternal fertility, but also negatively affects size and weight, as well as tracheal development of the F1-generation, lasting from embryonic stage until adulthood. These results not only underline the need for studies investigating the effects of maternal vaping on offspring health, but also propose this established model for analyzing molecular mechanisms and signaling pathways mediating these intergenerational changes.

Monday, April 5th - Adult Neural Development and Function

Jain, K., Lavista-Llanos, S., Grabe, V., Hansson, B. S. and Wicher, D. (2021). Calmodulin regulates the olfactory performance in Drosophila melanogaster. Sci Rep 11(1): 3747. PubMed ID: 33580172
Insect odorant receptors (ORs) detect volatile chemical cues with high sensitivity. These ORs operate as ligand-gated ion channels and are formed by heptahelical OrX and Orco (co-receptor) proteins. A highly conserved calmodulin (CaM) binding site (CBS) 'SAIKYWVER' within the second intracellular loop of Drosophila melanogaster Orco constitutes a target for regulating OR performance. This study asked how a point mutation K339N in this CBS affects the olfactory performance of Drosophila melanogaster. It was first asked how this mutation would affect the odor responses of olfactory sensory neurons (OSNs). Using Ca(2+) imaging in an ex-vivo antenna preparation, all OR (OrX/Orco) expressing neurons were activated using the synthetic agonist VUAA1. In a next attempt, the OR spectrum was restricted to Or22a expressing neurons (Or22a/Orco) and these OSNs were stimulated with the ligand ethyl hexanoate. In both approaches, it was found that flies carrying the K339N point mutation in Orco display a reduced olfactory response. It was also found that the mutation abolishes the capability of OSNs to sensitize by repeated weak odor stimuli. Next, it was asked whether OrcoK339N might affect the odor localization performance. Using a wind tunnel bioassay, it was found that odor localization in flies carrying the OrcoK339N mutation was severely diminished.
DeAngelis, M. W., Coolon, J. D. and Johnson, R. I. (2021). Comparative transcriptome analyses of the Drosophila pupal eye. G3 (Bethesda) 11(1). PubMed ID: 33561221
Tissue function is dependent on correct cellular organization and behavior. As a result, the identification and study of genes that contribute to tissue morphogenesis is of paramount importance to the fields of cell and developmental biology. The Drosophila melanogaster pupal eye that has a highly stereotyped arrangement of cells. In addition, the pupal eye is postmitotic that allows for the study of tissue morphogenesis independent from any effects of proliferation. While the changes in cell morphology and organization that occur throughout pupal eye development are well documented, less is known about the corresponding transcriptional changes that choreograph these processes. To identify these transcriptional changes, wild-type Canton S pupal eyes were dissected, and RNA-sequencing was performed. This analyses identified differential expression of many loci that are documented regulators of pupal eye morphogenesis and contribute to multiple biological processes including signaling, axon projection, adhesion, and cell survival. Differential expression of genes not previously implicated in pupal eye morphogenesis were identified such as components of the Toll pathway, several non-classical cadherins, and components of the muscle sarcomere, which could suggest these loci function as novel patterning factors
Fernandez-Hernandez, I., Marsh, E. B. and Bonaguidi, M. A. (2021). Mechanosensory neuron regeneration in adult Drosophila. Development 148(5). PubMed ID: 33597190
Auditory and vestibular mechanosensory hair cells do not regenerate following injury or aging in the adult mammalian inner ear, inducing irreversible hearing loss and balance disorders for millions of people. Research on model systems showing replacement of mechanosensory cells can provide mechanistic insights into developing new regenerative therapies. This study developed lineage tracing systems to reveal the generation of mechanosensory neurons in the Johnston's organ (JO) of intact adult Drosophila, which are the functional counterparts to hair cells in vertebrates. New JO neurons develop cilia and target central brain circuitry. Unexpectedly, mitotic recombination clones point to JO neuron self-replication as a likely source of neuronal plasticity. This mechanism is further enhanced upon treatment with experimental and ototoxic compounds. These findings introduce a new platform to expedite research on mechanisms and compounds mediating mechanosensory cell regeneration, with nascent implications for hearing and balance restoration.
Kan, L., Ott, S., Joseph, B., Park, E. S., Dai, W., Kleiner, R. E., Claridge-Chang, A. and Lai, E. C. (2021). A neural m(6)A/Ythdf pathway is required for learning and memory in Drosophila. Nat Commun 12(1): 1458. PubMed ID: 33674589
Epitranscriptomic modifications can impact behavior. This study used Drosophila melanogaster to study N(6)-methyladenosine (m(6)A), the most abundant modification of mRNA. Proteomic and functional analyses confirm its nuclear (Ythdc1) and cytoplasmic (Ythdf) YTH domain proteins as major m(6)A binders. Assays of short term memory in m(6)A mutants reveal neural-autonomous requirements of m(6)A writers working via Ythdf, but not Ythdc1. Furthermore, m(6)A/Ythdf operate specifically via the mushroom body, the center for associative learning. m(6)A from wild-type and Mettl3 mutant heads was mapped, allowing robust discrimination of Mettl3-dependent m(6)A sites that are highly enriched in 5' UTRs. Genomic analyses indicate that Drosophila m(6)A is preferentially deposited on genes with low translational efficiency and that m(6)A does not affect RNA stability. Nevertheless, functional tests indicate a role for m(6)A/Ythdf in translational activation. Altogether, this molecular genetic analyses and tissue-specific m(6)A maps reveal selective behavioral and regulatory defects for the Drosophila Mettl3/Ythdf pathway.
Jafari, S. and Alenius, M. (2021). Odor response adaptation in Drosophila-a continuous individualization process. Cell Tissue Res 383(1): 143-148. PubMed ID: 33492517
Olfactory perception is very individualized in humans and also in Drosophila. The process that individualize olfaction is adaptation that across multiple time scales and mechanisms shape perception and olfactory-guided behaviors. Olfactory adaptation occurs both in the central nervous system and in the periphery. Central adaptation occurs at the level of the circuits that process olfactory inputs from the periphery where it can integrate inputs from other senses, metabolic states, and stress. This study focused on the periphery and how the fast, slow, and persistent (lifelong) adaptation mechanisms in the olfactory sensory neurons individualize the Drosophila olfactory system.
Ichinose, T., Kanno, M., Wu, H., Yamagata, N., Sun, H., Abe, A. and Tanimoto, H. (2021). Mushroom body output differentiates memory processes and distinct memory-guided behaviors. Curr Biol. PubMed ID: 33476556
The mushroom body (MB) of Drosophila melanogaster has multiple functions in controlling memory and behavior. This study systematically probed the behavioral contribution of each type of MB output neuron (MBON) by blocking during acquisition, retention, or retrieval of reward or punishment memories. The contribution was evaluated using two conditioned responses: memory-guided odor choice and odor source attraction. Quantitative analysis revealed that these conditioned odor responses are controlled by different sets of MBONs. The valence of memory, rather than the transition of memory steps, has a larger impact on the patterns of required MBONs. Moreover, it was found that the glutamatergic MBONs forming recurrent circuits commonly contribute to appetitive memory acquisition, suggesting a pivotal role of this circuit motif for reward processing. These results provide principles how the MB output circuit processes associative memories of different valence and controls distinct memory-guided behaviors.

Friday, April 2nd - Disease models

Hebbar, S., Lehmann, M., Behrens, S., Halsig, C., Leng, W., Yuan, M., Winkler, S. and Knust, E. (2021). Mutations in the splicing regulator Prp31 lead to retinal degeneration in Drosophila. Biol Open 10(1). PubMed ID: 33495354
Retinitis pigmentosa (RP) is a clinically heterogeneous disease affecting 1.6 million people worldwide. The second-largest group of genes causing autosomal dominant RP in human encodes regulators of the splicing machinery. Yet, how defects in splicing factor genes are linked to the aetiology of the disease remains largely elusive. To explore possible mechanisms underlying retinal degeneration caused by mutations in regulators of the splicing machinery, mutations were induced in Drosophila Prp31, the orthologue of human PRPF31, mutations in which are associated with RP11. Flies heterozygous mutant for Prp31 are viable and develop normal eyes and retina. However, photoreceptors degenerate under light stress, thus resembling the human disease phenotype. Degeneration is associated with increased accumulation of the visual pigment rhodopsin 1 and increased mRNA levels of twinfilin, a gene associated with rhodopsin trafficking. Reducing rhodopsin levels by raising animals in a carotenoid-free medium not only attenuates rhodopsin accumulation, but also retinal degeneration. Given a similar importance of proper rhodopsin trafficking for photoreceptor homeostasis in human, results obtained in flies presented in this study will also contribute to further unravel molecular mechanisms underlying the human disease.
Hrizo, S. L., Eicher, S. L., Myers, T. D., McGrath, I., Wodrich, A. P. K., Venkatesh, H., Manjooran, D., Swoger, S., Gagnon, K., Bruskin, M., Lebedev, M. V., Zheng, S., Vitantonio, A., Kim, S., Lamb, Z. J., Vogt, A., Ruzhnikov, M. R. Z. and Palladino, M. J. (2021). Identification of protein quality control regulators using a Drosophila model of TPI deficiency. Neurobiol Dis 152: 105299. PubMed ID: 33600953
Triosephosphate isomerase (TPI) deficiency (Df) is a rare recessive metabolic disorder that manifests as hemolytic anemia, locomotor impairment, and progressive neurodegeneration. Drosophila with the recessive TPI (sugarkill) allele (a.k.a. sgk or M81T) exhibit progressive locomotor impairment, neuromuscular impairment and reduced longevity, modeling the human disorder. TPI(sugarkill) produces a functional protein that is degraded by the proteasome. Molecular chaperones, such as Hsp70 and Hsp90, have been shown to contribute to the regulation of TPI(sugarkill) degradation. In addition, stabilizing the mutant protein through chaperone modulation results in improved TPI deficiency phenotypes. To identify additional regulators of TPI(sugarkill) degradation, a genome-wide RNAi screen was performed that targeted known and predicted quality control proteins in the cell to identify novel factors that modulate TPI(sugarkill) turnover. Of the 430 proteins screened, 25 regulators of TPI(sugarkill) were identified. Proteins involved in co-translational protein quality control and ribosome function were isolated in the screen, suggesting that TPI(sugarkill) may undergo co-translational selection for polyubiquitination and proteasomal degradation as a nascent polypeptide. The proteins identified in this study may reveal novel pathways for the degradation of a functional, cytosolic protein by the ubiquitin proteasome system and define therapeutic pathways for TPI Df and other biomedically important diseases.
Gambini, J., Gimeno-Mallench, L., Olaso-Gonzalez, G., Mastaloudis, A., Traber, M. G., Monleon, D., Borras, C. and Vina, J. (2021). Moderate Red Wine Consumption Increases the Expression of Longevity-Associated Genes in Controlled Human Populations and Extends Lifespan in Drosophila melanogaster. Antioxidants (Basel) 10(2). PubMed ID: 33669360
The beneficial effects of moderate red wine consumption on cardiovascular health are well known. The composition of red wine includes several compounds, such as the phytoestrogen resveratrol, that exert these beneficial effects, although not all the mechanisms by which they act are known. The aim of this work was to study the effect of red wine consumption on longevity-related genes in controlled human populations, such as cloistered nuns. The expression of catalase, manganese-superoxide dismutase, Sirt1, and p53 was increased in peripheral blood mononuclear cells after 14 days of moderate red wine consumption. This increase was accompanied by an enhanced metabolic wellness: fatty acids, cholesterol, branched chain amino acids (isoleucine and leucine), ketone bodies (acetoacetate), bacterial co-metabolites (trimethylamine), and cellular antioxidants (taurine) contributed to a change in metabolic profile after moderate red wine consumption by the nuns. No serious unwanted side effects were observed. Finally, the effect of moderate red wine consumption was tested on longevity in a controlled animal population, such as D. melanogaster, and found that it increased average life span by 7%. In conclusion, moderate red wine consumption increases the expression of key longevity-related genes and improves metabolic health in humans and increases longevity in flies.
Grazioli, P., Parodi, C., Mariani, M., Bottai, D., Di Fede, E., Zulueta, A., Avagliano, L., Cereda, A., Tenconi, R., Wierzba, J., Adami, R., Iascone, M., Ajmone, P. F., Vaccari, T., Gervasini, C., Selicorni, A. and Massa, V. (2021). Lithium as a possible therapeutic strategy for Cornelia de Lange syndrome. Cell Death Discov 7(1): 34. PubMed ID: 33597506
Cornelia de Lange Syndrome (CdLS) is a rare developmental disorder affecting a multitude of organs including the central nervous system, inducing a variable neurodevelopmental delay. CdLS malformations derive from the deregulation of developmental pathways, inclusive of the canonical WNT pathway. This study has evaluated MRI anomalies and behavioral and neurological clinical manifestations in CdLS patients. Importantly, a significant association was observed between behavioral disturbance and structural abnormalities in brain structures of hindbrain embryonic origin. Considering the cumulative evidence on the cohesin-WNT-hindbrain shaping cascade, possible ameliorative effects of chemical activation of the canonical WNT pathway with lithium chloride was explored in different models: (I) Drosophila melanogaster CdLS model showing a significant rescue of mushroom bodies morphology in the adult flies; (II) mouse neural stem cells restoring physiological levels in proliferation rate and differentiation capabilities toward the neuronal lineage; (III) lymphoblastoid cell lines from CdLS patients and healthy donors restoring cellular proliferation rate and inducing the expression of CyclinD1. This work supports a role for WNT-pathway regulation of CdLS brain and behavioral abnormalities and a consistent phenotype rescue by lithium in experimental models.
Hunt, L. C., Schadeberg, B., Stover, J., Haugen, B., Pagala, V., Wang, Y. D., Puglise, J., Barton, E. R., Peng, J. and Demontis, F. (2021). Antagonistic control of myofiber size and muscle protein quality control by the ubiquitin ligase UBR4 during aging. Nat Commun 12(1): 1418. PubMed ID: 33658508
Sarcopenia is a degenerative condition that consists in age-induced atrophy and functional decline of skeletal muscle cells (myofibers). A common hypothesis is that inducing myofiber hypertrophy should also reinstate myofiber contractile function but such model has not been extensively tested. This study found that the levels of the ubiquitin ligase UBR4 increase in skeletal muscle with aging, and that UBR4 increases the proteolytic activity of the proteasome. Importantly, muscle-specific UBR4 loss rescues age-associated myofiber atrophy in mice. However, UBR4 loss reduces the muscle specific force and accelerates the decline in muscle protein quality that occurs with aging in mice. Similarly, hypertrophic signaling induced via muscle-specific loss of UBR4/poe and of ESCRT members (HGS/Hrs, STAM, USP8) that degrade ubiquitinated membrane proteins compromises muscle function and shortens lifespan in Drosophila by reducing protein quality control. Altogether, these findings indicate that these ubiquitin ligases antithetically regulate myofiber size and muscle protein quality control.
Hurley, E. P. and Staveley, B. E. (2021). Inhibition of Ref(2)P, the Drosophila homologue of the p62/SQSTM1 gene, increases lifespan and leads to a decline in motor function. BMC Res Notes 14(1): 53. PubMed ID: 33557921
Sequestosome 1 (p62/SQSTM1) is a multifunctional scaffold/adaptor protein encoded by the p62/SQSTM1 gene with function in cellular homeostasis. Mutations in the p62/SQSTM1 gene have been known to be associated with patients with amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and Parkinson disease (PD). The aim of the present study was to create a novel model of human neurogenerative disease in Drosophila melanogaster by altering the expression of Ref(2)P, the Drosophila orthologue of the human p62/SQSTM1 gene. Ref(2)P expression was altered in all neurons, the dopaminergic neurons and in the motor neurons, with longevity and locomotor function assessed over time. Inhibition of Ref(2)P resulted in a significantly increased median lifespan in the motor neurons, followed by a severe decline in motor skills. Inhibition of Ref(2)P in the dopaminergic neurons resulted in a significant, but minimal increase in median lifespan, accompanied by a drastic decline in locomotor function. Inhibition of Ref(2)P in the ddc-Gal4-expressing neurons resulted in a significant increase in median lifespan, while dramatically reducing motor function.

Thursday, April 1st - Evolution

Grainger, T. N., Rudman, S. M., Schmidt, P. and Levine, J. M. (2021). Competitive history shapes rapid evolution in a seasonal climate. Proc Natl Acad Sci U S A 118(6). PubMed ID: 33536336
Eco-evolutionary dynamics will play a critical role in determining species' fates as climatic conditions change. Unfortunately, there is little understanding of how rapid evolutionary responses to climate play out when species are embedded in the competitive communities that they inhabit in nature. The effects of rapid evolution in response to interspecific competition were tested on subsequent ecological and evolutionary trajectories in a seasonally changing climate using a field-based evolution experiment with Drosophila melanogaster. Populations of D. melanogaster were either exposed, or not exposed, to interspecific competition with an invasive competitor, Zaprionus indianus, over the summer. Yhese populations' ecological trajectories (abundances) and evolutionary trajectories (heritable phenotypic change) were then quantified when exposed to a cooling fall climate. It was found that competition with Z. indianus in the summer affected the subsequent evolutionary trajectory of D. melanogaster populations in the fall, after all interspecific competition had ceased. Specifically, flies with a history of interspecific competition evolved under fall conditions to be larger and have lower cold fecundity and faster development than flies without a history of interspecific competition. Surprisingly, this divergent fall evolutionary trajectory occurred in the absence of any detectible effect of the summer competitive environment on phenotypic evolution over the summer or population dynamics in the fall. This study demonstrates that competitive interactions can leave a legacy that shapes evolutionary responses to climate even after competition has ceased, and more broadly, that evolution in response to one selective pressure can fundamentally alter evolution in response to subsequent agents of selection.
Houle, D. and Cheng, C. (2021). Predicting the evolution of sexual dimorphism in gene expression. Mol Biol Evol. PubMed ID: 33512493
Sexual dimorphism in gene expression is likely to be the underlying source of dimorphism in a variety of traits. Many analyses implicitly make the assumption that dimorphism only evolves when selection favors different phenotypes in the two sexes, although theory makes clear that it can also evolve as an indirect response to other kinds of selection. Furthermore, previous analyses consider the evolution of a single transcript or trait at a time, ignoring the genetic covariance with other transcripts and traits. This study first shows which aspects of the genetic-variance covariance matrix, G, affect dimorphism when these assumptions about selection are relaxed. Gene expression data from Drosophila melanogaster were analyzed with these predictions in mind. Dimorphism of gene expression for individual transcripts shows the signature of both direct selection for dimorphism and indirect responses to selection. To account for the effect of measurement error on evolutionary predictions, a G matrix was estimated for eight linear combinations of expression traits. Sex-specific genetic variances in female- and male-biased transcription, as well as one relatively unbiased combination, were quite unequal, ensuring that most forms of selection on these traits will have large effects on dimorphism. Predictions of response to selection based on the whole G matrix showed that sexually concordant and antagonistic selection are equally capable of changing sexual dimorphism. In addition, the indirect responses of dimorphism due to cross-trait covariances were quite substantial. The assumption that sexual dimorphism in transcription is an adaptation could be incorrect in many specific cases.
Iranmehr, A., Stobdan, T., Zhou, D., Zhao, H., Kryazhimskiy, S., Bafna, V. and Haddad, G. G. (2021). Multiple mechanisms drive genomic adaptation to extreme O(2) levels in Drosophila melanogaster. Nat Commun 12(1): 997. PubMed ID: 33579965
To detect the genomic mechanisms underlying evolutionary dynamics of adaptation in sexually reproducing organisms, this study analyze multigenerational whole genome sequences of Drosophila melanogaster adapting to extreme O(2) conditions over an experiment conducted for nearly two decades. Methods to analyze time-series genomics data and predict adaptive mechanisms were developed. This study report a remarkable level of synchronicity in both hard and soft selective sweeps in replicate populations as well as the arrival of favorable de novo mutations that constitute a few asynchronized sweeps. Additionally direct experimental observations were made of rare recombination events that combine multiple alleles on to a single, better-adapted haplotype. Based on the analyses of the genes in genomic intervals, this study provides a deeper insight into the mechanisms of genome adaptation that allow complex organisms to survive harsh environments.
Zhao, Y., Lu, G. A., Yang, H., Lin, P., Liufu, Z., Tang, T. and Xu, J. (2020). Run or die in the evolution of new microRNAs - Testing the Red Queen hypothesis on de novo new genes. Mol Biol Evol. PubMed ID: 33306129
The Red Queen hypothesis depicts evolution as the continual struggle to adapt. According to this hypothesis, new genes, especially those originating from non-genic sequences (i.e., de novo genes), are eliminated unless they evolve continually in adaptation to a changing environment. This study analyzed two Drosophila de novo miRNAs that are expressed in a testis-specific manner with very high rates of evolution in their DNA sequence. These miRNAs were knocked out in two sibling species, and their contributions to different fitness components were investigated. It was observed that the fitness contributions of miR-975 in D. simulans seem positive, in contrast to its neutral contributions in D. melanogaster, while miR-983 appears to have negative contributions in both species, as the fitness of the knockout mutant increases. As predicted by the Red Queen hypothesis, the fitness difference of these de novo miRNAs indicates their different fates.
Bonchuk, A., Boyko, K., Fedotova, A., Nikolaeva, A., Lushchekina, S., Khrustaleva, A., Popov, V. and Georgiev, P. (2021). Structural basis of diversity and homodimerization specificity of zinc-finger-associated domains in Drosophila. Nucleic Acids Res 49(4): 2375-2389. PubMed ID: 33638995
In arthropods, zinc finger-associated domains (ZADs) are found at the N-termini of many DNA-binding proteins with tandem arrays of Cys2-His2 zinc fingers (ZAD-C2H2 proteins). ZAD-C2H2 proteins undergo fast evolutionary lineage-specific expansion and functional diversification. This study shows that all ZADs from Drosophila melanogaster form homodimers, but only certain ZADs with high homology can also heterodimerize. CG2712, for example, is unable to heterodimerize with its paralog, the previously characterized insulator protein Zw5, with which it shares 46% homology. A crystal structure was obtained of CG2712 protein's ZAD domain that, in spite of a low sequence homology, has similar spatial organization with the only known ZAD structure (from Grauzone protein). Steric clashes prevented the formation of heterodimers between Grauzone and CG2712 ZADs. Using detailed structural analysis, site-directed mutagenesis, and molecular dynamics simulations, this study demonstrated that rapid evolutionary acquisition of interaction specificity was mediated by the more energy-favorable formation of homodimers in comparison to heterodimers, and that this specificity was achieved by multiple amino acid substitutions resulting in the formation or breaking of stabilizing interactions. It is speculated that specific homodimerization of ZAD-C2H2 proteins is important for their architectural role in genome organization.
Garud, N. R., Messer, P. W. and Petrov, D. A. (2021). Detection of hard and soft selective sweeps from Drosophila melanogaster population genomic data. PLoS Genet 17(2): e1009373. PubMed ID: 33635910
Whether hard sweeps or soft sweeps dominate adaptation has been a matter of much debate. Recently, haplotype homozygosity statistics were developed that (i) can detect both hard and soft sweeps with similar power and (ii) can classify the detected sweeps as hard or soft. The application of this method to population genomic data from a natural population of Drosophila melanogaster (DGRP) allowed rediscovery of three known cases of adaptation at the loci Ace, Cyp6g1, and CHKov1 known to be driven by soft sweeps, and additional candidate loci were detected for recent and strong sweeps. Surprisingly, all of the top 50 candidates showed patterns much more consistent with soft rather than hard sweeps. Recently, this work has been criticized by suggesting that all the candidate loci detected by the haplotype statistics, including the positive controls, are unlikely to be sweeps at all and that instead these haplotype patterns can be more easily explained by complex neutral demographic models. This criticism also claimed that these neutral non-sweeps are likely to be hard instead of soft sweeps. This study reanalyze the DGRP data using a range of complex admixture demographic models and reconfirmed the original published results suggesting that the majority of recent and strong sweeps in D. melanogaster are first likely to be true sweeps, and second, that they do appear to be soft. Furthermore, ways to take this work forward are discussed given that most demographic models employed in such analyses are necessarily too simple to capture the full demographic complexity, while more realistic models are unlikely to be inferred correctly because they require a large number of free parameters.
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