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Current papers in developmental biology and gene function


Tuesday, May 31st, 2022 - Chromatin & DNA

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Mitchell, C., Becker, V., DeLoach, J., Nestore, E., Bolterstein, E. and Kohl, K. P. (2022). The Drosophila Mutagen-Sensitivity Gene mus109 Encodes DmDNA2. Genes (Basel) 13(2). PubMed ID: 35205357
The identification of mutants through forward genetic screens is the backbone of Drosophila genetics research, yet many mutants identified through these screens have yet to be mapped to the Drosophila genome. This is especially true of mutants that have been identified as mutagen-sensitive (mus), but have not yet been mapped to their associated molecular locus. This study addressed the need for additional mus gene identification by determining the locus and exploring the function of the X-linked mutagen-sensitive gene mus109 using three available mutant alleles: mus109(D1), mus109(D2), and mus109(lS). After first confirming that all three mus109 alleles were sensitive to methyl methanesulfonate (MMS) using complementation analysis, deletion mapping to narrow the candidate genes for mus109. Through DNA sequencing, it was possible to determine that mus109 is the uncharacterized gene CG2990, which encodes the Drosophila ortholog of the highly conserved DNA2 protein that is important for DNA replication and repair. The sequence and structure of DNA2 was used to predict the impact of the mus109 allele mutations on the final gene product. Together, these results provide a tool for researchers to further investigate the role of DNA2 in DNA repair processes in Drosophila.
Hafer, T. L., Patra, S., Tagami, D. and Kohwi, M. (2022). Enhancer of trithorax/polycomb, Corto, regulates timing of hunchback gene relocation and competence in Drosophila neuroblasts. Neural Dev 17(1): 3. PubMed ID: 35177098
Hunchback (Hb), the first of the series, specifies early-born neuronal identity. Neuroblast competence to generate early-born neurons is terminated when the hb gene relocates to the neuroblast nuclear lamina, rendering it refractory to activation in descendent neurons. This study identified Corto, an enhancer of Trithorax/Polycomb (ETP) protein, as a new regulator of neuroblast competence. The GAL4/UAS system was used to drive persistent misexpression of Hb in neuroblast 7-1 (NB7-1) to examine the role of Corto in neuroblast competence. Immuno-DNA Fluorescence in situ hybridization (DNA FISH) was used in whole embryos to track the position of the hb gene locus specifically in neuroblasts across developmental time. Finally, immunostaining was used in whole embryos to examine Corto's role in repression of Hb and a known target gene, Abdominal B (Abd-B). In corto mutants, the hb gene relocation to the neuroblast nuclear lamina was found to be delayed and the early competence window is extended. The delay in gene relocation occurs after hb transcription is already terminated in the neuroblast and is not due to prolonged transcriptional activity. Further, it was found that Corto genetically interacts with Posterior Sex Combs (Psc), a core subunit of polycomb group complex 1 (PRC1), to terminate early competence. Loss of Corto does not result in derepression of Hb or its Hox target, Abd-B, specifically in neuroblasts. These results show that in neuroblasts, Corto genetically interacts with PRC1 to regulate timing of nuclear architecture reorganization.
Andreev, V. I., Yu, C., Wang, J., Schnabl, J., Tirian, L., Gehre, M., Handler, D., Duchek, P., Novatchkova, M., Baumgartner, L., Meixner, K., Sienski, G., Patel, D. J. and Brennecke, J. (2022). Panoramix SUMOylation on chromatin connects the piRNA pathway to the cellular heterochromatin machinery. Nat Struct Mol Biol 29(2): 130-142. PubMed ID: 35173350
Nuclear Argonaute proteins, guided by small RNAs, mediate sequence-specific heterochromatin formation. The molecular principles that link Argonaute-small RNA complexes to cellular heterochromatin effectors on binding to nascent target RNAs are poorly understood. This study explained the mechanism by which the PIWI-interacting RNA (piRNA) pathway connects to the heterochromatin machinery in Drosophila. This study found that Panoramix, a corepressor required for piRNA-guided heterochromatin formation, is SUMOylated on chromatin in a Piwi-dependent manner. SUMOylation, together with an amphipathic LxxLL motif in Panoramix's intrinsically disordered repressor domain, are necessary and sufficient to recruit Small ovary (Sov), a multi-zinc-finger protein essential for general heterochromatin formation and viability. Structure-guided mutations that eliminate the Panoramix-Sov interaction or that prevent SUMOylation of Panoramix uncouple Sov from the piRNA pathway, resulting in viable but sterile flies in which Piwi-targeted transposons are derepressed. Thus, Piwi engages the heterochromatin machinery specifically at transposon loci by coupling recruitment of a corepressor to nascent transcripts with its SUMOylation.
Kolesnikova, T. D., Pokholkova, G. V., Dovgan, V. V., Zhimulev, I. F. and Schubert, V. (2022). Super-resolution microscopy reveals stochastic initiation of replication in Drosophila polytene chromosomes. Chromosome Res. PubMed ID: 35226231
Studying the probability distribution of replication initiation along a chromosome is a huge challenge. Drosophila polytene chromosomes in combination with super-resolution microscopy provide a unique opportunity for analyzing the probabilistic nature of replication initiation at the ultrastructural level. This study developed a method for synchronizing S-phase induction among salivary gland cells. An analysis of the replication label distribution in the first minutes of S phase and in the following hours after the induction revealed the dynamics of replication initiation. Spatial super-resolution structured illumination microscopy allowed identifying multiple discrete replication signals and to investigate the behavior of replication signals in the first minutes of the S phase at the ultrastructural level. Replication initiation zones were identified where initiation occurs stochastically. These zones differ significantly in the probability of replication initiation per time unit. There are zones in which initiation occurs on most strands of the polytene chromosome in a few minutes. In other zones, the initiation on all strands takes several hours. Compact bands are free of replication initiation events, and the replication runs from outer edges to the middle, where band shapes may alter.
Kyrchanova, O. V., Postika, N. Y., Sokolov, V. V. and Georgiev, P. G. (2022). Fragments of the Fab-3 and Fab-4 Boundaries of the Drosophila melanogaster Bithorax Complex That Include CTCF Sites Are not Effective Insulators. Dokl Biochem Biophys 502(1): 21-24. PubMed ID: 35275301
The segment-specific regulatory domains of the Bithorax complex (BX-C), which consists of three homeotic genes Ubx, abd-A and Abd-B, are separated by boundaries that function as insulators. Most of the boundaries contain binding sites for the architectural protein CTCF, which is conserved for higher eukaryotes. Previous work has shown that the CTCF sites determine the insulator activity of the boundaries of the Abd-B regulatory region. In this study, it was shown that fragments of the Fab-3 and Fab-4 boundaries of the abd-A regulatory region, containing CTCF binding sites, are not effective insulators.
Deger, N., Cao, X., Selby, C. P., Gulec, S., Kawara, H., Dewey, E. B., Wang, L., Yang, Y., Archibald, S., Selcuk, B., Adebali, O., Sekelsky, J., Sancar, A. and Liu, Z. (2022). CSB-independent, XPC-dependent transcription-coupled repair in Drosophila. Proc Natl Acad Sci U S A 119(9). PubMed ID: 35217627
An early study reported that Drosophila lacks the transcription-coupled repair (TCR) form of nucleotide excision repair. This conclusion was seemingly supported by the Drosophila genome sequencing project, which revealed that Drosophila lacks a homolog to CSB, which is known to be required for TCR in mammals and yeasts. However, by using excision repair sequencing (XR-seq) genome-wide repair mapping technology, it was recently found that the Drosophila S2 cell line performs TCR comparable to human cells. This study extended this work to Drosophila at all its developmental stages. It as find TCR takes place throughout the life cycle of the organism. Moreover, it was found that in contrast to humans and other multicellular organisms previously studied, the XPC repair factor is required for both global and transcription-coupled repair in Drosophila.

Friday, May 27th, 2022 - RNA and Transposons

Minervini, C. F., Berloco, M. F., Marsano, R. M. and Viggiano, L. (2022). The Ribosomal Protein RpL22 Interacts In Vitro with 5'-UTR Sequences Found in Some Drosophila melanogaster Transposons. Genes (Basel) 13(2). PubMed ID: 35205350
Mobility of eukaryotic transposable elements (TEs) are finely regulated to avoid an excessive mutational load caused by their movement. The transposition of retrotransposons is usually regulated through the interaction of host- and TE-encoded proteins, with non-coding regions (LTR and 5'-UTR) of the transposon. Examples of new potent cis-acting sequences, identified and characterized in the non-coding regions of retrotransposons, include the insulator of gypsy and Idefix, and the enhancer of ZAM of Drosophila melanogaster. Recently it has been shown that in the 5'-UTR of the LTR-retrotransposon ZAM there is a sequence structured in tandem-repeat capable of operating as an insulator both in Drosophila (S2R(+)) and human cells (HEK293). This study tested the hypothesis that tandem repeated 5'-UTR of a different LTR-retrotransposon could accommodate similar regulatory elements. The comparison of the 5'-UTR of some LTR-transposons allowed identification of a shared motif of 13 bp, called Transposable Element Redundant Motif (TERM). Surprisingly, it was demonstrated, by Yeast One-Hybrid assay, that TERM interacts with the D. melanogaster ribosomal protein RpL22. The Drosophila RpL22 has additional Ala-, Lys- and Pro-rich sequences at the amino terminus, which resembles the carboxy-terminal portion of histone H1 and histone H5. For this reason, it has been hypothesized that RpL22 might have two functions, namely the role in organizing the ribosome, and a potential regulatory role involving DNA-binding similar to histone H1, which represses transcription in Drosophila. This paper shows, by two independent sets of experiments, that DmRpL22 is able to directly and specifically bind DNA of Drosophila melanogaster.
Gallicchio, L., Griffiths-Jones, S. and Ronshaugen, M. (2022). miR-9a regulates levels of both rhomboid mRNA and protein in the early Drosophila melanogaster embryo. G3 (Bethesda) 12(4). PubMed ID: 35143618
MicroRNAs can have subtle and combinatorial effects on the levels of the targets and pathways they act on. Studying the consequences of a single microRNA knockout often proves difficult as many such knockouts exhibit phenotypes only under stress conditions. This has often led to the hypothesis that microRNAs buffer the effects of intrinsic and environmental stochasticity on gene expression. Observing and understanding this buffering effect entails quantitative analysis of microRNA and target expression in single cells. To this end, this study has employed single-molecule fluorescence in situ hybridization, immunofluorescence, and high-resolution confocal microscopy to investigate the effects of miR-9a loss on the expression of the serine-protease Rhomboid in Drosophila melanogaster early embryos. A single-cell quantitative approach shows that spatially, the rhomboid mRNA pattern is identical in WT and miR-9a knockout embryos. However, the number of mRNA molecules per cell is higher when miR-9a is absent, and the level and temporal accumulation of Rhomboid protein shows a more dramatic increase in the miR-9a knockout. Specifically, accumulation of Rhomboid protein was seen in miR-9a mutants by stage 5, much earlier than in WT. The data, therefore, show that miR-9a functions in the regulation of rhomboid mRNA and protein levels. While further work is required to establish whether rhomboid is a direct target of miR-9 in Drosophila, these results further establish the miR-9 family microRNAs as conserved regulators of timing in neurogenic processes. This study shows the power of single-cell quantification as an experimental tool to study phenotypic consequences of microRNA mis-regulation.
Isaacson, J. R., Berg, M. D., Charles, B., Jagiello, J., Villen, J., Brandl, C. J. and Moehring, A. J. (2022). A Novel Mistranslating tRNA Model in Drosophila melanogaster has Diverse, Sexually Dimorphic Effects. G3 (Bethesda). PubMed ID: 35143655
Transfer RNAs (tRNAs) are the adaptor molecules required for reading the genetic code and producing proteins. tRNA variants can lead to genome-wide mistranslation, the misincorporation of amino acids not specified by the standard genetic code into nascent proteins. While genome sequencing has identified putative mistranslating tRNA variants in human populations, little is known regarding how mistranslation affects multicellular organisms. This study created a multicellular model of mistranslation by integrating a serine tRNA variant that mistranslates serine for proline (tRNASerUGG, G26A) into the Drosophila melanogaster genome. Mistranslation was confirmed via mass spectrometry, and it was found that tRNASerUGG, G26A misincorporates serine for proline at a frequency of ∼ 0.6% per codon. tRNASerUGG, G26A extends development time and decreases the number of flies that reach adulthood. While both sexes of adult flies containing tRNASerUGG, G26A present with morphological deformities and poor climbing performance, these effects are more pronounced in female flies and the impact on climbing performance is exacerbated by age. This model will enable studies into the synergistic effects of mistranslating tRNA variants and disease-causing alleles.
Kokolo, M. and Elias, M. B. (2022). P68 RNA Helicase (DDX5) is Required for the Formation of Various Specific and Mature miRNA Active RISC Complexes. Microrna. PubMed ID: 35184719
DEAD box RNA helicases catalyze the ATP-dependent unwinding of double-stranded RNA. In addition, they are required for protein displacement and remodelling of RNA or RNA/protein complexes. P68 RNA helicase regulates the alternative splicing of the important proto-oncogene H-Ras, and numerous studies have shown that p68 RNA helicase is probably involved in miRNA biogenesis, mainly through Drosha and RISC/DICER complexes. This study was aimed to determine how p68 RNA helicase affects the activity of a selected mature miRNAs. This set included miR-342, miR-330, miR-138 and miR-206, miR-126 and miR-335, and let-7a, which are known to be related to cancer processes. The miRNA levels were analysed in stable HeLa cells containing p68 RNA helicase RNAi induced by doxycycline (DOX). This study shows that p68 RNA helicase downregulation increases accumulation of the mature miRNAs miR-126, let-7a, miR-206 and miR-138. Interestingly, the accumulation of these mature miRNAs does not downregulate their known protein targets, thus suggesting that p68 RNA helicase is required for mature miRNA active RISC complex activity. Furthermore, it was demonstrated that this requirement is conserved, as Drosophila p68 RNA helicase can complete the p68 RNA helicase depleted activity in human cells. Dicer and Drosha proteins are not affected by downregulation of p68 RNA helicase despite the fact that Dicer is also localized in the nucleus when p68 RNA helicase activity is reduced. It is concluded that p68 RNA helicase regulates a set of miRNAs related to cancer processes.
Green, K. M., Miller, S. L., Malik, I. and Todd, P. K. (2022). Non-canonical initiation factors modulate repeat-associated non-AUG translation. Hum Mol Genet. PubMed ID: 35220421
Repeat associated non-AUG (RAN) translation of expanded repeat mutation mRNA produces toxic peptides in neurons of patients suffering from neurodegenerative diseases. Recent findings indicate that RAN translation in diverse model systems is not inhibited by cellular stressors that impair global translation through phosphorylation of the alpha subunit of eIF2, the essential eukaryotic translation initiation factor that brings the initiator tRNA to the 40S ribosome. Using in vitro, cell-based, and Drosophila models, this study examined the role of alternative ternary complex factors that may function in place of eIF2, including eIF2A, eIF2D, DENR, and MCTS1. Among these factors, DENR knockdown had the greatest inhibitory effect on RAN translation of expanded GGGGCC and CGG repeat reporters, and its reduction improved survival of Drosophila expressing expanded GGGGCC repeats. Taken together, these data support a role for alternative initiation factors in RAN translation and suggest these may serve as novel therapeutic targets in neurodegenerative disease.
Zuniga, G., Levy, S., Ramirez, P., De Mange, J., Gonzalez, E., Gamez, M. and Frost, B. (2022). Tau-induced deficits in nonsense-mediated mRNA decay contribute to neurodegeneration. Alzheimers Dement. PubMed ID: 35416419
While brains of patients with Alzheimer's disease and related tauopathies have evidence of altered RNA processing, a mechanistic understanding is not available of how altered RNA processing arises in these disorders and if such changes are causally linked to neurodegeneration. Using Drosophila melanogaster models of tauopathy, this study found that overall activity of nonsense-mediated mRNA decay (NMD), a key RNA quality-control mechanism, is reduced. Genetic manipulation of NMD machinery significantly modifies tau-induced neurotoxicity, suggesting that deficits in NMD are causally linked to neurodegeneration. Mechanistically, this study found that deficits in NMD are a consequence of aberrant RNA export and RNA accumulation within nuclear envelope invaginations in tauopathy. A pharmacological activator of NMD was identified that suppresses neurodegeneration in tau transgenic Drosophila, indicating that tau-induced deficits in RNA quality control are druggable. These studies suggest that NMD activators should be explored for their potential therapeutic value to patients with tauopathies.

Thursday, May 26th - Synapse and Vesicles

Certel, S. J., McCabe, B. D. and Stowers, R. S. (2022). A conditional GABAergic synaptic vesicle marker for Drosophila. J Neurosci Methods 372: 109540. PubMed ID: 35219770
Throughout the animal kingdom, GABA is the principal inhibitory neurotransmitter of the nervous system. It is essential for maintaining the homeostatic balance between excitation and inhibition required for the brain to operate normally. Identification of GABAergic neurons and their GABA release sites are thus essential for understanding how the brain regulates the excitability of neurons and the activity of neural circuits responsible for numerous aspects of brain function including information processing, locomotion, learning, memory, and synaptic plasticity, among others. Since the structure and features of GABA synapses are critical to understanding their function within specific neural circuits of interest, this study developed and characterized a conditional marker of GABAergic synaptic vesicles for Drosophila, 9XV5-vGAT. 9XV5-vGAT is validated for conditionality of expression, specificity for localization to synaptic vesicles, specificity for expression in GABAergic neurons, and functionality. Its utility for GABAergic neurotransmitter phenotyping and identification of GABA release sites was verified for ellipsoid body neurons of the central complex. In combination with previously reported conditional SV markers for acetylcholine and glutamate, 9XV5-vGAT was used to demonstrate fast neurotransmitter phenotyping of subesophageal ganglion neurons. This method is an alternative to single cell transcriptomics for neurotransmitter phenotyping and can be applied to any neurons of interest represented by a binary transcription system driver. A conditional GABAergic synaptic vesicle marker has been developed and validated for GABA neurotransmitter phenotyping and subcellular localization of GABAergic synaptic vesicles.
Chaudhry, N., Sica, M., Surabhi, S., Hernandez, D. S., Mesquita, A., Selimovic, A., Riaz, A., Lescat, L., Bai, H., Macintosh, G. C. and Jenny, A. (2022). Lamp1 mediates lipid transport, but is dispensable for autophagy in Drosophila. Autophagy: 1-16. PubMed ID: 35266854
The endolysosomal system not only is an integral part of the cellular catabolic machinery that processes and recycles nutrients for synthesis of biomaterials, but also acts as signaling hub to sense and coordinate the energy state of cells with growth and differentiation. Lysosomal dysfunction adversely influences vesicular transport-dependent macromolecular degradation and thus causes serious problems for human health. In mammalian cells, loss of the lysosome associated membrane proteins LAMP1 and LAMP2 strongly affects autophagy and cholesterol trafficking. This study shows that the previously uncharacterized Drosophila Lamp1 is a bona fide ortholog of vertebrate LAMP1 and LAMP2. Surprisingly and in contrast to lamp1 lamp2 double-mutant mice, Drosophila Lamp1 is not required for viability or autophagy, suggesting that fly and vertebrate LAMP proteins acquired distinct functions, or that autophagy defects in lamp1 lamp2 mutants may have indirect causes. However, Lamp1 deficiency results in an increase in the number of acidic organelles in flies. Furthermore, Lamp1 mutant larvae were found to have defects in lipid metabolism as they show elevated levels of sterols and diacylglycerols (DAGs). Because DAGs are the main lipid species used for transport through the hemolymph (blood) in insects, these results indicate broader functions of Lamp1 in lipid transport. These findings make Drosophila an ideal model to study the role of LAMP proteins in lipid assimilation without the confounding effects of their storage and without interfering with autophagic processes.
Du, S., Wang, G., Zhang, Z., Ma, C., Gao, N. and Xiao, J. (2022). Structural insights into how GlcNAc-1-phosphotransferase directs lysosomal protein transport. J Biol Chem 298(3): 101702. PubMed ID: 35148990
GlcNAc-1-phosphotransferase catalyzes the initial step in the formation of the mannose-6-phosphate tag that labels ∼60 lysosomal proteins for transport. Mutations in GlcNAc-1-phosphotransferase are known to cause lysosomal storage disorders such as mucolipidoses. However, the molecular mechanism of GlcNAc-1-phosphotransferase activity remains unclear. Mammalian GlcNAc-1-phosphotransferases are α2β2γ2 hexamers in which the core catalytic α- and β-subunits are derived from the GNPTAB (N-acetylglucosamine-1-phosphate transferase subunits alpha and beta) gene. This study presents the cryo-electron microscopy structure of the Drosophila melanogaster GNPTAB homolog, DmGNPTAB. Four conserved regions were found located far apart in the sequence that fold into the catalytic domain, which exhibits structural similarity to that of the UDP-glucose glycoprotein glucosyltransferase. Comparison with UDP-glucose glycoprotein glucosyltransferase also revealed a putative donor substrate-binding site, and the functional requirements of critical residues in human GNPTAB were validated using GNPTAB-knockout cells. Finally, this study showed that DmGNPTAB forms a homodimer that is evolutionarily conserved and that perturbing the dimer interface undermines the maturation and activity of human GNPTAB. These results provide important insights into GlcNAc-1-phosphotransferase function and related diseases.
Martelli, F., Hernandes, N. H., Zuo, Z., Wang, J., Wong, C. O., Karagas, N. E., Roessner, U., Rupasinghe, T., Robin, C., Venkatachalam, K., Perry, T., Batterham, P. and Bellen, H. J. (2022). Low doses of the organic insecticide spinosad trigger lysosomal defects, elevated ROS, lipid dysregulation, and neurodegeneration in flies. Elife 11. PubMed ID: 35191376
Large-scale insecticide application is a primary weapon in the control of insect pests in agriculture. However, a growing body of evidence indicates that it is contributing to the global decline in population sizes of many beneficial insect species. Spinosad emerged as an organic alternative to synthetic insecticides and is considered less harmful to beneficial insects, yet its mode of action remains unclear. Using Drosophila, this study showed that low doses of spinosad antagonize its neuronal target, the nicotinic acetylcholine receptor subunit α 6 (nAChRα6), reducing the cholinergic response. The nAChRα6 receptors are transported to lysosomes that become enlarged and increase in number upon low doses of spinosad treatment. Lysosomal dysfunction is associated with mitochondrial stress and elevated levels of reactive oxygen species (ROS) in the central nervous system where nAChRα6 is broadly expressed. ROS disturb lipid storage in metabolic tissues in an nAChRα6-dependent manner. Spinosad toxicity is ameliorated with the antioxidant N-acetylcysteine amide. Chronic exposure of adult virgin females to low doses of spinosad leads to mitochondrial defects, severe neurodegeneration, and blindness. These deleterious effects of low-dose exposures warrant rigorous investigation of its impacts on beneficial insects.
Wang, R., Miao, G., Shen, J. L., Fortier, T. M. and Baehrecke, E. H. (2022). ESCRT dysfunction compromises endoplasmic reticulum maturation and autophagosome biogenesis in Drosophila. Curr Biol 32(6): 1262-1274.e1264. PubMed ID: 35134326
Autophagy targets cytoplasmic materials for degradation and influences cell health. Organelle contact and trafficking systems provide membranes for autophagosome formation, but how different membrane systems are selected for use during autophagy remains unclear. This study reports a novel function of the endosomal sorting complex required for transport (ESCRT) in the regulation of endoplasmic reticulum (ER) coat protein complex II (COPII) vesicle formation that influences autophagy. The ESCRT functions in a pathway upstream of Vps13D to influence COPII vesicle transport, ER-Golgi intermediate compartment (ERGIC) assembly, and autophagosome formation. Atg9 functions downstream of the ESCRT to facilitate ERGIC and autophagosome formation. Interestingly, cells lacking either ESCRT or Vps13D functions exhibit dilated ER structures that are similar to cranio-lenticulo-sutural dysplasia patient cells with SEC23A mutations, which encodes a component of COPII vesicles. These data reveal a novel ESCRT-dependent pathway that influences the ERGIC and autophagosome formation.
Jouandin, P., Marelja, Z., Shih, Y. H., Parkhitko, A. A., Dambowsky, M., Asara, J. M., Nemazanyy, I., Dibble, C. C., Simons, M. and Perrimon, N. (2022). Lysosomal cystine mobilization shapes the response of TORC1 and tissue growth to fasting. Science 375(6582): eabc4203. PubMed ID: 35175796
Adaptation to nutrient scarcity involves an orchestrated response of metabolic and signaling pathways to maintain homeostasis. This study found that in the fat body of fasting Drosophila, lysosomal export of cystine coordinates remobilization of internal nutrient stores with reactivation of the growth regulator target of rapamycin complex 1 (TORC1). Mechanistically, cystine was reduced to cysteine and metabolized to acetyl-coenzyme A (acetyl-CoA) by promoting CoA metabolism. In turn, acetyl-CoA retained carbons from alternative amino acids in the form of tricarboxylic acid cycle intermediates and restricted the availability of building blocks required for growth. This process limited TORC1 reactivation to maintain autophagy and allowed animals to cope with starvation periods. It is proposed that cysteine metabolism mediates a communication between lysosomes and mitochondria, highlighting how changes in diet divert the fate of an amino acid into a growth suppressive program.

Wednesday, May 25th - Protein structure and function

Cotsworth, S., Jackson, C. J., Hallson, G., Fitzpatrick, K. A., Syrzycka, M., Coulthard, A. B., Bejsovec, A., Marchetti, M., Pimpinelli, S., Wang, S. J. H., Camfield, R. G., Verheyen, E. M., Sinclair, D. A., Honda, B. M. and Hilliker, A. J. (2022). Characterization of Gfat1 (zeppelin) and Gfat2, Essential Paralogous Genes Which Encode the Enzymes That Catalyze the Rate-Limiting Step in the Hexosamine Biosynthetic Pathway in Drosophila melanogaster. Cells 11(3). PubMed ID: 35159258
The zeppelin (zep) locus is known for its essential role in the development of the embryonic cuticle of Drosophila melanogaster. This study shows that zep encodes Gfat1 (Glutamine: Fructose-6-Phosphate Aminotransferase 1; CG12449), the enzyme that catalyzes the rate-limiting step in the hexosamine biosynthesis pathway (HBP). This conserved pathway diverts 2%-5% of cellular glucose from glycolysis and is a nexus of sugar (fructose-6-phosphate), amino acid (glutamine), fatty acid [acetyl-coenzymeA (CoA)], and nucleotide/energy (UDP) metabolism. The isolation and characterization of lethal mutants in the euchromatic paralog, Gfat2 (CG1345) is described, and it was demonstrated that ubiquitous expression of Gfat1(+) or Gfat2(+) transgenes can rescue lethal mutations in either gene. Gfat1 and Gfat2 show differences in mRNA and protein expression during embryogenesis and in essential tissue-specific requirements for Gfat1 and Gfat2, suggesting a degree of functional evolutionary divergence. An evolutionary, cytogenetic analysis of the two genes in six Drosophila species revealed Gfat2 is located within euchromatin in all six species. Gfat1 localizes to heterochromatin in three melanogaster-group species, and to euchromatin in the more distantly related species. It was also found that the pattern of flanking-gene microsynteny is highly conserved for Gfat1 and somewhat less conserved for Gfat2.
Lunetti, P., Gorgoglione, R., Curcio, R., Marra, F., Pignataro, A., Vozza, A., Riley, C. L., Capobianco, L., Palmieri, L., Dolce, V. and Fiermonte, G. (2022). Drosophila melanogaster Uncoupling Protein-4A (UCP4A) Catalyzes a Unidirectional Transport of Aspartate. Int J Mol Sci 23(3). PubMed ID: 35162943
Uncoupling proteins (UCPs) form a distinct subfamily of the mitochondrial carrier family (MCF) SLC25. Four UCPs, DmUCP4A-C and DmUCP5, have been identified in Drosophila melanogaster on the basis of their sequence homology with mammalian UCP4 and UCP5. In a Parkinson's disease model, DmUCP4A showed a protective role against mitochondrial dysfunction, by increasing mitochondrial membrane potential and ATP synthesis. To date, DmUCP4A is still an orphan of a biochemical function, although its possible involvement in mitochondrial uncoupling has been ruled out. This study shows that DmUCP4A expressed in bacteria and reconstituted in phospholipid vesicles catalyzes a unidirectional transport of aspartate, which is saturable and inhibited by mercurials and other mitochondrial carrier inhibitors to various degrees. Swelling experiments carried out in yeast mitochondria have demonstrated that the unidirectional transport of aspartate catalyzed by DmUCP4 is not proton-coupled. The biochemical function of DmUCP4A has been further confirmed in a yeast cell model, in which growth has required an efflux of aspartate from mitochondria. Notably, DmUCP4A is the first UCP4 homolog from any species to be biochemically characterized. In Drosophila melanogaster, DmUCP4A could be involved in the transport of aspartate from mitochondria to the cytosol, in which it could be used for protein and nucleotide synthesis, as well as in the biosynthesis of β-alanine and N-acetylaspartate, which play key roles in signal transmission in the central nervous system.
Duong, P. C., McCabe, T. C., Riley, G. F., Holmes, H. L., Piermarini, P. M., Romero, M. F. and Gillen, C. M. (2022). Sequence analysis and function of mosquito aeCCC2 and Drosophila Ncc83 orthologs. Insect Biochem Mol Biol 143: 103729. PubMed ID: 35150868
Dipteran insects have genes that code for two different Na(+)-dependent cation-chloride cotransporter (CCC) paralogs. Aedes aegypti aeNKCC1 is an ortholog of Drosophila melanogaster Ncc69, a bumetanide-sensitive Na(+)-K(+)-2Cl(-) cotransporter (NKCC). Aedes aegypti aeCCC2 and aeCCC3 are orthologs of Drosophila Ncc83. Prior work suggests that the transport properties of aeCCC2 differ from canonical NKCCs. In particular, Xenopus oocytes expressing aeCCC2 have increased Na(+)-dependent membrane currents compared to controls, whereas NKCCs are electroneutral. This study further evaluated the function and localization of aeCCC2 and Ncc83. In oocytes expressing aeCCC2 or Ncc83, membrane potential (V(m)) hyperpolarized upon Na(+) removal; following hypotonic exposure the change in V(m) was greater than it was in controls. In voltage-clamp experiments, membrane currents were concentration dependent on Na(+) with an apparent affinity (K(m)) of approximately 4.6 mM. In Malpighian tubules of larval and adult mosquitoes, aeCCC2 was localized along the basolateral aspect of principal cells. Sequence comparisons among transporters from Drosophila, Aedes, Anopheles, and Culex revealed 33 residues within the transmembrane domains (TMDs) that are fully conserved within paralogs but that differ between orthologs of NKCC1 and orthologs of aeCCC2/Ncc83. These residues are distributed across all 12 TMDs. These results provide a foundation for further exploration of the structural basis for functional differences between insect Na(+)-dependent CCCs.
Moussalem, D., Auge, B., Di Stefano, L., Osman, D., Gobert, V. and Haenlin, M. (2021). Two Isoforms of serpent Containing Either One or Two GATA Zinc Fingers Provide Functional Diversity During Drosophila Development. Front Cell Dev Biol 9: 795680. PubMed ID: 35178397
GATA transcription factors play crucial roles in various developmental processes in organisms ranging from flies to humans. In mammals, GATA factors are characterized by the presence of two highly conserved domains, the N-terminal (N-ZnF) and the C-terminal (C-ZnF) zinc fingers. The Drosophila GATA factor Serpent (Srp) is produced in different isoforms that contains either both N-ZnF and C-ZnF (SrpNC) or only the C-ZnF (SrpC). This study investigated the functional roles ensured by each of these isoforms during Drosophila development. Using the CRISPR/Cas9 technique, new mutant fly lines were generated deleted for one (ΔsrpNC) or the other (ΔsrpC) encoded isoform, and a third one with a single point mutation in the N-ZnF that alters its interaction with its cofactor, the Drosophila FOG homolog U-shaped (Ush). Analysis of these mutants revealed that the Srp zinc fingers are differentially required for Srp to fulfill its functions. While SrpC is essential for embryo to adult viability, SrpNC, which is the closest conserved isoform to that of vertebrates, is not. However, to ensure its specific functions in larval hematopoiesis and fertility, Srp requires the presence of both N- and C-ZnF (SrpNC) and interaction with its cofactor Ush. These results also reveal that in vivo the presence of N-ZnF restricts rather than extends the ability of GATA factors to regulate the repertoire of C-ZnF bound target genes.
Telonis-Scott, M., Ali, Z., Hangartner, S. and Sgro, C. M. (2021). Temporal specific coevolution of Hsp70 and co-chaperone stv expression in Drosophila melanogaster under selection for heat tolerance. J Therm Biol 102: 103110. PubMed ID: 34863477
This study examined Hsp70 and co-chaperone stv isoform transcript expression in Australian D. melanogaster lines selected for static heat tolerance and observed a temporal and stv isoform specific, coordinated transcriptional selection response with Hsp70, suggesting that increased chaperone output accompanied increased heat tolerance. It was hypothesize that the coordinated evolutionary response of Hsp70 and stv may have arisen as a correlated response resulting from a shared regulatory hierarchy. This work highlights the complexity and specificity of the heat shock response in D. melanogaster. The coevolution of Hsp70 and stv provide new avenues to examine the common mechanisms underpinning direct and correlated phenotypic responses to selection for heat tolerance.
Chowdhury, R., Abboud, M. I., Wiley, J., Tumber, A., Markolovic, S. and Schofield, C. J. (2022). Conservation of the unusual dimeric JmjC fold of JMJD7 from Drosophila melanogaster to humans. Sci Rep 12(1): 6065. PubMed ID: 35410347
The JmjC family of 2-oxoglutarate dependent oxygenases catalyse a range of hydroxylation and demethylation reactions in humans and other animals. Jumonji domain-containing 7 (JMJD7) is a JmjC (3S)-lysyl-hydroxylase that catalyses the modification of Developmentally Regulated GTP Binding Proteins 1 and 2 (DRG1 and 2); JMJD7 has also been reported to have histone endopeptidase activity. This paper report biophysical and biochemical studies on JMJD7 from Drosophila melanogaster (dmJMJD7). Notably, crystallographic analyses reveal that the unusual dimerization mode of JMJD7, which involves interactions between both the N- and C-terminal regions of both dmJMJD7 monomers and disulfide formation, is conserved in human JMJD7 (hsJMJD7). The results further support the assignment of JMJD7 as a lysyl hydroxylase and will help enable the development of selective inhibitors for it and other JmjC oxygenases.

Tuesday, May 24th - Enhancers and Transcriptional Regulation

Nim, H. T., Dang, L., Thiyagarajah, H., Bakopoulos, D., See, M., Charitakis, N., Sibbritt, T., Eichenlaub, M. P., Archer, S. K., Fossat, N., Burke, R. E., Tam, P. P. L., Warr, C. G., Johnson, T. K. and Ramialison, M. (2021). A cis-regulatory-directed pipeline for the identification of genes involved in cardiac development and disease. Genome Biol 22(1): 335. PubMed ID: 34906219
Congenital heart diseases are the major cause of death in newborns, but the genetic etiology of this developmental disorder is not fully known. The conventional approach to identify the disease-causing genes focuses on screening genes that display heart-specific expression during development. However, this approach would have discounted genes that are expressed widely in other tissues but may play critical roles in heart development. This study reports an efficient pipeline of genome-wide gene discovery based on the identification of a cardiac-specific cis-regulatory element signature that points to candidate genes involved in heart development and congenital heart disease. With this pipeline, 76% of the known cardiac developmental genes were retrived and 35 novel genes were predicted that previously had no known connectivity to heart development. Functional validation of these novel cardiac genes by RNAi-mediated knockdown of the conserved orthologs in Drosophila cardiac tissue reveals that disrupting the activity of 71% of these genes leads to adult mortality. Among these genes, RpL14, RpS24, and Rpn8 are associated with heart phenotypes. This pipeline has enabled the discovery of novel genes with roles in heart development. This workflow, which relies on screening for non-coding cis-regulatory signatures, is amenable for identifying developmental and disease genes for an organ without constraining to genes that are expressed exclusively in the organ of interest.
Bourbon, H. G., Benetah, M. H., Guillou, E., Mojica-Vazquez, L. H., Baanannou, A., Bernat-Fabre, S., Loubiere, V., Bantignies, F., Cavalli, G. and Boube, M. (2022). A shared ancient enhancer element differentially regulates the bric-a-brac tandem gene duplicates in the developing Drosophila leg. PLoS Genet 18(3): e1010083. PubMed ID: 35294439
Gene duplications and transcriptional enhancer emergence/modifications are thought having greatly contributed to phenotypic innovations during animal evolution. Nevertheless, little is known about how enhancers evolve after gene duplication and how regulatory information is rewired between duplicated genes. The Drosophila melanogaster bric-a-brac (bab) complex, comprising the tandem paralogous genes bab1 and bab2, provides a paradigm to address these issues. Previous work characterized an intergenic enhancer (named LAE) regulating bab2 expression in the developing legs. This study shows that bab2 regulators binding directly the LAE also govern bab1 expression in tarsal cells. LAE excision by CRISPR/Cas9-mediated genome editing reveals that this enhancer appears involved but not strictly required for bab1 and bab2 co-expression in leg tissues. Instead, the LAE enhancer is critical for paralog-specific bab2 expression along the proximo-distal leg axis. Chromatin features and phenotypic rescue experiments indicate that LAE functions partly redundantly with leg-specific regulatory information overlapping the bab1 transcription unit. Phylogenomics analyses indicate that (i) the bab complex originates from duplication of an ancestral singleton gene early on within the Cyclorrhapha dipteran sublineage, and (ii) LAE sequences have been evolutionarily-fixed early on within the Brachycera suborder thus predating the gene duplication event. This work provides new insights on enhancers, particularly about their emergence, maintenance and functional diversification during evolution.
Fernandes, G., Tran, H., Andrieu, M., Diaw, Y., Perez Romero, C., Fradin, C., Coppey, M., Walczak, A. M. and Dostatni, N. (2022). Synthetic reconstruction of the hunchback promoter specifies the role of Bicoid, Zelda and Hunchback in the dynamics of its transcription. Elife 11. PubMed ID: 35363606
For over 40 years, the Bicoid-hunchback (Bcd-hb) system in the fruit fly embryo has been used as a model to study how positional information in morphogen concentration gradients is robustly translated into step-like responses. A body of quantitative comparisons between theory and experiment have since questioned the initial paradigm that the sharp hb transcription pattern emerges solely from diffusive biochemical interactions between the Bicoid transcription factor and the gene promoter region. Several alternative mechanisms have been proposed, such as additional sources of positional information, positive feedback from Hb proteins or out-of-equilibrium transcription activation. By using the MS2-MCP RNA-tagging system and analysing in real time, the transcription dynamics of synthetic reporters for Bicoid and/or its two partners Zelda and Hunchback, this study showed that all the early hb expression pattern features and temporal dynamics are compatible with an equilibrium model with a short decay length Bicoid activity gradient as a sole source of positional information. Meanwhile, Bicoid's partners speed-up the process by different means: Zelda lowers the Bicoid concentration threshold required for transcriptional activation while Hunchback reduces burstiness and increases the polymerase firing rate.
Bellec, M., Dufourt, J., Hunt, G., Lenden-Hasse, H., Trullo, A., Zine El Aabidine, A., Lamarque, M., Gaskill, M. M., Faure-Gautron, H., Mannervik, M., Harrison, M. M., Andrau, J. C., Favard, C., Radulescu, O. and Lagha, M. (2022). The control of transcriptional memory by stable mitotic bookmarking. Nat Commun 13(1): 1176. PubMed ID: 35246556
To maintain cellular identities during development, gene expression profiles must be faithfully propagated through cell generations. The reestablishment of gene expression patterns upon mitotic exit is mediated, in part, by transcription factors (TF) mitotic bookmarking. However, the mechanisms and functions of TF mitotic bookmarking during early embryogenesis remain poorly understood. This study took advantage of the naturally synchronized mitoses of Drosophila early embryos, providing evidence that GAGA pioneer factor (GAF) acts as a stable mitotic bookmarker during zygotic genome activation. During mitosis, GAF remains associated to a large fraction of its interphase targets, including at cis-regulatory sequences of key developmental genes with both active and repressive chromatin signatures. GAF mitotic targets are globally accessible during mitosis and are bookmarked via histone acetylation (H4K8ac). By monitoring the kinetics of transcriptional activation in living embryos, this study reports that GAF binding establishes competence for rapid activation upon mitotic exit.
Pasquier, C. and Robichon, A. (2022). Temporal and sequential order of nonoverlapping gene networks unraveled in mated female Drosophila. Life Sci Alliance 5(2). PubMed ID: 34844981
This study reanalyzed available datasets of gene expression changes in female Drosophila head induced by mating. Mated females present metabolic phenotypic changes and display behavioral characteristics that are not observed in virgin females, such as repulsion to male sexual aggressiveness, fidelity to food spots selected for oviposition, and restriction to the colonization of new niches. This study characterized gene networks that play a role in female brain plasticity after mating using AMINE, a novel algorithm to find dysregulated modules of interacting genes. The uncovered networks of altered genes revealed a strong specificity for each successive period of life span after mating in the female head, with little conservation between them. This finding highlights a temporal order of recruitment of waves of interconnected genes which are apparently transiently modified: the first wave disappears before the emergence of the second wave in a reversible manner and ends with few consolidated gene expression changes at day 20. This analysis might document an extended field of a programmatic control of female phenotypic traits by male seminal fluid.
Loganathan, R., Levings, D. C., Kim, J. H., Wells, M. B., Chiu, H., Wu, Y., Slattery, M. and Andrew, D. J. (2022). Ribbon boosts ribosomal protein gene expression to coordinate organ form and function. J Cell Biol 221(4). PubMed ID: 35195669
Cell growth is well defined for late (postembryonic) stages of development, but evidence for early (embryonic) cell growth during postmitotic morphogenesis is limited. This study reports early cell growth as a key characteristic of tubulogenesis in the Drosophila embryonic salivary gland (SG) and trachea. A BTB/POZ domain nuclear factor, Ribbon (Rib), mediates this early cell growth. Rib binds the transcription start site of nearly every SG-expressed ribosomal protein gene (RPG) and is required for full expression of all RPGs tested. Rib binding to RPG promoters in vitro is weak and not sequence specific, suggesting that specificity is achieved through cofactor interactions. Accordingly, this study demonstrates Rib's ability to physically interact with each of the three known regulators of RPG transcription. Surprisingly, Rib-dependent early cell growth in another tubular organ, the embryonic trachea, is not mediated by direct RPG transcription. These findings support a model of early cell growth customized by transcriptional regulatory networks to coordinate organ form and function.

Monday, May 23rd - Larval and Adult Neural Development and Function

Ketkar, M. D., Gur, B., Molina-Obando, S., Ioannidou, M., Martelli, C. and Silies, M. (2022). First-order visual interneurons distribute distinct contrast and luminance information across ON and OFF pathways to achieve stable behavior. Elife 11. PubMed ID: 35263247
The accurate processing of contrast is the basis for all visually guided behaviors. Visual scenes with rapidly changing illumination challenge contrast computation because photoreceptor adaptation is not fast enough to compensate for such changes. Yet, human perception of contrast is stable even when the visual environment is quickly changing, suggesting rapid post receptor luminance gain control. Similarly, in the fruit fly Drosophila, such gain control leads to luminance invariant behavior for moving OFF stimuli. This study shows that behavioral responses to moving ON stimuli also utilize a luminance gain, and that ON-motion guided behavior depends on inputs from three first-order interneurons L1, L2, and L3. Each of these neurons encodes contrast and luminance differently and distributes information asymmetrically across both ON and OFF contrast-selective pathways. Behavioral responses to both ON and OFF stimuli rely on a luminance-based correction provided by L1 and L3, wherein L1 supports contrast computation linearly, and L3 non-linearly amplifies dim stimuli. Therefore, L1, L2, and L3 are not specific inputs to ON and OFF pathways but the lamina serves as a separate processing layer that distributes distinct luminance and contrast information across ON and OFF pathways to support behavior in varying conditions.
Jonaitis, J., MacLeod, J. and Pulver, S. R. (2022). Localization of muscarinic acetylcholine receptor-dependent rhythm-generating modules in the Drosophila larval locomotor network. J Neurophysiol 127(4): 1098-1116. PubMed ID: 35294308
Mechanisms of rhythm generation have been extensively studied in motor systems that control locomotion over terrain in limbed animals; however, much less is known about rhythm generation in soft-bodied terrestrial animals. This study explored how muscarinic acetylcholine receptor (mAChR)-modulated rhythm-generating networks are distributed in the central nervous system (CNS) of soft-bodied Drosophila larvae. Fictive motor patterns were measured in isolated CNS preparations, using a combination of Ca(2+) imaging and electrophysiology while manipulating mAChR signaling pharmacologically. Bath application of the mAChR agonist oxotremorine potentiated bilaterally asymmetric activity in anterior thoracic regions and promoted bursting in posterior abdominal regions. Application of the mAChR antagonist scopolamine suppressed rhythm generation in these regions and blocked the effects of oxotremorine. Oxotremorine triggered fictive forward crawling in preparations without brain lobes. Oxotremorine also potentiated rhythmic activity in isolated posterior abdominal CNS segments as well as isolated anterior brain and thoracic regions, but it did not induce rhythmic activity in isolated anterior abdominal segments. Bath application of scopolamine to reduced preparations lowered baseline Ca(2+) levels and abolished rhythmic activity. Overall, these results suggest that mAChR signaling plays a role in enabling rhythm generation at multiple sites in the larval CNS. This work furthers understanding of motor control in soft-bodied locomotion and provides a foundation for study of rhythm-generating networks in an emerging genetically tractable locomotor system.
Lacoste, J., Soula, H., Burg, A., Audibert, A., Darnat, P., Gho, M. and Louvet-Vallee, S. (2022). A neural progenitor mitotic wave is required for asynchronous axon outgrowth and morphology. Elife 11. PubMed ID: 35254258
Spatiotemporal mechanisms generating neural diversity are fundamental for understanding neural processes. This study investigated how neural diversity arises from neurons coming from identical progenitors. In the dorsal thorax of Drosophila, rows of mechanosensory organs originate from the division of sensory organ progenitor (SOPs). In each row of the notum, an anteromedial located central SOP divides first, then neighbouring SOPs divide, and so on. This centrifugal wave of mitoses depends on cell-cell inhibitory interactions mediated by SOP cytoplasmic protrusions and Scabrous, a secreted protein interacting with the Delta/Notch complex. Furthermore, when this mitotic wave was reduced, axonal growth was more synchronous, axonal terminals had a complex branching pattern and fly behaviour was impaired. The temporal order of progenitor divisions influences the birth order of sensory neurons, axon branching and impact on grooming behaviour. These data support the idea that developmental timing controls axon wiring neural diversity.
Jeong, E. M., Kwon, M., Cho, E., Lee, S. H., Kim, H., Kim, E. Y. and Kim, J. K. (2022). Systematic modeling-driven experiments identify distinct molecular clockworks underlying hierarchically organized pacemaker neurons. Proc Natl Acad Sci U S A 119(8). PubMed ID: 35193959
In metazoan organisms, circadian (∼24 h) rhythms are regulated by pacemaker neurons organized in a master-slave hierarchy. Although it is widely accepted that master pacemakers and slave oscillators generate rhythms via an identical negative feedback loop of transcription factor CLOCK (CLK) and repressor PERIOD (PER), their different roles imply heterogeneity in their molecular clockworks. Indeed, in Drosophila, defective binding between CLK and PER disrupts molecular rhythms in the master pacemakers, small ventral lateral neurons (sLN(v)s), but not in the slave oscillator, posterior dorsal neuron 1s (DN1(p)s). This study developed a systematic and expandable approach that unbiasedly searches the source of the heterogeneity in molecular clockworks from time-series data. In combination with in vivo experiments, it was found that sLN(v)s exhibit higher synthesis and turnover of PER and lower CLK levels than DN1(p)s. Importantly, light shift analysis reveals that due to such a distinct molecular clockwork, sLN(v)s can obtain paradoxical characteristics as the master pacemaker, generating strong rhythms that are also flexibly adjustable to environmental changes. These results identify the different characteristics of molecular clockworks of pacemaker neurons that underlie hierarchical multi-oscillator structure to ensure the rhythmic fitness of the organism.
Lin, C. H., Senapati, B., Chen, W. J., Bansal, S. and Lin, S. (2022). Semaphorin 1a-mediated dendritic wiring of the Drosophila mushroom body extrinsic neurons. Proc Natl Acad Sci U S A 119(12): e2111283119. PubMed ID: 35286204
The adult Drosophila mushroom body (MB) is one of the most extensively studied neural circuits. However, how its circuit organization is established during development is unclear. This study provides an initial characterization of the assembly process of the extrinsic neurons (dopaminergic neurons and MB output neurons) that target the vertical MB lobes. The cellular mechanisms guiding the neurite targeting of these extrinsic neurons were probed, and it was demonstrate that Semaphorin 1a is required in several MB output neurons for their dendritic innervations to three specific MB lobe zones. This study reveals several intriguing molecular and cellular principles governing assembly of the MB circuit.
Klapoetke, N. C., Nern, A., Rogers, E. M., Rubin, G. M., Reiser, M. B. and Card, G. M. (2022). A functionally ordered visual feature map in the Drosophila brain. Neuron. PubMed ID: 35290791
Topographic maps, the systematic spatial ordering of neurons by response tuning, are common across species. In Drosophila, the lobula columnar (LC) neuron types project from the optic lobe to the central brain, where each forms a glomerulus in a distinct position. However, the advantages of this glomerular arrangement are unclear. This study examine the functional and spatial relationships of 10 glomeruli using single-neuron calcium imaging. Novel detectors were discovered for objects smaller than the lens resolution (LC18) and for complex line motion (LC25). Glomeruli are spatially clustered by selectivity for looming versus drifting object motion and ordered by size tuning to form a topographic visual feature map. Furthermore, connectome analysis shows that downstream neurons integrate from sparse subsets of possible glomeruli combinations, which are biased for glomeruli encoding similar features. LC neurons are thus an explicit example of distinct feature detectors topographically organized to facilitate downstream circuit integration.

Friday, May 20th, Signaling

Alves, A. N., Sgro, C. M., Piper, M. D. W. and Mirth, C. K. (2022). Target of Rapamycin Drives Unequal Responses to Essential Amino Acid Depletion for Egg Laying in Drosophila Melanogaster. Front Cell Dev Biol 10: 822685. PubMed ID: 35252188
Nutrition shapes a broad range of life-history traits, ultimately impacting animal fitness. A key fitness-related trait, female fecundity is well known to change as a function of diet. In particular, the availability of dietary protein is one of the main drivers of egg production, and in the absence of essential amino acids egg laying declines. However, it is unclear whether all essential amino acids have the same impact on phenotypes like fecundity. Using a holidic diet, this study fed adult female Drosophila melanogaster diets that contained all necessary nutrients except one of the 10 essential amino acids and assessed the effects on egg production. For most essential amino acids, depleting a single amino acid induced as rapid a decline in egg production as when there were no amino acids in the diet. However, when either methionine or histidine were excluded from the diet, egg production declined more slowly. Next, this study tested whether GCN2 and TOR mediated this difference in response across amino acids. While mutations in GCN2 did not eliminate the differences in the rates of decline in egg laying among amino acid drop-out diets, it was found that inhibiting TOR signalling caused egg laying to decline rapidly for all drop-out diets. TOR signalling does this by regulating the yolk-forming stages of egg chamber development. These results suggest that amino acids differ in their ability to induce signalling via the TOR pathway. This is important because if phenotypes differ in sensitivity to individual amino acids, this generates the potential for mismatches between the output of a pathway and the animal's true nutritional status.
Cho, B., Song, S., Wan, J. Y. and Axelrod, J. D. (2022). Prickle isoform participation in distinct polarization events in the Drosophila eye. PLoS One 17(2): e0262328. PubMed ID: 35148314
Planar cell polarity (PCP) signaling regulates several polarization events during development of ommatidia in the Drosophila eye, including directing chirality by polarizing a cell fate choice and determining the direction and extent of ommatidial rotation. The pksple isoform of the PCP protein Prickle is known to participate in the R3/R4 cell fate decision, but the control of other polarization events and the potential contributions of the three Pk isoforms have not been clarified. By characterizing expression and subcellular localization of individual isoforms together with re-analyzing isoform specific phenotypes, this study showed that the R3/R4 fate decision, its coordination with rotation direction, and completion of rotation to a final ±90° rotation angle are separable polarization decisions with distinct Pk isoform requirements and contributions. Both pksple and pkpk can enforce robust R3/R4 fate decisions, but only pksple can correctly orient them along the dorsal-ventral axis. In contrast, pksple and pkpk can fully and interchangeably sustain coordination of rotation direction and rotation to completion. It is proposed that expression dynamics and competitive interactions determine isoform participation in these processes. It is proposed that the selective requirement for pksple to orient the R3/R4 decision and their interchangeability for coordination and completion of rotation reflects their previously described differential interaction with the Fat/Dachsous system which is known to be required for orientation of R3/R4 decisions but not for coordination or completion of rotation.
Kumari, A., Ghosh, A., Kolay, S. and Raghu, P. (2022). Septins tune lipid kinase activity and PI(4,5)P(2) turnover during G-protein-coupled PLC signalling in vivo. Life Sci Alliance 5(6). PubMed ID: 35277468
Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)] hydrolysis by phospholipase C (PLC) is a conserved mechanism of signalling. Given the low abundance of PI(4,5)P(2), its hydrolysis needs to be coupled to resynthesis to ensure continued PLC activity; however, the mechanism by which depletion is coupled to resynthesis remains unknown. PI(4,5)P(2) synthesis is catalyzed by the phosphorylation of phosphatidylinositol 4 phosphate (PI4P) by phosphatidylinositol 4 phosphate 5 kinase (PIP5K). In Drosophila photoreceptors, photon absorption is transduced into PLC activity and during this process, PI(4,5)P(2) is resynthesized by a PIP5K. However, the mechanism by which PIP5K activity is coupled to PI(4,5)P(2) hydrolysis is unknown. This study identified a unique isoform dPIP5K(L), that is both necessary and sufficient to mediate PI(4,5)P(2) synthesis during phototransduction. Depletion of PNUT, a non-redundant subunit of the septin family, enhances dPIP5K(L) activity in vitro and PI(4,5)P(2) resynthesis in vivo; co-depletion of dPIP5K(L) reverses the enhanced rate of PI(4,5)P(2) resynthesis in vivo. Thus, this work defines a septin-mediated mechanism through which PIP5K activity is coupled to PLC-mediated PI(4,5)P(2) hydrolysis.
Ghosh, S., Leng, W., Wilsch-Brauninger, M., Barrera-Velazquez, M., Leopold, P. and Eaton, S. (2022). A local insulin reservoir in Drosophila alpha cell homologs ensures developmental progression under nutrient shortage. Curr Biol. PubMed ID: 35316653
Insulin/insulin-like growth factor (IGF) signaling (IIS) controls many aspects of development and physiology. In Drosophila, a conserved family of insulin-like peptides called Dilps is produced by brain neurosecretory cells, and it regulates organismal growth and developmental timing. To accomplish these systemic functions, the Dilps are secreted into the general circulation, and they signal to peripheral tissues in an endocrine fashion. This study describes the local uptake and storage of Dilps in the corpora cardiaca (CC), an endocrine organ composed of alpha cell homologs known to produce the glucagon-like adipokinetic hormone (AKH). Dilp uptake by the CC relies on the expression of an IGF-binding protein called ImpL2. Following their uptake, immunogold staining demonstrates that Dilps are co-packaged with AKH in dense-core vesicles for secretion. In response to nutrient shortage, this specific Dilp reservoir is released and activates IIS in a paracrine manner in the prothoracic gland. This stimulates the production of the steroid hormone ecdysone and initiates entry into pupal development. This study has therefore uncovered a sparing mechanism whereby insulin stores in CC serve to locally activate IIS and the production of ecdysone in the PG, accelerating developmental progression in adverse food conditions.
Li, Z., Qian, W., Song, W., Zhao, T., Yang, Y., Wang, W., Wei, L., Zhao, D., Li, Y., Perrimon, N., Xia, Q. and Cheng, D. (2022). A salivary gland-secreted peptide regulates insect systemic growth. Cell Rep 38(8): 110397. PubMed ID: 35196492
Insect salivary glands have been shown to function in pupal attachment and food lubrication by secreting factors into the lumen via an exocrine way. This study found in Drosophila that a salivary gland-derived secreted factor (Sgsf) peptide regulates systemic growth via an endocrine way. Sgsf is specifically expressed in salivary glands and secreted into the hemolymph. Sgsf knockout or salivary gland-specific Sgsf knockdown decrease the size of both the body and organs, phenocopying the effects of genetic ablation of salivary glands, while salivary gland-specific Sgsf overexpression increases their size. Sgsf promotes systemic growth by modulating the secretion of the insulin-like peptide Dilp2 from the brain insulin-producing cells (IPCs) and affecting mechanistic target of rapamycin (mTOR) signaling in the fat body. Altogether, this study demonstrates that Sgsf mediates the roles of salivary glands in Drosophila systemic growth, establishing an endocrine function of salivary glands.
Ding, X., Li, Z., Peng, K., Zou, R., Wu, C., Lin, G., Li, W. and Xue, L. (2022). Snail regulates Hippo signalling-mediated cell proliferation and tissue growth in Drosophila. Open Biol 12(3): 210357. PubMed ID: 35259952
Snail (Sna) plays a pivotal role in epithelia-mesenchymal transition and cancer metastasis, yet its functions in normal tissue development remain elusive. Using Drosophila as a model organism, this study identified Sna as an essential regulator of Hippo signalling-mediated cell proliferation and tissue growth. First, Sna is necessary and sufficient for impaired Hippo signalling-induced cell proliferation and tissue overgrowth. Second, Sna is necessary and sufficient for the expression of Hippo pathway target genes. Third, genetic epistasis data indicate Sna acts downstream of Yki in the Hippo signalling. Finally, Sna is physiologically required for tissue growth in normal development. Mechanistically, Yki activates the transcription of sna, whose protein product binds to Scalloped (Sd) and promotes Sd-dependent cell proliferation. Thus, this study uncovered a previously unknown physiological function of Sna in normal tissue development and revealed the underlying mechanism by which Sna modulates Hippo signalling-mediated cell proliferation and tissue growth.

Thursday, May 19th - Adult Physiology

Fang, Y., Chen, B., Liu, Z., Gong, A. Y., Gunning, W. T., Ge, Y., Malhotra, D., Gohara, A. F., Dworkin, L. D. and Gong, R. (2022). Age-related GSK3beta overexpression drives podocyte senescence and glomerular aging. J Clin Invest 132(4). PubMed ID: 35166234
As life expectancy continues to increase, clinicians are challenged by age-related renal impairment that involves podocyte senescence and glomerulosclerosis. There is now compelling evidence that lithium has a potent antiaging activity that ameliorates brain aging and increases longevity in Drosophila and Caenorhabditis elegans. As the major molecular target of lithium action and a multitasking protein kinase recently implicated in a variety of renal diseases, glycogen synthase kinase 3β (GSK3β) is overexpressed and hyperactive with age in glomerular podocytes, correlating with functional and histological signs of kidney aging. Moreover, podocyte-specific ablation of GSK3β substantially attenuated podocyte senescence and glomerular aging in mice. Mechanistically, key mediators of senescence signaling, such as p16INK4A and p53, contain high numbers of GSK3β consensus motifs, physically interact with GSK3β, and act as its putative substrates. In addition, therapeutic targeting of GSK3β by microdose lithium later in life reduced senescence signaling and delayed kidney aging in mice. Furthermore, in psychiatric patients, lithium carbonate therapy inhibited GSK3β activity and mitigated senescence signaling in urinary exfoliated podocytes and was associated with preservation of kidney function. Thus, GSK3β appears to play a key role in podocyte senescence by modulating senescence signaling and may be an actionable senostatic target to delay kidney aging.
Karam, C. S., Williams, B. L., Morozova, I., Yuan, Q., Panarsky, R., Zhang, Y., Hodgkinson, C. A., Goldman, D., Kalachikov, S. and Javitch, J. A. (2022). Functional Genomic Analysis of Amphetamine Sensitivity in Drosophila. Front Psychiatry 13: 831597. PubMed ID: 35250674
Abuse of psychostimulants, including amphetamines (AMPHs), is a major public health problem with profound psychiatric, medical, and psychosocial complications. The actions of these drugs at the dopamine transporter (DAT) play a critical role in their therapeutic efficacy as well as their liability for abuse and dependence. To date, however, the mechanisms that mediate these actions are not well-understood, and therapeutic interventions for AMPH abuse have been limited. Drug exposure can induce broad changes in gene expression that can contribute to neuroplasticity and effect long-lasting changes in neuronal function. Identifying genes and gene pathways perturbed by drug exposure is essential to understanding of the molecular basis of drug addiction. This study used Drosophila as a model to examine AMPH-induced transcriptional changes that are DAT-dependent, as those would be the most relevant to the stimulatory effects of the drug. Using this approach, it was found that genes involved in the control of mRNA translation to be significantly upregulated in response to AMPH in a DAT-dependent manner. To further prioritize genes for validation, this study explored functional convergence between these genes and genes identified in a genome-wide association study of AMPH sensitivity using the Drosophila Genetic Reference Panel. A number of these genes were validated by showing that they act specifically in dopamine neurons to mediate the behavioral effects of AMPH. Taken together, these data establish Drosophila as a powerful model that enables the integration of behavioral, genomic and transcriptomic data, followed by rapid gene validation, to investigate the molecular underpinnings of psychostimulant action.
Kapali, G. P., Callier, V., Gascoigne, S. J. L., Harrison, J. F. and Shingleton, A. W. (2022). The steroid hormone ecdysone regulates growth rate in response to oxygen availability. Sci Rep 12(1): 4730. PubMed ID: 35304878
In almost all animals, physiologically low oxygen (hypoxia) during development slows growth and reduces adult body size. The developmental mechanisms that determine growth under hypoxic conditions are, however, poorly understood. This study shows that the growth and body size response to moderate hypoxia (10% O(2)) in Drosophila melanogaster is systemically regulated via the steroid hormone ecdysone. Hypoxia increases level of circulating ecdysone and inhibition of ecdysone synthesis ameliorates the negative effect of low oxygen on growth. This study also shows that the effect of ecdysone on growth under hypoxia is through suppression of the insulin/IGF-signaling pathway, via increased expression of the insulin-binding protein Imp-L2. These data indicate that growth suppression in hypoxic Drosophila larvae is accomplished by a systemic endocrine mechanism that overlaps with the mechanism that slows growth at low nutrition. This suggests the existence of growth-regulatory mechanisms that respond to general environmental perturbation rather than individual environmental factors.
Emtenani, S., Martin, E. T., Gyoergy, A., Bicher, J., Genger, J. W., Köcher, T., Akhmanova, M., Guarda, M., Roblek, M., Bergthaler, A., Hurd, T. R., Rangan, P. and Siekhaus, D. E. (2022). Macrophage mitochondrial bioenergetics and tissue invasion are boosted by an Atossa-Porthos axis in Drosophila. Embo j: e109049. PubMed ID: 35319107
Cellular metabolism must adapt to changing NADPH glyoxylate reductase demands to enable homeostasis. During immune responses or cancer metastasis, cells leading migration into challenging environments require an energy boost, but what controls this capacity is unclear. A previously uncharacterized nuclear protein, Atossa (encoded by CG9005) was studied that supports macrophage invasion into the germband of Drosophila by controlling cellular metabolism. First, nuclear Atossa increases mRNA levels of Porthos, a DEAD-box protein, and of two metabolic enzymes, lysine-α-ketoglutarate reductase (LKR/SDH) and NADPH glyoxylate reductase (GR/HPR), thus enhancing mitochondrial bioenergetics. Then Porthos supports ribosome assembly and thereby raises the translational efficiency of a subset of mRNAs, including those affecting mitochondrial functions, the electron transport chain, and metabolism. Mitochondrial respiration measurements, metabolomics, and live imaging indicate that Atossa and Porthos power up OxPhos and energy production to promote the forging of a path into tissues by leading macrophages. Since many crucial physiological responses require increases in mitochondrial energy output, this previously undescribed genetic program may modulate a wide range of cellular behaviors.
Charroux, B. and Royet, J. (2022).. Gut-derived peptidoglycan remotely inhibits bacteria dependent activation of SREBP by Drosophila adipocytes. PLoS Genet 18(3): e1010098. PubMed ID: 35245295
Bacteria that colonize eukaryotic gut have profound influences on the physiology of their host. In Drosophila, many of these effects are mediated by adipocytes that combine immune and metabolic functions. Enteric infection with some bacteria species was shown to triggers the activation of the SREBP lipogenic protein in surrounding enterocytes but also in remote fat body cells and in ovaries, an effect that requires insulin signaling. By activating the NF-κB pathway, the cell wall peptidoglycan produced by the same gut bacteria remotely, and cell-autonomously, represses SREBP activation in adipocytes. It was shown that by reducing the level of peptidoglycan, the gut born PGRP-LB amidase balances host immune and metabolic responses of the fat body to gut-associated bacteria. In the absence of such modulation, uncontrolled immune pathway activation prevents SREBP activation and lipid production by the fat body.
Gera, J., Budakoti, P., Suhag, M., Mandal, L. and Mandal, S. (2022). Physiological ROS controls Upd3-dependent modeling of ECM to support cardiac function in Drosophila. Sci Adv 8(7): eabj4991. PubMed ID: 35179958
Despite their highly reactive nature, reactive oxygen species (ROS) at the physiological level serve as signaling molecules regulating diverse biological processes. While ROS usually act autonomously, they also function as local paracrine signals by diffusing out of the cells producing them. Using in vivo molecular genetic analyses in Drosophila, this study provides evidence for ROS-dependent paracrine signaling that does not entail ROS release. Elevated levels of physiological ROS within the pericardial cells activate a signaling cascade transduced by Ask1, c-Jun N-terminal kinase, and p38 to regulate the expression of the cytokine Unpaired 3 (Upd3). Upd3 released by the pericardial cells controls fat body-specific expression of the extracellular matrix (ECM) protein Pericardin, essential for cardiac function and healthy life span. Therefore, this work reveals an unexpected inter-organ communication circuitry wherein high physiological levels of ROS regulate cytokine-dependent modulation of cardiac ECM with implications in normal and pathophysiological conditions.

Wednesday, May 18th - Disease Models

Farrugia, M., Vassallo, N. and Cauchi, R. J. (2022). Disruption of Survival Motor Neuron in Glia Impacts Survival but has no Effect on Neuromuscular Function in Drosophila. Neuroscience 491: 32-42. PubMed ID: 35314252
Increasing evidence points to the involvement of cell types other than motor neurons in both amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA), the predominant motor neuron disease in adults and infants, respectively. The contribution of glia to ALS pathophysiology is well documented. Studies have since focused on evaluating the contribution of glia in SMA. This study made use of the Drosophila model to ask whether the survival motor neuron (Smn) protein, the causative factor for SMA, is required selectively in glia. The specific loss of Smn function in glia during development reduced survival to adulthood but did not affect motoric performance or neuromuscular junction (NMJ) morphology in flies. In contrast, gain rather than loss of ALS-linked TDP-43, FUS or C9orf72 function in glia induced significant defects in motor behaviour in addition to reduced survival. Furthermore, glia-specific gain of TDP-43 function caused both NMJ defects and muscle atrophy. Smn together with Gemins 2-8 and Unrip, form the Smn complex which is indispensable for the assembly of spliceosomal small nuclear ribonucleoproteins (snRNPs). This study shows that glial-selective perturbation of Smn complex components or disruption of key snRNP biogenesis factors pICln and Tgs1, induce deleterious effects on adult fly viability but, similar to Smn reduction, had no negative effect on neuromuscular function. These findings suggest that the role of Smn in snRNP biogenesis as part of the Smn complex is required in glia for the survival of the organism, underscoring the importance of glial cells in SMA disease formation.
da Costa Silva, J. R., Fujimura, P. T., Batista, L. L., Malta, S. M., Filho, R. M., Silva, M. H., de Souza, A. G., Silva, A. P. M., Borges, L. D. F., Bastos, V. A. F., Cossolin, J. F. S., Serrao, J. E., Bonetti, A. M., Junior, L. C. O. and Ueira-Vieira, C. (2022). Differential gene expression by RNA-seq during Alzheimer's disease-like progression in the Drosophila melanogaster model. Neurosci Res. PubMed ID: 35219723
Alzheimer's disease (AD) is characterized by progressive, irreversible loss of memory and cognitive function. Drosophila melanogaster and other animal models are used to study several diseases, in order to elucidate unknown mechanisms and develop potential therapies. Molecular studies require biological samples and, for neuropathologies such as AD biopsy of the human brain, are invasive and potentially damaging. The solution is to use animal models, such as D. melanogaster, which is a model organism that can replace mammalian organisms in such studies. This study evaluated the climbing ability and differential gene expression during AD progression due to the amylodoigenic pathway using RNA-seq, and an in silico analysis of a fruit fly AD-like GFP (Green Fluorescent Protein) model was performed with GFP expression in the pan-neural elav driver. A total of 1388 genes were differentially expressed in all analyzed groups. The main pathways related to those Differentially Expressed Genes (DEGs) during aging and AD progression were evaluated using the fly genes and human orthologs, in order to link genomic information to higher-order functional information with gene pathway mapping. Pathways were identified as present in all analyzed groups, such as metabolic pathways, ribosomal pathways, proteasome pathways and immune system pathways. Some of the genes were validated by qPCR. Knockdown of CG17754 gene by RNAi promoted degeneration in the fly eye, validating these findings in vivo. The identification of similarities in molecular pathways between the transgenic fly AD-like GFP model and mammals related to AD provides new insights into the use of this fly in screening novel anti-AD drugs.
Jarabo, P., de Pablo, C., Gonzalez-Blanco, A. and Casas-Tinto, S. (2022). Circadian Gene cry Controls Tumorigenesis through Modulation of Myc Accumulation in Glioblastoma Cells. Int J Mol Sci 23(4). PubMed ID: 35216153
Glioblastoma (GB) is the most frequent malignant brain tumor among adults and currently there is no effective treatment. This aggressive tumor grows fast and spreads through the brain causing death in 15 months. GB cells display a high mutation rate and generate a heterogeneous population of tumoral cells that are genetically distinct. Thus, the contribution of genes and signaling pathways relevant for GB progression is of great relevance. This study used a Drosophila model of GB that reproduces the features of human GB and describes the upregulation of the circadian gene cry in GB patients and in a Drosophila GB model. The contribution of cry to the expansion of GB cells and the neurodegeneration and premature death caused by GB was study, and it was determined that cry is required for GB progression. Moreover, it wa determined that the PI3K pathway regulates cry expression in GB cells, and in turn, cry is necessary and sufficient to promote Myc accumulation in GB. These results contribute to understanding the mechanisms underlying GB malignancy and lethality, and describe a novel role of Cry in GB cells.
Escobedo, S. E., Stanhope, S. C., Dong, Z. and Weake, V. M. (2022). Aging and Light Stress Result in Overlapping and Unique Gene Expression Changes in Photoreceptors. Genes (Basel) 13(2). PubMed ID: 35205309
The extended photoreceptor cell lifespan, in addition to its high metabolic needs due to phototransduction, makes it critical for these neurons to continually respond to the stresses associated with aging by mounting an appropriate gene expression response. This study sought to untangle the more general neuronal age-dependent transcriptional signature of photoreceptors with that induced by light stress. To do this, flies were aged or exposed to various durations of blue light, followed by photoreceptor nuclei-specific transcriptome profiling. Using this approach, genes were identified that are both common and uniquely regulated by aging and light induced stress. Whereas both age and blue light induce expression of DNA repair genes and a neuronal-specific signature of death, both conditions result in downregulation of phototransduction. Interestingly, blue light uniquely induced genes that directly counteract the overactivation of the phototransduction signaling cascade. Lastly, unique gene expression changes in aging photoreceptors included the downregulation of genes involved in membrane potential homeostasis and mitochondrial function, as well as the upregulation of immune response genes. It is proposed that light stress contributes to the aging transcriptome of photoreceptors, but that there are also other environmental or intrinsic factors involved in age-associated photoreceptor gene expression signatures.
Hayashi, Y., Kashio, S., Murotomi, K., Hino, S., Kang, W., Miyado, K., Nakao, M., Miura, M., Kobayashi, S. and Namihira, M. (2022). Biosynthesis of S-adenosyl-methionine enhances aging-related defects in Drosophila oogenesis. Sci Rep 12(1): 5593. PubMed ID: 35379840
Tissue aging is a major cause of aging-related disabilities and a shortened life span. Understanding how tissue aging progresses and identifying the factors underlying tissue aging are crucial; however, the mechanism of tissue aging is not fully understood. This study shows that the biosynthesis of S-adenosyl-methionine (SAM), the major cellular donor of methyl group for methylation modifications, potently accelerates the aging-related defects during Drosophila oogenesis. An aging-related increase in the SAM-synthetase (Sam-S) levels in the germline leads to an increase in ovarian SAM levels. Sam-S-dependent biosynthesis of SAM controls aging-related defects in oogenesis through two mechanisms, decreasing the ability to maintain germline stem cells and accelerating the improper formation of egg chambers. Aging-related increases in SAM commonly occur in mouse reproductive tissue and the brain. Therefore, these results raise the possibility suggesting that SAM is the factor related to tissue aging beyond the species and tissues.
Lee, H. Y., Lee, S. H. and Min, K. J. (2022). The Increased Abundance of Commensal Microbes Decreases Drosophila melanogaster Lifespan through an Age-Related Intestinal Barrier Dysfunction. Insects 13(2). PubMed ID: 35206792
Commensal microbiota live in their host with a symbiotic relationship that affects the host's health and physiology. Many studies showed that microbial load and composition were changed by aging and have observed that increasing the abundance and changing the composition of commensal microbes had detrimental effects on host lifespan. It was hypothesized that dysbiosis of the intestinal microbiota leads to systemic effects in aging flies as a result of the increased intestinal permeability. The fruit fly laboratory strain w(1118) was used as a model system. The incidence of intestinal dysfunction was increased with age, and intestinal dysfunction increased the permeability of the fly intestine to resident microbes. The lifespan of flies with an intestinal barrier dysfunction was increased by removal of the microbes. Interestingly, some bacteria were also found in the hemolymph of flies with intestinal barrier dysfunction. These findings suggest the possibility that, as the host ages, there is an increase in intestinal permeability, which leads to an increased intestinal microbial load and a reduction in the host lifespan. These data therefore indicate a connection between commensal microbes and host lifespan.

Tuesday, May 17th - Adult Neural Development and Function

Gu, P., Wang, F., Shang, Y., Liu, J., Gong, J., Xie, W., Han, J. and Xiang, Y. (2022). Nociception and hypersensitivity involve distinct neurons and molecular transducers in Drosophila. Proc Natl Acad Sci U S A 119(12): e2113645119. PubMed ID: 35294287
Acute nociception is essential for survival by warning organisms against potential dangers, whereas tissue injury results in a nociceptive hypersensitivity state that is closely associated with debilitating disease conditions, such as chronic pain. Transient receptor potential (Trp) ion channels expressed in nociceptors detect noxious thermal and chemical stimuli to initiate acute nociception. The existing hypersensitivity model suggests that under tissue injury and inflammation, the same Trp channels in nociceptors are sensitized through transcriptional and posttranslational modulation, leading to nociceptive hypersensitivity. Unexpectedly and different from this model, this study found that in Drosophila larvae, acute heat nociception and tissue injury-induced hypersensitivity involve distinct cellular and molecular mechanisms. Specifically, isoform of TrpA1, TrpA1-D in peripheral sensory neurons, mediates acute heat nociception, whereas isoform TrpA1-C in a cluster of larval brain neurons transduces the heat stimulus under the allodynia state. As a result, interfering with synaptic transmission of these brain neurons or genetic targeting of TrpA1-C blocks heat allodynia but not acute heat nociception. TrpA1-C and TrpA1-D are two splicing variants of TrpA1 channels and are coexpressed in these brain neurons. It was further shown that Gq-phospholipase C signaling, downstream of the proalgesic neuropeptide Tachykinin, differentially modulates these two TrpA1 isoforms in the brain neurons by selectively sensitizing heat responses of TrpA1-C but not TrpA1-D. Together, our studies provide evidence that nociception and noncaptive sensitization could be mediated by distinct sensory neurons and molecular sensors.
Grover, D., Chen, J. Y., Xie, J., Li, J., Changeux, J. P. and Greenspan, R. J. (2022). Differential mechanisms underlie trace and delay conditioning in Drosophila.. Nature 603(7900): 302-308. PubMed ID: 35173333
Two forms of associative learning-delay conditioning and trace conditioning-have been widely investigated in humans and higher-order mammals. In delay conditioning, an unconditioned stimulus (for example, an electric shock) is introduced in the final moments of a conditioned stimulus (for example, a tone), with both ending at the same time. In trace conditioning, a 'trace' interval separates the conditioned stimulus and the unconditioned stimulus. Trace conditioning therefore relies on maintaining a neural representation of the conditioned stimulus after its termination (hence making distraction possible), to learn the conditioned stimulus-unconditioned stimulus contingency; this makes it more cognitively demanding than delay conditioning. By combining virtual-reality behaviour with neurogenetic manipulations and in vivo two-photon brain imaging, this study shows that visual trace conditioning and delay conditioning in Drosophila mobilize R2 and R4m ring neurons in the ellipsoid body. In trace conditioning, calcium transients during the trace interval show increased oscillations and slower declines over repeated training, and both of these effects are sensitive to distractions. Dopaminergic activity accompanies signal persistence in ring neurons, and this is decreased by distractions solely during trace conditioning. Finally, dopamine D1-like and D2-like receptor signalling in ring neurons have different roles in delay and trace conditioning; dopamine D1-like receptor 1 mediates both forms of conditioning, whereas the dopamine D2-like receptor is involved exclusively in sustaining ring neuron activity during the trace interval of trace conditioning.
Haussmann, I. U., Wu, Y., Nallasivan, M. P., Archer, N., Bodi, Z., Hebenstreit, D., Waddell, S., Fray, R. and Soller, M. (2022). CMTr cap-adjacent 2'-O-ribose mRNA methyltransferases are required for reward learning and mRNA localization to synapses. Nat Commun 13(1): 1209. PubMed ID: 35260552
Cap-adjacent nucleotides of animal, protist and viral mRNAs can be O-methylated at the 2' position of the ribose (cOMe). The functions of cOMe in animals, however, remain largely unknown. This study shows that the two cap methyltransferases (CMTr1 and CMTr2) of Drosophila can methylate the ribose of the first nucleotide in mRNA. Double-mutant flies lack cOMe but are viable. Consistent with prominent neuronal expression, they have a reward learning defect that can be rescued by conditional expression in mushroom body neurons before training. Among CMTr targets are cell adhesion and signaling molecules. Many are relevant for learning, and are also targets of Fragile X Mental Retardation Protein (FMRP). Like FMRP, cOMe is required for localization of untranslated mRNAs to synapses and enhances binding of the cap binding complex in the nucleus. Hence, thie study reveals a mechanism to co-transcriptionally prime mRNAs by cOMe for localized protein synthesis at synapses.
Jang, S., Choi, B., Lim, C., Lee, B. and Cho, K. S. (2022).. Roles of Drosophila fatty acid-binding protein in development and behavior. Biochem Biophys Res Commun 599: 87-92. PubMed ID: 35176630
Fatty acid-binding proteins (FABPs) are lipid chaperones that mediate the intracellular dynamics of the hydrophobic molecules that they physically bind to. FABPs are implicated in sleep and psychiatric disorders, as well as in various cellular processes, such as cell proliferation and survival. FABP is well conserved in insects, and Drosophila has one FABP ortholog, dFabp, in its genome. Although dFabp appears to be evolutionarily conserved in some brain functions, little is known about its development and physiological function. This study investigated the function of dFabp in Drosophila development and behavior. Knockdown or overexpression of dFabp in the developing brain, wing, and eye resulted in developmental defects, such as decreased survival, altered cell proliferation, and increased apoptosis. Glia-specific knockdown of dFabp affected neuronal development, and neuronal regulation of dFabp affected glial cell proliferation. Moreover, the behavioral phenotypes (circadian rhythm and locomotor activity) of flies with regulated dFabp expression in glia and flies with regulated dFabp expression in neurons were very similar. Collectively, these results suggest that dFabp is involved in the development of various tissues and brain functions to control behavior and is a mediator of neuron-glia interactions in the Drosophila nervous system.
Han, Y., Peng, L. and Wang, T. (2022). Tadr is an axonal histidine transporter required for visual neurotransmission in Drosophila. Elife 11. PubMed ID: 35229720
Neurotransmitters are generated by de novo synthesis and are essential for sustained, high-frequency synaptic transmission. Histamine, a monoamine neurotransmitter, is synthesized through decarboxylation of histidine by histidine decarboxylase (Hdc). However, little is known about how histidine is presented to Hdc as a precursor. This study identified a specific histidine transporter, TADR (torn and diminished rhabdomeres), which is required for visual transmission in Drosophila. Both TADR and Hdc localized to neuronal terminals, and mutations in tadr reduced levels of histamine, thus disrupting visual synaptic transmission and phototaxis behavior. These results demonstrate that a specific amino acid transporter provides precursors for monoamine neurotransmitters, providing the first genetic evidence that a histidine amino acid transporter plays a critical role in synaptic transmission. These results suggest that TADR-dependent local de novo synthesis of histamine is required for synaptic transmission.
Groschner, L. N., Malis, J. G., Zuidinga, B. and Borst, A. (2022). A biophysical account of multiplication by a single neuron. Nature 603(7899): 119-123. PubMed ID: 35197635
Nonlinear, multiplication-like operations carried out by individual nerve cells greatly enhance the computational power of a neural system, but understanding of their biophysical implementation is scant. This study pursued this problem in the Drosophila melanogaster ON motion vision circuit, in which the membrane potentials of direction-selective T4 neurons and of their columnar input elements were recorded in response to visual and pharmacological stimuli in vivo. Electrophysiological measurements and conductance-based simulations provide evidence for a passive supralinear interaction between two distinct types of synapse on T4 dendrites. This multiplication-like nonlinearity arises from the coincidence of cholinergic excitation and release from glutamatergic inhibition. The latter depends on the expression of the glutamate-gated chloride channel GluClα in T4 neurons, which sharpens the directional tuning of the cells and shapes the optomotor behaviour of the animals. Interacting pairs of shunting inhibitory and excitatory synapses have long been postulated as an analogue approximation of a multiplication, which is integral to theories of motion detection, sound localization and sensorimotor control.

Monday, May 16 - Behavior

Iyengar, A. S., Kulkarni, R. and Sheeba, V. (2022). Under warm ambient conditions, Drosophila melanogaster suppresses nighttime activity via the neuropeptide pigment dispersing factor. Genes Brain Behav 21(4): e12802. PubMed ID: 35285135
Rhythmic locomotor behaviour of flies is controlled by an endogenous time-keeping mechanism, the circadian clock, and is influenced by environmental temperatures. Flies inherently prefer cool temperatures around 25°C, and under such conditions, time their locomotor activity to occur at dawn and dusk. Under relatively warmer conditions such as 30°C, flies shift their activity into the night, advancing their morning activity bout into the early morning, before lights-ON, and delaying their evening activity into early night. The molecular basis for such temperature-dependent behavioural modulation has been associated with core circadian clock genes, but the neuronal basis is not yet clear. Under relatively cool temperatures such as 25°C, the role of the circadian pacemaker ventrolateral neurons (LNvs), along with a major neuropeptide secreted by them, pigment dispersing factor (PDF), has been showed in regulating various aspects of locomotor activity rhythms. However, the role of the LNvs and PDF in warm temperature-mediated behavioural modulation has not been explored. This study shows that flies lacking proper PDF signalling or the LNvs altogether, cannot suppress their locomotor activity resulting in loss of sleep during the middle of the night, and thus describe a novel role for PDF signalling and the LNvs in behavioural modulation under warm ambient conditions. In a rapidly warming world, such behavioural plasticity may enable organisms to respond to harsh temperatures in the environment.
Eick, A. K., Ogueta, M., Buhl, E., Hodge, J. J. L. and Stanewsky, R. (2022). The opposing chloride cotransporters KCC and NKCC control locomotor activity in constant light and during long days. Curr Biol 32(6): 1420-1428.e1424. PubMed ID: 35303416
Cation chloride cotransporters (CCCs) regulate intracellular chloride ion concentration ([Cl(-)](i)) within neurons, which can reverse the direction of the neuronal response to the neurotransmitter GABA. Na(+) K(+) Cl(-) (NKCC) and K(+) Cl(-) (KCC) cotransporters transport Cl(-) into or out of the cell, respectively. When NKCC activity dominates, the resulting high [Cl(-)](i) can lead to an excitatory and depolarizing response of the neuron upon GABA(A) receptor opening, while KCC dominance has the opposite effect. This inhibitory-to-excitatory GABA switch has been linked to seasonal adaption of circadian clock function to changing day length, and its dysregulation is associated with neurodevelopmental disorders such as epilepsy. In Drosophila melanogaster, constant light normally disrupts circadian clock function and leads to arrhythmic behavior. This study demonstrates a function for CCCs in regulating Drosophila locomotor activity and GABA responses in circadian clock neurons because alteration of CCC expression in circadian clock neurons elicits rhythmic behavior in constant light. The same effects were observed after downregulation of the Wnk and Fray kinases, which modulate CCC activity in a [Cl(-)](i)-dependent manner. Patch-clamp recordings from the large LNv clock neurons show that downregulation of KCC results in a more positive GABA reversal potential, while KCC overexpression has the opposite effect. Finally, KCC and NKCC downregulation reduces or increases morning behavioral activity during long photoperiods, respectively. In summary, these results support a model in which the regulation of [Cl(-)](i) by a KCC/NKCC/Wnk/Fray feedback loop determines the response of clock neurons to GABA, which is important for adjusting behavioral activity to constant light and long-day conditions.
Dweck, H. K. M., Talross, G. J. S., Luo, Y., Ebrahim, S. A. M. and Carlson, J. R. (2022). Ir56b is an atypical ionotropic receptor that underlies appetitive salt response in Drosophila. Curr Biol. PubMed ID: 35294865
Salt taste is one of the most ancient of all sensory modalities. However, the molecular basis of salt taste remains unclear in invertebrates. This study shows that the response to low, appetitive salt concentrations in Drosophila depends on Ir56b, an atypical member of the ionotropic receptor (Ir) family. Ir56b acts in concert with two coreceptors, Ir25a and Ir76b. Mutation of Ir56b virtually eliminates an appetitive behavioral response to salt. Ir56b is expressed in neurons that also sense sugars via members of the Gr (gustatory receptor) family. Misexpression of Ir56b in bitter-sensing neurons confers physiological responses to appetitive doses of salt. Ir56b is unique among tuning Irs in containing virtually no N-terminal region, a feature that is evolutionarily conserved. Moreover, Ir56b is a "pseudo-pseudogene": its coding sequence contains a premature stop codon that can be replaced with a sense codon without loss of function. This stop codon is conserved among many Drosophila species but is absent in a number of species associated with cactus in arid regions. Thus, Ir56b serves the evolutionarily ancient function of salt detection in neurons that underlie both salt and sweet taste modalities.
Duckhorn, J. C., Cande, J., Metkus, M. C., Song, H., Altamirano, S., Stern, D. L. and Shirangi, T. R. (2022). Regulation of Drosophila courtship behavior by the Tlx/tailless-like nuclear receptor, dissatisfaction.. Curr Biol. PubMed ID: 35245457
Sexually dimorphic courtship behaviors in Drosophila melanogaster develop from the activity of the sexual differentiation genes, doublesex (dsx) and fruitless (fru), functioning with other regulatory factors that have received little attention. The dissatisfaction (dsf) gene encodes an orphan nuclear receptor homologous to vertebrate Tlx and Drosophila tailless that is critical for the development of several aspects of female- and male-specific sexual behaviors. This study reports the pattern of dsf expression in the central nervous system and show that the activity of sexually dimorphic abdominal interneurons that co-express dsf and dsx is necessary and sufficient for vaginal plate opening in virgin females, ovipositor extrusion in mated females, and abdominal curling in males during courtship. dsf activity results in different neuroanatomical outcomes in females and males, promoting and suppressing, respectively, female development and function of these neurons depending upon the sexual state of dsx expression. It is positted that dsf and dsx interact to specify sex differences in the neural circuitry for dimorphic abdominal behaviors.
Meda, N., Menti, G. M., Megighian, A. and Zordan, M. A. (2022). A heuristic underlies the search for relief in Drosophila melanogaster. Ann N Y Acad Sci 1510(1): 158-166. PubMed ID: 34928521
Humans rely on multiple types of sensory information to make decisions, and strategies that shorten decision-making time by taking into account fewer but essential elements of information are preferred to strategies that require complex analyses. Such shortcuts to decision making are known as heuristics. The identification of heuristic principles in species phylogenetically distant to humans would shed light on the evolutionary origin of speed-accuracy trade-offs and offer the possibility for investigating the brain representations of such trade-offs, urgency and uncertainty. By performing experiments on spatial learning in the invertebrate Drosophila melanogaster, this study showed that the fly's search strategies conform to a spatial heuristic-the nearest neighbor rule-to avoid bitter taste (a negative stimulation). That is, Drosophila visits a salient location closest to its current position to stop the negative stimulation; only if this strategy proves unsuccessful does the fly use other learned associations to avoid bitter taste. Characterizing a heuristic in D. melanogaster supports the view that invertebrates can, when making choices, operate on economic principles, as well as the conclusion that heuristic decision making dates to at least 600 million years ago.
Dhar, G., Paikra, S. K. and Mishra, M. (2022). Aminoglycoside treatment alters hearing-related genes and depicts behavioral defects in Drosophila. Arch Insect Biochem Physiol 110(1): e21871. PubMed ID: 35150449
The hearing organ of Drosophila is present within the second segment of antennae. The hearing organ of Drosophila (Johnston's organ [JO]) shares much structural, developmental, and functional similarity with the vertebrate hearing organ (Organ of Corti). JO is evolving as a potential model system to examine the hearing-associated defects in vertebrates. In the vertebrates, aminoglycosides like gentamicin, kanamycin, and neomycin have been known to cause defects in the hearing organ. However, a complete mechanism of toxicity is not known. Taking the evolutionary conservation into account the current study aims to test various concentrations of aminoglycoside on the model organism, Drosophila melanogaster. The current study uses the oral route to check the toxicity of various aminoglycosides at different concentrations (50, 100, 150, 200, and 250 μg ml(-) (1) ). In Drosophila, many foreign particles enter the body through the gut via food. The aminoglycoside treated third instar larvae show defective crawling and sound avoidance behavior. The adult flies release lower amounts of acetylcholine esterase and higher amounts of reactive oxygen species than control untreated animals, accompanied by defective climbing and aggressive behavior. All these behavioral defects are further confirmed by the altered expression level of hearing genes such as nompC, inactive, nanchung, pyrexia. All the behavioral and genetic defects are reported as a readout of aminoglycoside toxicity.

Friday, May 13th - Methods

Marr, E. and Potter, C. J. (2021). Base Editing of Somatic Cells Using CRISPR-Cas9 in Drosophila. Crispr j 4(6): 836-845. PubMed ID: 34813372
Cas9 and a guide RNA (gRNA) function to target specific genomic loci for generation of a double-stranded break. Catalytic dead versions of Cas9 (dCas9) no longer cause double-stranded breaks and instead can serve as molecular scaffolds to target additional enzymatic proteins to specific genomic loci. To generate mutations in selected genomic residues, dCas9 can be used for genomic base editing by fusing a cytidine deaminase (CD) to induce C > T (or G>A) mutations at targeted sites. This study tested base editing in Drosophila by expressing a transgenic Drosophila base editor (based on the mammalian BE2) that consists of a fusion protein of CD, dCas9, and uracil glycosylase inhibitor. Transgenic lines expressing gRNAs were used along with pan-tissue expression of the Drosophila base editor (Actin5C-BE2) and found high rates of base editing at multiple targeted loci in the 20 bp target sequence. Highest rates of conversion of C > T were found in positions 3-9 of the gRNA-targeted site, with conversion reaching ∼100% of targeted DNA in somatic tissues. Surprisingly, the simultaneous use of two gRNAs targeting a genomic region spaced ∼50 bp apart led to mutations between the two gRNA targets, implicating a method to broaden the available sites accessible to targeting. These results indicate base editing is efficient in Drosophila, and could be used to induce point mutations at select loci.
Kilwein, M. D. and Welte, M. A. (2021). Visualizing Cytoskeleton-Dependent Trafficking of Lipid-Containing Organelles in Drosophila Embryos. J Vis Exp(178). PubMed ID: 34958089
Early Drosophila embryos are large cells containing a vast array of conventional and embryo-specific organelles. During the first three hours of embryogenesis, these organelles undergo dramatic movements powered by actin-based cytoplasmic streaming and motor-driven trafficking along microtubules. The development of a multitude of small, organelle-specific fluorescent probes (FPs) makes it possible to visualize a wide range of different lipid-containing structures in any genotype, allowing live imaging without requiring a genetically encoded fluorophore. This protocol shows how to inject vital dyes and molecular probes into Drosophila embryos to monitor the trafficking of specific organelles by live imaging. This approach is demonstrated by labeling lipid droplets (LDs) and following their bulk movement by particle image velocimetry (PIV). This protocol provides a strategy amenable to the study of other organelles, including lysosomes, mitochondria, yolk vesicles, and the ER, and for tracking the motion of individual LDs along microtubules. Using commercially available dyes brings the benefits of separation into the violet/blue and far-red regions of the spectrum. By multiplex co-labeling of organelles and/or cytoskeletal elements via microinjection, all the genetic resources in Drosophila are available for trafficking studies without the need to introduce fluorescently tagged proteins. Unlike genetically encoded fluorophores, which have low quantum yields and bleach easily, many of the available dyes allow for rapid and simultaneous capture of several channels with high photon yields.
Sanchez-Mirasierra, I., Hernandez-Diaz, S., Ghimire, S., Montecinos-Oliva, C. and Soukup, S. F. (2021). Macros to Quantify Exosome Release and Autophagy at the Neuromuscular Junction of Drosophila Melanogaster. Front Cell Dev Biol 9: 773861. PubMed ID: 34869373
Automatic quantification of image parameters is a powerful and necessary tool to explore and analyze crucial cell biological processes. This article describes two ImageJ/Fiji automated macros to approach the analysis of synaptic autophagy and exosome release from 2D confocal images. Emerging studies point out that exosome biogenesis and autophagy share molecular and organelle components. Indeed, the crosstalk between these two processes may be relevant for brain physiology, neuronal development, and the onset/progression of neurodegenerative disorders. In this context, this study studied the macros "Autophagoquant" and "Exoquant" to assess the quantification of autophagosomes and exosomes at the neuronal presynapse of the Neuromuscular Junction (NMJ) in Drosophila melanogaster using confocal microscopy images. The Drosophila NMJ is a valuable model for the study of synapse biology, autophagy, and exosome release. By use of Autophagoquant and Exoquant, researchers can have an unbiased, standardized, and rapid tool to analyze autophagy and exosomal release in Drosophila NMJ.
Warecki, B., Bast, I. and Sullivan, W. (2022). Visualizing the Dynamics of Cell Division by Live Imaging Drosophila Larval Brain Squashes. Methods Mol Biol 2415: 37-46. PubMed ID: 34972944
The dramatic changes of subcellular structures during mitosis are best visualized by live imaging. In general, live imaging requires the expression of proteins of interest fused to fluorophores and a model system amenable to live microscopy. Drosophila melanogaster is an attractive model in which to perform live imaging because of the numerous transgenic stocks bearing fluorescently tagged transgenes as well as the ability to precisely manipulate gene expression. Traditionally, the early Drosophila embryo has been used for live fluorescent analysis of mitotic events such as spindle formation and chromosome segregation. More recent studies demonstrate that the Drosophila third instar neuroblasts have a number of properties that make them well suited for live analysis: (1) neuroblasts are distinct cells surrounded by plasma membranes; (2) neuroblasts undergo a complete cell cycle, consisting of G1, S, G2, and M phases; and (3) neuroblasts gene expression is not influenced by maternal load, and so the genetics are therefore relatively more simple. Finally, the Drosophila neuroblast is arguably the best system for live imaging asymmetric stem cell divisions. This paper details a method for live imaging Drosophila larval neuroblasts.
Felix, M. and Prasad, M. (2022). Mapping Asymmetry in Collective Cell Migration: Lessons from Border Cells in Drosophila Oogenesis. Methods Mol Biol 2438: 483-494. PubMed ID: 35147959
Asymmetry in the migrating group of cells is critical for efficient directed movement observed in normal development and in pathological conditions like tumor cell metastasis. This is conspicuously detected at the level of polarized protrusions and differential localization of various polarity proteins in collectively moving clusters. Over the years, border cell migration in Drosophila oogenesis has emerged as an excellent model system for studying polarity in the migrating group of cells. This paper reports two protocols employing live cell imaging and tissue immunohistochemistry to evaluate the polarity in migrating border cell clusters.
Liu, K., Deng, S., Ye, C., Yao, Z., Wang, J., Gong, H., Liu, L. and He, X. (2021). Mapping single-cell-resolution cell phylogeny reveals cell population dynamics during organ development. Nat Methods 18(12): 1506-1514. PubMed ID: 34857936
Mapping the cell phylogeny of a complex multicellular organism relies on somatic mutations accumulated from zygote to adult. Available cell barcoding methods can record about three mutations per barcode, enabling only low-resolution mapping of the cell phylogeny of complex organisms. This study developed SMALT, a substitution mutation-aided lineage-tracing system that outperforms the available cell barcoding methods in mapping cell phylogeny. This study applied SMALT to Drosophila melanogaster and obtained on average more than 20 mutations on a three-kilobase-pair barcoding sequence in early-adult cells. The obtained cell phylogenies enabled a population genetic analysis that estimates the longitudinal dynamics of the number of actively dividing parental cells (Np) in each organ through development. The Np dynamics revealed the trajectory of cell births and provided insight into the balance of symmetric and asymmetric cell division.

Thursday, May 12 - Gonad

Martin, E. T., Blatt, P., Nguyen, E., Lahr, R., Selvam, S., Yoon, H. A. M., Pocchiari, T., Emtenani, S., Siekhaus, D. E., Berman, A., Fuchs, G. and Rangan, P. (2022). A translation control module coordinates germline stem cell differentiation with ribosome biogenesis during Drosophila oogenesis. Dev Cell 57(7): 883-900. PubMed ID: 35413237
Ribosomal defects perturb stem cell differentiation, and this is the cause of ribosomopathies. How ribosome levels control stem cell differentiation is not fully known. This study discovered that three DExD/H-box proteins govern ribosome biogenesis (RiBi) and Drosophila oogenesis. Loss of these DExD/H-box proteins, which were named Aramis, Athos, and Porthos, aberrantly stabilizes p53, arrests the cell cycle, and stalls germline stem cell (GSC) differentiation. Aramis controls cell-cycle progression by regulating translation of mRNAs that contain a terminal oligo pyrimidine (TOP) motif in their 5' UTRs. TOP motifs confer sensitivity to ribosome levels that are mediated by La-related protein (Larp). One such TOP-containing mRNA codes for novel nucleolar protein 1 (Non1), a conserved p53 destabilizing protein. Upon a sufficient ribosome concentration, Non1 is expressed, and it promotes GSC cell-cycle progression via p53 degradation. Thus, a previously unappreciated TOP motif in Drosophila responds to reduced RiBi to co-regulate the translation of ribosomal proteins and a p53 repressor, coupling RiBi to GSC differentiation.
Gandara, A. C. P. and Drummond-Barbosa, D. (2022). Warm and cold temperatures have distinct germline stem cell lineage effects during Drosophila oogenesis. Development 149(5). PubMed ID: 35156684
Despite their medical and economic relevance, it remains largely unknown how suboptimal temperatures affect adult insect reproduction. This study reports an in-depth analysis of how chronic adult exposure to suboptimal temperatures affects oogenesis using the model insect Drosophila melanogaster. In adult females maintained at 18°C (cold) or 29°C (warm), relative to females at the 25°C control temperature, egg production was reduced through distinct cellular mechanisms. Chronic 18°C exposure improved germline stem cell maintenance, survival of early germline cysts and oocyte quality, but reduced follicle growth with no obvious effect on vitellogenesis. By contrast, in females at 29°C, germline stem cell numbers and follicle growth were similar to those at 25°C, while early germline cyst death and degeneration of vitellogenic follicles were markedly increased and oocyte quality plummeted over time. Finally, this study also showed that these effects are largely independent of diet, male factors or canonical temperature sensors. These findings are relevant not only to cold-blooded organisms, which have limited thermoregulation, but also potentially to warm-blooded organisms, which are susceptible to hypothermia, heatstroke and fever.
Hayashi, Y., Kashio, S., Murotomi, K., Hino, S., Kang, W., Miyado, K., Nakao, M., Miura, M., Kobayashi, S. and Namihira, M. (2022). Biosynthesis of S-adenosyl-methionine enhances aging-related defects in Drosophila oogenesis. Sci Rep 12(1): 5593. PubMed ID: 35379840
Tissue aging is a major cause of aging-related disabilities and a shortened life span. Understanding how tissue aging progresses and identifying the factors underlying tissue aging are crucial; however, the mechanism of tissue aging is not fully understood. This study showed that the biosynthesis of S-adenosyl-methionine (SAM), the major cellular donor of methyl group for methylation modifications, potently accelerates the aging-related defects during Drosophila oogenesis. An aging-related increase in the SAM-synthetase (Sam-S) levels in the germline leads to an increase in ovarian SAM levels. Sam-S-dependent biosynthesis of SAM controls aging-related defects in oogenesis through two mechanisms, decreasing the ability to maintain germline stem cells and accelerating the improper formation of egg chambers. Aging-related increases in SAM commonly occur in mouse reproductive tissue and the brain. Therefore, these results raise the possibility suggesting that SAM is the factor related to tissue aging beyond the species and tissues.
Alsous, J. I., Rozman, J., Marmion, R. A., Kosmrlj, A. and Shvartsman, S. Y. (2021). Clonal dominance in excitable cell networks. Nat Phys 17(12): 1391-1395. PubMed ID: 35242199
Clonal dominance arises when the descendants (clones) of one or a few founder cells contribute disproportionally to the final structure during collective growth. In contexts such as bacterial growth, tumorigenesis, and stem cell reprogramming, this phenomenon is often attributed to pre-existing propensities for dominance, while in stem cell homeostasis, neutral drift dynamics are invoked. The mechanistic origin of clonal dominance during development, where it is increasingly documented, is less understood. This study investigated this phenomenon in the Drosophila melanogaster follicle epithelium, a system in which the joint growth dynamics of cell lineage trees can be reconstructed. This study demonstrated that clonal dominance can emerge spontaneously, in the absence of pre-existing biases, as a collective property of evolving excitable networks through coupling of divisions among connected cells. Similar mechanisms have been identified in forest fires and evolving opinion networks; the spatial coupling of excitable units explains a critical feature of the development of the organism, with implications for tissue organization and dynamics.
Bhaskar, P. K., Southard, S., Baxter, K. and Van Doren, M. (2022). Germline sex determination regulates sex-specific signaling between germline stem cells and their niche. Cell Rep 39(1): 110620. PubMed ID: 35385723
Establishing germ cell sexual identity is critical for development of male and female germline stem cells (GSCs) and production of sperm or eggs. Germ cells depend on signals from the somatic gonad to determine sex, but in organisms such as flies, mice, and humans, the sex chromosome genotype of the germ cells is also important for germline sexual development. How somatic signals and germ-cell-intrinsic cues combine to regulate germline sex determination is thus a key question. This study found that JAK/STAT signaling in the GSC niche promotes male identity in germ cells, in part by activating the chromatin reader Phf7. Further, it was found that JAK/STAT signaling is blocked in XX (female) germ cells through the action of the sex determination gene Sex lethal to preserve female identity. Thus, an important function of germline sexual identity is to control how GSCs respond to signals in their niche environment.
To, V., Kim, H. J., Jang, W., Sreejith, P. and Kim, C. (2021). Lin28 and Imp are Required for Stability of Bowl Transcripts in Hub Cells of the Drosophila Testis. Dev Reprod 25(4): 313-319. PubMed ID: 35141457
Hub cells comprise a niche for germline stem cells and cyst stem cells in the Drosophila testis. Hub cells arise from common somatic gonadal precursors in embryos, but the mechanism of their specification is still poorly understood. This study found that RNA binding proteins Lin28 and Imp mediate transcript stability of Bowl, a known hub specification factor; Bowl transcripts were reduced in the testis of Lin28 and Imp mutants, and also when RNA-mediated interference against Lin28 or Imp was expressed in hub cells. In tissue culture Luciferase assays involving the Bowl 3'UTR, stability of Luc reporter transcripts depended on the Bowl 3'UTR and required Lin28 and Imp. These findings suggest that proper Bowl function during hub cell specification requires Lin28 and Imp in the testis hub cells.

Wednesday, May 11th - Disease Models

Chi, W., Iyengar, A. S. R., Fu, W., Liu, W., Berg, A. E., Wu, C. F. and Zhuang, X. (2022). Drosophila carrying epilepsy-associated variants in the vitamin B6 metabolism gene PNPO display allele- and diet-dependent phenotypes. Proc Natl Acad Sci U S A 119(9). PubMed ID: 35217610
Pyridox(am)ine 5 (') -phosphate oxidase (PNPO) catalyzes the rate-limiting step in the synthesis of pyridoxal 5 (') -phosphate (PLP), the active form of vitamin B6 required for the synthesis of neurotransmitters gamma-aminobutyric acid (GABA) and the monoamines. Pathogenic variants in PNPO have been increasingly identified in patients with neonatal epileptic encephalopathy and early-onset epilepsy. These patients often exhibit different types of seizures and variable comorbidities. Recently, the PNPO gene has also been implicated in epilepsy in adults. It is unclear how these phenotypic variations are linked to specific PNPO alleles and to what degree diet can modify their expression. Using CRISPR-Cas9, four knock-in Drosophila alleles, h(WT) , h(R) (116) (Q) , h(D) (33) (V) , and h(R) (95) (H) were generated, in which the endogenous Drosophila PNPO (sugarlethal) was replaced by wild-type human PNPO complementary DNA (cDNA) and three epilepsy-associated variants. These knock-in flies exhibited a wide range of phenotypes, including developmental impairments, abnormal locomotor activities, spontaneous seizures, and shortened life span. These phenotypes are allele dependent, varying with the known biochemical severity of these mutations and characterized molecular defects. Diet treatments further diversified the phenotypes among alleles, and PLP supplementation at larval and adult stages prevented developmental impairments and seizures in adult flies, respectively. Furthermore, it was found that h(R95H) had a significant dominant-negative effect, rendering heterozygous flies susceptible to seizures and premature death. Together, these results provide biological bases for the various phenotypes resulting from multifunction of PNPO, specific molecular and/or genetic properties of each PNPO variant, and differential allele-diet interactions.
Chung, H. L., Rump, P., Lu, D., Glassford, M. R., Mok, J. W., Fatih, J., Basal, A., Marcogliese, P. C., Kanca, O., Rapp, M., Fock, J. M., Kamsteeg, E. J., Lupski, J. R., Larson, A., Haninbal, M. C., Bellen, H. and Harel, T. (2022). De novo variants in EMC1 lead to neurodevelopmental delay and cerebellar degeneration and affect glial function in Drosophila. Hum Mol Genet. PubMed ID: 35234901
The endoplasmic reticulum (ER)-membrane protein complex (EMC) is a multi-protein transmembrane complex composed of 10 subunits that functions as a membrane-protein chaperone. Variants in EMC1 lead to neurodevelopmental delay and cerebellar degeneration. Multiple families with biallelic variants have been published, yet to date, only a single report of a monoallelic variant has been described, and functional evidence is sparse. Exome sequencing was used to investigate the genetic cause underlying severe developmental delay in three unrelated children. EMC1 variants were modeled in Drosophila, using loss-of-function (LoF) and overexpression studies. Glial-specific and neuronal-specific assays were used to determine whether the dysfunction was specific to one cell type. Exome sequencing identified de novo variants in EMC1 in three individuals affected by global developmental delay, hypotonia, seizures, visual impairment, and cerebellar atrophy. All variants were located at Pro582 or Pro584. Drosophila studies indicated that imbalance of EMC1-either overexpression or knockdown-results in pupal lethality and suggest that the tested homologous variants are LoF alleles. In addition, glia-specific gene dosage, overexpression or knockdown, of EMC1 led to lethality, whereas neuron-specific alterations were tolerated. This study established de novo monoallelic EMC1 variants as causative of a neurological disease trait by providing functional evidence in a Drosophila model. The identified variants failed to rescue the lethality of a null allele. Variations in dosage of the wild-type EMC1, specifically in glia, lead to pupal lethality, which is hypothesized to result from the altered stoichiometry of the multi-subunit protein complex EMC.
Bierzynska, A., Bull, K., Miellet, S., Dean, P., Neal, C., Colby, E., McCarthy, H. J., Hegde, S., Sinha, M. D., Bugarin Diz, C., Stirrups, K., Megy, K., Mapeta, R., Penkett, C., Marsh, S., Forrester, N., Afzal, M., Stark, H., BioResource, N., Williams, M., Welsh, G. I., Koziell, A. B., Hartley, P. S. and Saleem, M. A. (2022). Exploring the relevance of NUP93 variants in steroid-resistant nephrotic syndrome using next generation sequencing and a fly kidney model. Pediatr Nephrol. PubMed ID: 35211795
Variants in genes encoding nuclear pore complex (NPC) proteins are a newly identified cause of paediatric steroid-resistant nephrotic syndrome (SRNS). Recent reports describing NUP93 variants suggest these could be a significant cause of paediatric onset SRNS. This study report NUP93 cases in the UK and demonstrate in vivo functional effects of Nup93 depletion in a fly (Drosophila melanogaster) nephrocyte model. Three hundred thirty-seven paediatric SRNS patients from the National cohort of patients with Nephrotic Syndrome (NephroS) were whole exome and/or whole genome sequenced. Patients were screened for over 70 genes known to be associated with Nephrotic Syndrome (NS). D. melanogaster Nup93 knockdown was achieved by RNA interference using nephrocyte-restricted drivers. Six novel homozygous and compound heterozygous NUP93 variants were detected in 3 sporadic and 2 familial paediatric onset SRNS characterised histologically by focal segmental glomerulosclerosis (FSGS) and progressing to kidney failure by 12 months from clinical diagnosis. Silencing of the two orthologs of human NUP93 expressed in D. melanogaster, Nup93-1, and Nup93-2 resulted in significant signal reduction of up to 82% in adult pericardial nephrocytes with concomitant disruption of NPC protein expression. Additionally, nephrocyte morphology was highly abnormal in Nup93-1 and Nup93-2 silenced flies surviving to adulthood. This study expanded the spectrum of NUP93 variants detected in paediatric onset SRNS and demonstrate its incidence within a national cohort. Silencing of either D. melanogaster Nup93 ortholog caused a severe nephrocyte phenotype, signaling an important role for the nucleoporin complex in podocyte biology.
Chaouhan, H. S., Li, X., Sun, K. T., Wang, I. K., Yu, T. M., Yu, S. H., Chen, K. B., Lin, W. Y. and Li, C. Y. (2022). Calycosin Alleviates Paraquat-Induced Neurodegeneration by Improving Mitochondrial Functions and Regulating Autophagy in a Drosophila Model of Parkinson's Disease. Antioxidants (Basel) 11(2). PubMed ID: 35204105
Parkinson's disease (PD) is the second most common age-related neurodegenerative disorder with limited clinical treatments. The occurrence of PD includes both genetic and environmental toxins, such as the pesticides paraquat (PQ), as major contributors to PD pathology in both invertebrate and mammalian models. Calycosin, an isoflavone phytoestrogen, has multiple pharmacological properties, including neuroprotective activity. However, the paucity of information regarding the neuroprotective potential of calycosin on PQ-induced neurodegeneration led to an exploration of whether calycosin can mitigate PD-like phenotypes and the underlying molecular mechanisms. A PQ-induced PD model in Drosophila was used as a cost-effective in vivo screening platform to investigate the neuroprotective efficacy of natural compounds on PD. Calycosin showed a protective role in preventing dopaminergic (DA) neuronal cell death in PQ-exposed Canton S flies. Calycosin-fed PQ-exposed flies exhibit significant resistance against PQ-induced mortality and locomotor deficits in terms of reduced oxidative stress, loss of DA neurons, the depletion of dopamine content, and phosphorylated JNK-caspase-3 levels. Additionally, mechanistic studies show that calycosin administration improves PQ-induced mitochondrial dysfunction and stimulates mitophagy and general autophagy with reduced pS6K and p4EBP1 levels, suggestive of a maintained energy balance between anabolic and catabolic processes, resulting in the inhibition of neuronal cell death. Collectively, this study substantiates the protective effect of calycosin against PQ-induced neurodegeneration by improving DA neurons' survival and reducing apoptosis, likely via autophagy induction, and it is implicated as a novel therapeutic application against toxin-induced PD pathogenesis.
Baenas, N. and Wagner, A. E. (2022). Drosophila melanogaster as a Model Organism for Obesity and Type-2 Diabetes Mellitus by Applying High-Sugar and High-Fat Diets. Biomolecules 12(2). PubMed ID: 35204807
Several studies have been published introducing Drosophila melanogaster as a research model to investigate the effects of high-calorie diets on metabolic dysfunctions. However, differences between the use of high-sugar diets (HSD) and high-fat diets (HFD) to affect fly physiology, as well as the influence on sex and age, have been seldom described. Thus, the aim of the present work was to investigate and compare the effects of HSD (30% sucrose) and HFD (15% coconut oil) on symptoms of metabolic dysfunction related to obesity and type-2 diabetes mellitus, including weight gain, survival, climbing ability, glucose and triglycerides accumulation and expression levels of Drosophila insulin-like peptides (dIlps). Female and male flies were subjected to HSD and HFD for 10, 20 and 30 days. The obtained results showed clear differences in the effects of both diets on survival, glucose and triglyceride accumulation and dIlps expression, being gender and age determinant. The present study also suggested that weight gain does not seem to be an appropriate parameter to define fly obesity, since other characteristics appear to be more meaningful in the development of obesity phenotypes. Taken together, the results demonstrate a key role for both diets, HSD and HFD, to induce an obese fly phenotype with associated diseases. However, further studies are needed to elucidate the underlying molecular mechanisms how both diets differently affect fly metabolism.
Cha, S. J., Lee, S., Choi, H. J., Han, Y. J., Jeon, Y. M., Jo, M., Lee, S., Nahm, M., Lim, S. M., Kim, S. H., Kim, H. J. and Kim, K. (2022). Therapeutic modulation of GSTO activity rescues FUS-associated neurotoxicity via deglutathionylation in ALS disease models. Dev Cell 57(6): 783-798. PubMed ID: 35320731
Fused in sarcoma (FUS) is a DNA/RNA-binding protein that is involved in DNA repair and RNA processing. FUS is associated with neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). However, the molecular mechanisms underlying FUS-mediated neurodegeneration are largely unknown. Using a Drosophila model, this study showed that the overexpression of glutathione transferase omega 2 (GstO2) reduces cytoplasmic FUS aggregates and prevents neurodegenerative phenotypes, including neurotoxicity and mitochondrial dysfunction. A FUS glutathionylation site was found at the 447(th) cysteine residue in the RanBP2-type ZnF domain. The glutathionylation of FUS induces FUS aggregation by promoting phase separation. GstO2 reduced cytoplasmic FUS aggregation by deglutathionylation in Drosophila brains. Moreover, this study demonstrated that the overexpression of human GSTO1, the homolog of Drosophila GstO2, attenuates FUS-induced neurotoxicity and cytoplasmic FUS accumulation in mouse neuronal cells. Thus, the modulation of FUS glutathionylation might be a promising therapeutic strategy for FUS-associated neurodegenerative diseases.

Tuesday, May 10th - Evolution

Deppisch, P., Prutscher, J. M., Pegoraro, M., Tauber, E., Wegener, C. and Helfrich-Forster, C. (2022). Adaptation of Drosophila melanogaster to Long Photoperiods of High-Latitude Summers Is Facilitated by the ls-Timeless Allele. J Biol Rhythms 37(2): 185-201. PubMed ID: 35301885
Circadian clocks help animals to be active at the optimal time of the day whereby for most species the daily light-dark cycle is the most important zeitgeber for their circadian clock. In this respect, long arctic summer days are particularly challenging as light is present almost 24 h per day, and continuous light makes the circadian clocks of many animals arrhythmic. This is especially true for the fruit fly, Drosophila melanogaster, which possesses a very light-sensitive clock. The blue-light photoreceptor Cryptochrome (CRY) and the clock protein Timeless (TIM) are the light-sensitive components of the circadian clock and are responsible for constant light-induced arrhythmicity even at very low light intensities. Nevertheless, D. melanogaster was able to spread from its tropical origin and invade northern latitudes. This study tested whether a natural polymorphism at the timeless (tim) locus, s-tim and ls-tim, helped adaptation to very long photoperiods. The recently evolved natural allele, ls-tim, encodes a longer, less light sensitive form of TIM (L-TIM) in addition to the shorter (S-TIM) form, the only form encoded by the ancient s-tim allele. ls-tim has evolved in southeastern Italy and slowly spread to higher latitudes. L-TIM is known to interact less efficiently with CRY as compared with S-TIM. The locomotor activity patterns of ~40 wild s-tim and ls-tim isofemale lines caught at different latitudes was measured under simulated high-latitude summer light conditions (continuous light or long photoperiods with 20-h daily light). It was found that the ls-tim lines were significantly more rhythmic under continuous light than the s-tim lines. Importantly, the ls-tim lines can delay their evening activity under long photoperiods, a behavioral adaptation that appears to be optimal under high-latitude conditions. These observations suggest that the functional gain associated with ls-tim may drive the northern spread of this allele by directional selection.
Li, F., Rane, R. V., Luria, V., Xiong, Z., Chen, J., Li, Z., Catullo, R. A., Griffin, P. C., Schiffer, M., Pearce, S., Lee, S. F., McElroy, K., Stocker, A., Shirriffs, J., Cockerell, F., Coppin, C., Sgro, C. M., Karger, A., Cain, J. W., Weber, J. A., Santpere, G., Kirschner, M. W., Hoffmann, A. A., Oakeshott, J. G. and Zhang, G. (2022). Phylogenomic analyses of the genus Drosophila reveals genomic signals of climate adaptation. Mol Ecol Resour 22(4): 1559-1581. PubMed ID: 34839580
Many Drosophila species differ widely in their distributions and climate niches, making them excellent subjects for evolutionary genomic studies. A database was developed of high-quality assemblies for 46 Drosophila species and one closely related Zaprionus. Fifteen of the genomes were newly sequenced, and 20 were improved with additional sequencing. New or improved annotations were generated for all 47 species, assisted by new transcriptomes for 19. Phylogenomic analyses of these data resolved several previously ambiguous relationships, especially in the melanogaster species group. However, it also revealed significant phylogenetic incongruence among genes, mainly in the form of incomplete lineage sorting in the subgenus Sophophora but also including asymmetric introgression in the subgenus Drosophila. Using the phylogeny as a framework and taking into account these incongruences, the data was screened for genome-wide signals of adaptation to different climatic niches. First, phylostratigraphy revealed relatively high rates of recent novel gene gain in three temperate pseudoobscura and five desert-adapted cactophilic mulleri subgroup species. Second, it was found differing ratios of nonsynonymous to synonymous substitutions in several hundred orthologues between climate generalists and specialists, with trends for significantly higher ratios for those in tropical and lower ratios for those in temperate-continental specialists respectively than those in the climate generalists. Finally, resequencing natural populations of 13 species revealed tropics-restricted species generally had smaller population sizes, lower genome diversity and more deleterious mutations than the more widespread species. It is concluded that adaptation to different climates in the genus Drosophila has been associated with large-scale and multifaceted genomic changes.
Zhang, D., Leng, L., Chen, C., Huang, J., Zhang, Y., Yuan, H., Ma, C., Chen, H. and Zhang, Y. E. (2021). Dosage sensitivity and exon shuffling shape the landscape of polymorphic duplicates in Drosophila and humans. Nat Ecol Evol. PubMed ID: 34969986
Despite polymorphic duplicate genes' importance for the early stages of duplicate gene evolution, they are less studied than old gene duplicates. Two essential questions thus remain poorly addressed: how does dosage sensitivity, imposed by stoichiometry in protein complexes or by X chromosome dosage compensation, affect the emergence of complete duplicate genes? Do introns facilitate intergenic and intragenic chimaerism as predicted by the theory of exon shuffling? This study analysed new data for Drosophila and public data for humans, to characterize polymorphic duplicate genes with respect to dosage, exon-intron structures and allele frequencies. It was found that complete duplicate genes are under dosage constraint induced by protein stoichiometry but potentially tolerated by X chromosome dosage compensation. It was also found that in the intron-rich human genome, gene fusions and intragenic duplications extensively use intronic breakpoints generating in-frame proteins, in accordance with the theory of exon shuffling. Finally, it was found that only a small proportion of complete or partial duplicates are at high frequencies, indicating the deleterious nature of dosage or gene structural changes. Altogether, this study demonstrates how mechanistic factors including dosage sensitivity and exon-intron structure shape the short-term functional consequences of gene duplication.
Dobler, R., Charette, M., Kaplan, K., Turnell, B. R. and Reinhardt, K. (2022). Divergent natural selection alters male sperm competition success in Drosophila melanogaster. Ecol Evol 12(2): e8567. PubMed ID: 35222953
Sexually selected traits may also be subject to non-sexual selection. If optimal trait values depend on environmental conditions, then "narrow sense" (i.e., non-sexual) natural selection can lead to local adaptation, with fitness in a certain environment being highest among individuals selected under that environment. Such adaptation can, in turn, drive ecological speciation via sexual selection. To date, most research on the effect of narrow-sense natural selection on sexually selected traits has focused on precopulatory measures like mating success. However, postcopulatory traits, such as sperm function, can also be under non-sexual selection, and have the potential to contribute to population divergence between different environments. This study investigated the effects of narrow-sense natural selection on male postcopulatory success in Drosophila melanogaster. Two extreme environments, low oxygen (10%, hypoxic) or high CO(2) (5%, hypercapnic) were chosen to detect small effects. The sperm defensive (P1) and offensive (P2) capabilities of selected and control males were measured in the corresponding selection environment and under control conditions. Overall, selection under hypoxia decreased both P1 and P2, while selection under hypercapnia had no effect. Surprisingly, P1 for both selected and control males was higher under both ambient hypoxia and ambient hypercapnia, compared to control conditions, while P2 was lower under hypoxia. Limited evidence was found for local adaptation: the positive environmental effect of hypoxia on P1 was greater in hypoxia-selected males than in controls. The implications of our findings for the evolution of postcopulatory traits in response to non-sexual and sexual selection.
Huang, Y., Lack, J. B., Hoppel, G. T. and Pool, J. E. (2022). Gene regulatory evolution in cold-adapted fly populations neutralizes plasticity and may undermine genetic canalization. Genome Biol Evol. PubMed ID: 35380655
The relationships between adaptive evolution, phenotypic plasticity, and canalization remain incompletely understood. Theoretical and empirical studies have made conflicting arguments on whether adaptive evolution may enhance or oppose the plastic response. Gene regulatory traits offer excellent potential to study the relationship between plasticity and adaptation, and they can now be studied at the transcriptomic level. This study took advantage of three closely-related pairs of natural populations of Drosophila melanogaster from contrasting thermal environments that reflect three separate instances of cold tolerance evolution. The transcriptome-wide plasticity in gene expression levels and alternative splicing (intron usage) were measured between warm and cold laboratory environments. Suspected adaptive changes in both gene expression and alternative splicing tended to neutralize the ancestral plastic response. Further, the hypothesis was tested that adaptive evolution can lead to decanalization of selected gene regulatory traits. Strong evidence was found that suspected adaptive gene expression (but not splicing) changes in cold-adapted populations are more vulnerable to the genetic perturbation of inbreeding than putatively neutral changes. Some evidence was found that these patterns may reflect a loss of genetic canalization accompanying adaptation, although other processes including hitchhiking recessive deleterious variants may contribute as well. These findings augment our understanding of genetic and environmental effects on gene regulation in the context of adaptive evolution.
Jin, B., Barbash, D. A. and Castillo, D. M. (2022). Divergent selection on behavioural and chemical traits between reproductively isolated populations of Drosophila melanogaster. J Evol Biol. PubMed ID: 35411988
Speciation is driven by traits that can act to prevent mating between nascent lineages, including male courtship and female preference for male traits. Mating barriers involving these traits evolve quickly because there is strong selection on males and females to maximize reproductive success, and the tight co-evolution of mating interactions can lead to rapid diversification of sexual behaviour. Populations of Drosophila melanogaster show strong asymmetrical reproductive isolation that is correlated with geographic origin. Using strains that capture natural variation in mating traits, two key questions were asked: which specific male traits are females selecting, and are these traits under divergent sexual selection? These questions have proven extremely challenging to answer, because even in closely related lineages males often differ in multiple traits related to mating behaviour. These questions were addressed by estimating selection gradients for male courtship and cuticular hydrocarbons for two different female genotypes. Specific behaviours and particular cuticular hydrocarbons were identified that are under divergent sexual selection and could potentially contribute to premating reproductive isolation. Additionally, it is reported that a subset of these traits are plastic; males adjust these traits based on the identity of the female genotype they interact with. These results suggest that even when male courtship is not fixed between lineages, ongoing selection can act on traits that are important for reproductive isolation.

Monday, May 9th - Larval and Adult Development

Courcoubetis, G., Xu, C., Nuzhdin, S. V. and Haas, S. (2022). Avalanches during epithelial tissue growth; Uniform Growth and a Drosophila eye disc model. PLoS Comput Biol 18(3): e1009952. PubMed ID: 35303738
Epithelial tissues constitute an exotic type of active matter with non-linear properties reminiscent of amorphous materials. In the context of a proliferating epithelium, modeled by the quasistatic vertex model, this study identified novel discrete tissue scale rearrangements, i.e. cellular rearrangement avalanches, which are a form of collective cell movement. During the avalanches, the vast majority of cells retain their neighbors, and the resulting cellular trajectories are radial in the periphery, a vortex in the core. After the onset of these avalanches, the epithelial area grows discontinuously. The avalanches are found to be stochastic, and their strength is correlated with the density of cells in the tissue. Overall, avalanches redistribute accumulated local spatial pressure along the tissue. Furthermore, the distribution of avalanche magnitudes is found to obey a power law, with an exponent consistent with sheer induced avalanches in amorphous materials. To understand the role of avalanches in organ development, epithelial growth of the Drosophila eye disc during the third instar was simulated using a computational model, which includes both chemical and mechanistic signaling. During the third instar, the morphogenetic furrow (MF), a ~10 cell wide wave of apical area constriction propagates through the epithelium. These simulations are used to understand the details of the growth process, the effect of the MF on the growth dynamics on the tissue scale, and to make predictions for experimental observations. The avalanches are found to depend on the strength of the apical constriction of cells in the MF, with a stronger apical constriction leading to less frequent and more pronounced avalanches. The results herein highlight the dependence of simulated tissue growth dynamics on relaxation timescales, and serve as a guide for in vitro experiments.
Davis, J. R., Ainslie, A. P., Williamson, J. J., Ferreira, A., Torres-Sanchez, A., Hoppe, A., Mangione, F., Smith, M. B., Martin-Blanco, E., Salbreux, G. and Tapon, N. (2022). ECM degradation in the Drosophila abdominal epidermis initiates tissue growth that ceases with rapid cell-cycle exit. Curr Biol 32(6): 1285-1300.e1284. PubMed ID: 35167804
During development, multicellular organisms undergo stereotypical patterns of tissue growth in space and time. How developmental growth is orchestrated remains unclear, largely due to the difficulty of observing and quantitating this process in a living organism. Drosophila histoblast nests are small clusters of progenitor epithelial cells that undergo extensive growth to give rise to the adult abdominal epidermis and are amenable to live imaging. Our quantitative analysis of histoblast proliferation and tissue mechanics reveals that tissue growth is driven by cell divisions initiated through basal extracellular matrix degradation by matrix metalloproteases secreted by the neighboring larval epidermal cells. Laser ablations and computational simulations show that tissue mechanical tension does not decrease as the histoblasts fill the abdominal epidermal surface. During tissue growth, the histoblasts display oscillatory cell division rates until growth termination occurs through the rapid emergence of G0/G1 arrested cells, rather than a gradual increase in cell-cycle time as observed in other systems such as the Drosophila wing and mouse postnatal epidermis. Different developing tissues can therefore achieve their final size using distinct growth termination strategies. Thus, adult abdominal epidermal development is characterized by changes in the tissue microenvironment and a rapid exit from the cell cycle.
Jang, S., Choi, B., Lim, C., Lee, B. and Cho, K. S. (2022). Roles of Drosophila fatty acid-binding protein in development and behavior. Biochem Biophys Res Commun 599: 87-92. PubMed ID: 35176630
Fatty acid-binding proteins (FABPs) are lipid chaperones that mediate the intracellular dynamics of the hydrophobic molecules that they physically bind to. FABPs are implicated in sleep and psychiatric disorders, as well as in various cellular processes, such as cell proliferation and survival. FABP is well conserved in insects, and Drosophila has one FABP ortholog, dFabp, in its genome. Although dFabp appears to be evolutionarily conserved in some brain functions, little is known about its development and physiological function. This study investigated the function of dFabp in Drosophila development and behavior. Knockdown or overexpression of dFabp in the developing brain, wing, and eye resulted in developmental defects, such as decreased survival, altered cell proliferation, and increased apoptosis. Glia-specific knockdown of dFabp affected neuronal development, and neuronal regulation of dFabp affected glial cell proliferation. Moreover, the behavioral phenotypes (circadian rhythm and locomotor activity) of flies with regulated dFabp expression in glia and flies with regulated dFabp expression in neurons were very similar. Collectively, these results suggest that dFabp is involved in the development of various tissues and brain functions to control behavior and is a mediator of neuron-glia interactions in the Drosophila nervous system.
Nikonova, E., Mukherjee, A., Kamble, K., Barz, C., Nongthomba, U. and Spletter, M. L. (2022). Rbfox1 is required for myofibril development and maintaining fiber type-specific isoform expression in Drosophila muscles. Life Sci Alliance 5(4). PubMed ID: 34996845
Protein isoform transitions confer muscle fibers with distinct properties and are regulated by differential transcription and alternative splicing. RNA-binding Fox protein 1 (Rbfox1) can affect both transcript levels and splicing, and is known to contribute to normal muscle development and physiology in vertebrates, although the detailed mechanisms remain obscure. This study reports that Rbfox1 contributes to the generation of adult muscle diversity in Drosophila Rbfox1 is differentially expressed among muscle fiber types, and RNAi knockdown causes a hypercontraction phenotype that leads to behavioral and eclosion defects. Misregulation of fiber type-specific gene and splice isoform expression, notably loss of an indirect flight muscle-specific isoform of Troponin-I that is critical for regulating myosin activity, leads to structural defects. It was further shown that Rbfox1 directly binds the 3'-UTR of target transcripts, regulates the expression level of myogenic transcription factors myocyte enhancer factor 2 and Salm, and both modulates expression of and genetically interacts with the CELF family RNA-binding protein Bruno1 (Bru1). Rbfox1 and Bru1 co-regulate fiber type-specific alternative splicing of structural genes, indicating that regulatory interactions between FOX and CELF family RNA-binding proteins are conserved in fly muscle. Rbfox1 thus affects muscle development by regulating fiber type-specific splicing and expression dynamics of identity genes and structural proteins.
Konstantinides, N., Holguera, I., ...., Walldorf, U., Roussos, P. and Desplan, C. (2022). A complete temporal transcription factor series in the fly visual system. Nature 604(7905): 316-322. PubMed ID: 35388222
The brain consists of thousands of neuronal types that are generated by stem cells producing different neuronal types as they age. In Drosophila, this temporal patterning is driven by the successive expression of temporal transcription factors (tTFs). This study used single-cell mRNA sequencing to identify the complete series of tTFs that specify most Drosophila optic lobe neurons. It was verified that tTFs regulate the progression of the series by activating the next tTF(s) and repressing the previous one(s), and also identify more complex mechanisms of regulation. Moreover, the temporal window of origin and birth order of each neuronal type in the medulla was established Finally, this study describes the first steps of neuronal differentiation and shows that these steps are conserved in humans. That terminal differentiation genes, such as neurotransmitter-related genes, are present as transcripts, but not as proteins, in immature larval neurons.
Truman, J. W. and Riddiford, L. M. (2022). Chinmo is the larval member of the molecular trinity that directs Drosophila metamorphosis . Proc Natl Acad Sci U S A 119(15): e2201071119. PubMed ID: 35377802
The genome of insects with complete metamorphosis contains the instructions for making three distinct body forms, that of the larva, of the pupa, and of the adult. However, the molecular mechanisms by which each gene set is called forth and stably expressed are poorly understood. A half century ago, it was proposed that there was a set of three master genes that inhibited each other's expression and enabled the expression of genes for each respective stage. This study shows that the transcription factor chinmo is essential for maintaining the larval stage in Drosophila, and with two other regulatory genes, broad and E93, makes up the trinity of mutually repressive master genes that underlie insect metamorphosis.

Friday, May 6th - Larval and Adult Neural Development and Function

Bajar, B. T., Phi, N. T., Isaacman-Beck, J., Reichl, J., Randhawa, H. and Akin, O. (2022). A discrete neuronal population coordinates brain-wide developmental activity. Nature 602(7898): 639-646. PubMed ID: 35140397
In vertebrates, stimulus-independent activity accompanies neural circuit maturation throughout the developing brain. The recent discovery of similar activity in the developing Drosophila central nervous system suggests that developmental activity is fundamental to the assembly of complex brains. How such activity is coordinated across disparate brain regions to influence synaptic development at the level of defined cell types is not well understood. This study shows that neurons expressing the cation channel transient receptor potential gamma (Trpγ) relay and pattern developmental activity throughout the Drosophila brain. In trpγ mutants, activity is attenuated globally, and both patterns of activity and synapse structure are altered in a cell-type-specific manner. Less than 2% of the neurons in the brain express Trpγ. These neurons arborize throughout the brain, and silencing or activating them leads to loss or gain of brain-wide activity. Together, these results indicate that this small population of neurons coordinates brain-wide developmental activity. It is proposed that stereotyped patterns of developmental activity are driven by a discrete, genetically specified network to instruct neural circuit assembly at the level of individual cells and synapses. This work establishes the fly brain as an experimentally tractable system for studying how activity contributes to synapse and circuit formation.
Aryal, B., Dhakal, S., Shrestha, B. and Lee, Y. (2022). Molecular and neuronal mechanisms for amino acid taste perception in the Drosophila labellum. Curr Biol 32(6): 1376-1386. PubMed ID: 35176225
Amino acids are essential nutrients that act as building blocks for protein synthesis. Recent studies in Drosophila have demonstrated that glycine, phenylalanine, and threonine elicit attraction, whereas tryptophan elicits aversion at ecologically relevant concentrations. This study demonstrated that eight amino acids, including arginine, glycine, alanine, serine, phenylalanine, threonine, cysteine, and proline, differentially stimulate feeding behavior by activating sweet-sensing gustatory receptor neurons (GRNs) in L-type and S-type sensilla. In turn, this process is mediated by three GRs (GR5a, GR61a, and GR64f), as well as two broadly required ionotropic receptors (IRs), IR25a and IR76b. However, GR5a, GR61a, and GR64f are only required for sensing amino acids in the sweet-sensing GRNs of L-type sensilla. This suggests that amino acid sensing in different type sensilla occurs through dual mechanisms. Furthermore, the findings indicated that ecologically relevant high concentrations of arginine, lysine, proline, valine, tryptophan, isoleucine, and leucine elicit aversive responses via bitter-sensing GRNs, which are mediated by three IRs (IR25a, IR51b, and IR76b). More importantly, these results demonstrate that arginine, lysine, and proline induce biphasic responses in a concentration-dependent manner. Therefore, amino acid detection in Drosophila occurs through two classes of receptors that activate two sets of sensory neurons in physiologically distinct pathways, which ultimately mediates attraction or aversion behaviors.
Xu, S., Sergeeva, A. P., Katsamba, P. S., Mannepalli, S., Bahna, F., Bimela, J., Zipursky, S. L., Shapiro, L., Honig, B. and Zinn, K. (2022). Affinity requirements for control of synaptic targeting and neuronal cell survival by heterophilic IgSF cell adhesion molecules. Cell Rep 39(1): 110618. PubMed ID: 35385751
Neurons in the developing brain express many different cell adhesion molecules (CAMs) on their surfaces. CAM-binding affinities can vary by more than 200-fold, but the significance of these variations is unknown. Interactions between the immunoglobulin superfamily CAM DIP-α and its binding partners, Dpr10 and Dpr6, control synaptic targeting and survival of Drosophila optic lobe neurons. This study designed mutations that systematically change interaction affinity and analyze function in vivo. Reducing affinity causes loss-of-function phenotypes whose severity scales with the magnitude of the change. Synaptic targeting is more sensitive to affinity reduction than is cell survival. Increasing affinity rescues neurons that would normally be culled by apoptosis. By manipulating CAM expression together with affinity, this study shows that the key parameter controlling circuit assembly is surface avidity, which is the strength of adherence between cell surfaces. It is concluded that CAM binding affinities and expression levels are finely tuned for function during development.
Chaturvedi, R., Stork, T., Yuan, C., Freeman, M. R. and Emery, P. (2022). Astrocytic GABA transporter controls sleep by modulating GABAergic signaling in Drosophila circadian neurons. Curr Biol. PubMed ID: 35303417
A precise balance between sleep and wakefulness is essential to sustain a good quality of life and optimal brain function. GABA is known to play a key and conserved role in sleep control, and GABAergic tone should, therefore, be tightly controlled in sleep circuits. This study examined the role of the astrocytic GABA transporter (Gat) in sleep regulation using Drosophila melanogaster. A hypomorphic Gat mutation (Gat33-1) increased sleep amount, decreased sleep latency, and increased sleep consolidation at night. Interestingly, sleep defects were suppressed when Gat33-1 was combined with a mutation disrupting wide-awake (wake), a gene that regulates the cell-surface levels of the GABA(A) receptor Resistance to dieldrin (Rdl) in the wake-promoting large ventral lateral neurons (l-LNvs). Moreover, RNAi knockdown of Rdl and its modulators dnlg4 and wake in these circadian neurons also suppressed Gat33-1 sleep phenotypes. Brain immunohistochemistry showed that GAT-expressing astrocytes were located near RDL-positive l-LNv cell bodies and dendritic processes. It is concluded that astrocytic GAT decreases GABAergic tone and RDL activation in arousal-promoting LNvs, thus determining proper sleep amount and quality in Drosophila.
Bajar, B. T., Phi, N. T., Randhawa, H. and Akin, O. (2022). Developmental neural activity requires neuron-astrocyte interactions. Dev Neurobiol 82(3): 235-244. PubMed ID: 35225404
Developmental neural activity is a common feature of neural circuit assembly. Although glia have established roles in synapse development, the contribution of neuron-glia interactions to developmental activity remains largely unexplored. This study shows that astrocytes are necessary for developmental activity during synaptogenesis in Drosophila. Using wide-field epifluorescence and two-photon imaging, it was shown that the glia of the central nervous system participate in developmental activity with type-specific patterns of intracellular calcium dynamics. Genetic ablation of astrocytes, but not of cortex or ensheathing glia, leads to severe attenuation of neuronal activity. Similarly, inhibition of neuronal activity results in the loss of astrocyte calcium dynamics. By altering these dynamics, this study showed that astrocytic calcium cycles can influence neuronal activity but are not necessary per se. Taken together, these results indicate that, in addition to their recognized role in the structural maturation of synapses, astrocytes are also necessary for the function of synapses during development.
Wang, G. (2022). Dopaminergic neurons mediate male Drosophila courtship motivational state. Biochem Biophys Res Commun 610: 23-29. PubMed ID: 35430448
Motivational states are important determinants of behavior. In Drosophila melanogaster, courtship behavior is robust and crucial for species continuation. However, the motivation of courtship behavior remains unexplored. This study first found the phenomenon that courtship behavior is modulated by motivational state. A male fly courts another male fly when it first courts a decapitated female fly, however, male-male courtship behavior rarely occurs under normal conditions. Therefore, in this phenomenon, the male fly's courtship motivational state is induced by its exposure to female flies. Blocking dopaminergic neurons synaptic transmission by expressing Tetanus toxin light chain (TNTe) decreases motivational state induced male-male courtship behavior without affecting male-female courtship behavior. Vision cues are another key component in sexually driven Drosophila male-male courtship behavior. This study has identified a base theory that the inner motivational state could eventually decide Drosophila behavior.

Thursday, May 5th - Disease Models

Aisenberg, W. H., McCray, B. A., Sullivan, J. M., Diehl, E., DeVine, L. R., Alevy, J., Bagnell, A. M., Carr, P., Donohue, J. K., Goretzki, B., Cole, R. N., Hellmich, U. A. and Sumner, C. J. (2022). Multiubiquitination of TRPV4 reduces channel activity independent of surface localization. J Biol Chem 298(4): 101826. PubMed ID: 35300980
Ubiquitin (Ub)-mediated regulation of plasmalemmal ion channel activity canonically occurs via stimulation of endocytosis. Whether ubiquitination can modulate channel activity by alternative mechanisms remains unknown. This study shows that the transient receptor potential vanilloid 4 (TRPV4) cation channel is multiubiquitinated within its cytosolic N-terminal and C-terminal intrinsically disordered regions (IDRs). Mutagenizing select lysine residues to block ubiquitination of the N-terminal but not C-terminal IDR resulted in a marked elevation of TRPV4-mediated intracellular calcium influx, without increasing cell surface expression levels. Conversely, enhancing TRPV4 ubiquitination via expression of an E3 Ub ligase reduced TRPV4 channel activity but did not decrease plasma membrane abundance. These results demonstrate Ub-dependent regulation of TRPV4 channel function independent of effects on plasma membrane localization. Consistent with ubiquitination playing a key negative modulatory role of the channel, gain-of-function neuropathy-causing mutations in the TRPV4 gene led to reduced channel ubiquitination in both cellular and Drosophila models of TRPV4 neuropathy, whereas increasing mutant TRPV4 ubiquitination partially suppressed channel overactivity. Together, these data reveal a novel mechanism via which ubiquitination of an intracellular flexible IDR domain modulates ion channel function independently of endocytic trafficking and identify a contributory role for this pathway in the dysregulation of TRPV4 channel activity by neuropathy-causing mutations.
Accogli, A., Lu, S., Musante, I., Scudieri, P., Rosenfeld, J. A., ..., Bellen, H. J., Lalani, S. R., Zara, F., Striano, P. and Salpietro, V. (2022). Loss of Neuron Navigator 2 Impairs Brain and Cerebellar Development. Cerebellum. PubMed ID: 35218524
Cerebellar hypoplasia and dysplasia encompass a group of clinically and genetically heterogeneous disorders frequently associated with neurodevelopmental impairment. The Neuron Navigator 2 (NAV2) gene (MIM: 607,026) encodes a member of the Neuron Navigator protein family, widely expressed within the central nervous system (CNS), and particularly abundant in the developing cerebellum. Evidence across different species supports a pivotal function of NAV2 in cytoskeletal dynamics and neurite outgrowth. Specifically, deficiency of Nav2 in mice leads to cerebellar hypoplasia with abnormal foliation due to impaired axonal outgrowth. However, little is known about the involvement of the NAV2 gene in human disease phenotypes. This study identified a female affected with neurodevelopmental impairment and a complex brain and cardiac malformations in which clinical exome sequencing led to the identification of NAV2 biallelic truncating variants. Through protein expression analysis and cell migration assay in patient-derived fibroblasts, evidence is provided linking NAV2 deficiency to cellular migration deficits. In model organisms, the overall CNS histopathology of the Nav2 hypomorphic mouse revealed developmental anomalies including cerebellar hypoplasia and dysplasia, corpus callosum hypo-dysgenesis, and agenesis of the olfactory bulbs. Lastly, this study shows that the NAV2 ortholog in Drosophila, sickie (sick) is widely expressed in the fly brain, and sick mutants are mostly lethal with surviving escapers showing neurobehavioral phenotypes. In summary, these results unveil a novel human neurodevelopmental disorder due to genetic loss of NAV2, highlighting a critical conserved role of the NAV2 gene in brain and cerebellar development across species.
Amstutz, J., Khalifa, A., Palu, R. and Jahan, K. (2022). Cluster-Based Analysis of Retinitis Pigmentosa Modifiers Using Drosophila Eye Size and Gene Expression Data. Genes (Basel) 13(2). PubMed ID: 35205430
The goal of this research is to computationally identify candidate modifiers for retinitis pigmentosa (RP), a group of rare genetic disorders that trigger the cellular degeneration of retinal tissue. RP being subject to phenotypic variation complicates diagnosis and treatment of the disease. In a previous study, modifiers of RP were identified by an association between genetic variation in the DNA sequence and variation in eye size in a well-characterized Drosophila model of RP. This study instead focusses on RNA expression data to identify candidate modifier genes whose expression is correlated with phenotypic variation in eye size. The proposed approach uses the K-Means algorithm to cluster 171 Drosophila strains based on their expression profiles for 18,140 genes in adult females. This algorithm is designed to investigate the correlation between Drosophila eye size and genetic expression and gather suspect genes from clusters with abnormally large or small eyes. The clustering algorithm was implemented using the R scripting language and successfully identified 10 suspected candidate modifiers for RP. This analysis was followed by a validation study that tested seven candidate modifiers and found that the loss of five of them significantly altered the degeneration phenotype and thus can be labeled as a bona fide modifier of disease.
Adedara, A. O., Babalola, A. D., Stephano, F., Awogbindin, I. O., Olopade, J. O., Rocha, J. B. T., Whitworth, A. J. and Abolaji, A. O. (2022). An assessment of the rescue action of resveratrol in parkin loss of function-induced oxidative stress in Drosophila melanogaster. Sci Rep 12(1): 3922. PubMed ID: 35273283
Loss-of-function mutations in parkin is associated with onset of juvenile Parkinson's disease (PD). Resveratrol is a polyphenolic stilbene with neuroprotective activity. This study evaluated the rescue action of resveratrol in parkin mutant D. melanogaster. The control flies (w1118) received diet-containing 2% ethanol (vehicle), while the PD flies received diets-containing resveratrol (15, 30 and 60 mg/kg diet) for 21 days to assess survival rate. Consequently, similar treatments were carried out for 10 days to evaluate locomotor activity, oxidative stress and antioxidant markers. mRNA levels were determined of Superoxide dismutase 1 (Sod1, an antioxidant gene) and ple, which encodes tyrosine hydroxylase, the rate-limiting step in dopamine synthesis. The data showed that resveratrol improved survival rate and climbing activity of PD flies compared to untreated PD flies. Additionally, resveratrol protected against decreased activities of acetylcholinesterase and catalase and levels of non-protein thiols and total thiols displayed by PD flies. Moreover, resveratrol mitigated against parkin mutant-induced accumulations of hydrogen peroxide, nitric oxide and malondialdehyde. Resveratrol attenuated downregulation of ple and Sod1 and reduction in mitochondrial fluorescence intensity displayed by PD flies. Overall, resveratrol alleviated oxidative stress and locomotor deficit associated with parkin loss-of-function mutation and therefore might be useful for the management of PD.
Wu, Q., Akhter, A., Pant, S., Cho, E., Zhu, J. X., Garner, A., Ohyama, T., Tajkhorshid, E., van Meyel, D. J. and Ryan, R. M. (2022). Ataxia-linked SLC1A3 mutations alter EAAT1 chloride channel activity and glial regulation of CNS function. J Clin Invest 132(7). PubMed ID: 35167492
Glutamate is the predominant excitatory neurotransmitter in the mammalian central nervous system (CNS). Excitatory amino acid transporters (EAATs) regulate extracellular glutamate by transporting it into cells, mostly glia, to terminate neurotransmission and to avoid neurotoxicity. EAATs are also chloride (Cl-) channels, but the physiological role of Cl- conductance through EAATs is poorly understood. Mutations of human EAAT1 (hEAAT1; see Drosophila Eaat1) have been identified in patients with episodic ataxia type 6 (EA6). One mutation showed increased Cl- channel activity and decreased glutamate transport, but the relative contributions of each function of hEAAT1 to mechanisms underlying the pathology of EA6 remain unclear. This study investigated the effects of 5 additional EA6-related mutations on hEAAT1 function in Xenopus laevis oocytes, and on CNS function in a Drosophila melanogaster model of locomotor behavior. The results indicate that mutations resulting in decreased hEAAT1 Cl- channel activity but with functional glutamate transport can also contribute to the pathology of EA6, highlighting the importance of Cl- homeostasis in glial cells for proper CNS function. This study also identified what is believed to be a novel mechanism involving an ectopic sodium (Na+) leak conductance in glial cells. Together, these results strongly support the idea that EA6 is primarily an ion channelopathy of CNS glia.
Akinade, T. C., Babatunde, O. O., Adedara, A. O., Adeyemi, O. E., Otenaike, T. A., Ashaolu, O. P., Johnson, T. O., Terriente-Felix, A., Whitworth, A. J. and Abolaji, A. O. (2022). Protective capacity of carotenoid trans-astaxanthin in rotenone-induced toxicity in Drosophila melanogaster. Sci Rep 12(1): 4594. PubMed ID: 35301354
Trans-astaxanthin (TA), a keto-carotenoid found in aquatic invertebrates, possesses anti-oxidative and anti-inflammatory activities. Rotenone is used to induce oxidative stress-mediated Parkinson's disease (PD) in animals. Probes were performed to see if TA would protect against rotenone-induced toxicity in Drosophila melanogaster. Trans-astaxanthin and rotenone were separately orally exposed to flies in the diet to evaluate longevity and survival rates, respectively. Consequently, the ameliorative actions of TA on rotenone -induced toxicity was evaluated in Drosophila after 7 days' exposure. Additionally, molecular docking of TA was performed against selected pro-inflammatory protein targets. It was observed that TA increased the lifespan of D. melanogaster by 36.36%. Moreover, TA ameliorated rotenone-mediated inhibition of Catalase, Glutathione-S-transferase and Acetylcholinesterase activities, and depletion of Total Thiols and Non-Protein Thiols contents. Trans-astaxanthin prevented behavioural dysfunction and accumulation of Hydrogen Peroxide, Malondialdehyde, Protein Carbonyls and Nitric Oxide in D. melanogaster. Trans-astaxanthin showed higher docking scores against the pro-inflammatory protein targets evaluated than the standard inhibitors. Conclusively, the structural features of TA might have contributed to its protective actions against rotenone-induced toxicity.

Wednesday, May 4th - Chromatin and DNA Repair

Walsh, B. S., Parratt, S. R., Snook, R. R., Bretman, A., Atkinson, D. and Price, T. A. R. (2022). Female fruit flies cannot protect stored sperm from high temperature damage. J Therm Biol 105: 103209. PubMed ID: 35393050
Recently, it has been demonstrated that heat-induced male sterility is likely to shape population persistence as climate change progresses. However, an under-explored possibility is that females may be able to successfully store and preserve sperm at temperatures that sterilise males, which could ameliorate the impact of male infertility on populations. This study tested whether females from two fruit fly species can protect stored sperm from a high temperature stress. Sperm carried by female Drosophila virilis are almost completely sterilised by high temperatures, whereas sperm carried by female Zaprionus indianus show only slightly reduced fertility. Heat-shocked D. virilis females can recover fertility when allowed to remate, suggesting that the delivered heat-shock is damaging stored sperm and not directly damaging females in this species. The temperatures required to reduce fertility of mated females are substantially lower than the temperatures required to damage mature sperm in males, suggesting that females are worse than males at protecting mature sperm. This suggests that female sperm storage is unlikely to ameliorate the impacts of high temperature fertility losses in males, and instead exacerbates fertility costs of high temperatures, representing an important determinant of population persistence during climate change.
Texada, M. J., Lassen, M., Pedersen, L. H., Koyama, T., Malita, A. and Rewitz, K. (2022). Insulin signaling couples growth and early maturation to cholesterol intake in Drosophila. Curr Biol 32(7): 1548-1562. PubMed ID: 35245460
Childhood obesity is associated with precocious puberty, but the assessment mechanism that links body fat to early maturation is unknown. During development, the intake of nutrients promotes signaling through insulin-like systems that govern the growth of cells and tissues and also regulates the timely production of the steroid hormones that initiate the juvenile-adult transition. This study shows that the dietary lipid cholesterol, which is required as a component of cell membranes and as a substrate for steroid biosynthesis, also governs body growth and maturation in Drosophila via promoting the expression and release of insulin-like peptides. This nutritional input acts via the nutrient sensor TOR, which is regulated by the Niemann-Pick-type-C 1 (Npc1) cholesterol transporter, in the glia of the blood-brain barrier and cells of the adipose tissue to remotely drive systemic insulin signaling and body growth. Furthermore, increasing intracellular cholesterol levels in the steroid-producing prothoracic gland strongly promotes endoreduplication, leading to an accelerated attainment of a nutritional checkpoint that normally ensures that animals do not initiate maturation prematurely. These findings, therefore, show that a Npc1-TOR signaling system couples the sensing of the lipid cholesterol with cellular and systemic growth control and maturational timing, which may help explain both the link between cholesterol and cancer as well as the connection between body fat (obesity) and early puberty.
Tian, Y., Tian, Y., Yu, G., Li, K., Du, Y., Yuan, Z., Gao, Y., Fan, X., Yang, D., Mao, X. and Yang, M. (2022). VhaAC39-1 regulates gut homeostasis and affects the health span in Drosophila. Mech Ageing Dev 204: 111673. PubMed ID: 35398002
Gut homeostasis is a dynamically balanced state to maintain intestinal health. Vacuolar ATPases (V-ATPases) are multi-subunit proton pumps that were driven by ATP hydrolysis. Several subunits of V-ATPases may be involved in the maintenance of intestinal pH and gut homeostasis in Drosophila. However, the specific role of each subunit in this process remains to be elucidated. This study knocked down the Drosophila gene VhaAC39-1 encoding the V0d1 subunit of V-ATPases to assess its function in gut homeostasis. Knockdown of VhaAC39-1 resulted in the loss of midgut acidity, the increase of the number of gut microbiota and the impairment of intestinal epithelial integrity in flies. The knockdown of VhaAC39-1 led to the hyperproliferation of intestinal stem cells, increasing the number of enteroendocrine cells, and activated IMD signaling pathway and JAK-STAT signaling pathway, inducing intestinal immune response of Drosophila. In addition, knockdown of VhaAC39-1 caused the disturbance of many physiological indicators such as food intake, triglyceride level and fecundity of flies, which ultimately led to the shortening of the life span of Drosophila. These results shed light on the gut homeostasis mechanisms which would help to identify interventions to promote healthy aging.

Ueda, K., Anderson-Baron, M. N., Haskins, J., Hughes, S. C. and Simmonds, A. J. (2022). Recruitment of Peroxin 14 to lipid droplets affects lipid storage in Drosophila. J Cell Sci 135(7). PubMed ID: 35274690
Both peroxisomes and lipid droplets regulate cellular lipid homeostasis. Direct inter-organellar contacts as well as novel roles for proteins associated with peroxisome or lipid droplets occur when cells are induced to liberate fatty acids from lipid droplets. This study has shown a non-canonical role for a subset of peroxisome-assembly [Peroxin (Pex)] proteins in this process in Drosophila. Transmembrane proteins Pex3, Pex13 and Pex14 were observed to surround newly formed lipid droplets. Trafficking of Pex14 to lipid droplets was enhanced by loss of Pex19, which directs insertion of transmembrane proteins like Pex14 into the peroxisome bilayer membrane. Accumulation of Pex14 around lipid droplets did not induce changes to peroxisome size or number, and co-recruitment of the remaining Peroxins was not needed to assemble peroxisomes observed. Increasing the relative level of Pex14 surrounding lipid droplets affected the recruitment of Hsl lipase. Fat body-specific reduction of these lipid droplet-associated Peroxins caused a unique effect on larval fat body development and affected their survival on lipid-enriched or minimal diets. This revealed a heretofore unknown function for a subset of Pex proteins in regulating lipid storage.
Strilbytska, O. M., Semaniuk, U. V., Strutynska, T. R., Burdyliuk, N. I., Tsiumpala, S., Bubalo, V. and Lushchak, O. (2022). Herbicide Roundup shows toxic effects in nontarget organism Drosophila. Arch Insect Biochem Physiol: e21893. PubMed ID: 35388481
Glyphosate-based herbicide Roundup, as the most employed herbicide used for multiple purposes in agriculture, adversely affects nontarget organisms. This study tested the effects of Roundup applied at larval and adult stages. Roundup caused developmental delay and increased larvae mortality. Roundup treatment reduced hemolymph glucose and glycogen levels in adult flies of both sexes at the highest concentration tested. Sex-dependent diverse effects were found in catalase and Cu,Zn superoxide dismutase (Cu,Zn-SOD) activities. Decreased aconitase activity, contents of thiols, and lipid peroxides were found after larval Roundup exposure. Furthermore, chronic exposure to adult flies decreased appetite, body weight, and shortened lifespan. Thus, these results suggest that high concentrations of Roundup are deleterious to both larvae and adults, resulting in a shift of the metabolism and antioxidant defense system in Drosophila melanogaster.
Chandler, J. A., Innocent, L. V., Martinez, D. J., Huang, I. L., Yang, J. L., Eisen, M. B. and Ludington, W. B. (2022). Microbiome-by-ethanol interactions impact Drosophila melanogaster fitness, physiology, and behavior. iScience 25(4): 104000. PubMed ID: 35313693
The gut microbiota can affect how animals respond to ingested toxins, such as ethanol, which is prevalent in the diets of diverse animals and often leads to negative health outcomes in humans. Ethanol is a complex dietary factor because it acts as a toxin, behavioral manipulator, and nutritional source, with both direct effects on the host as well as indirect ones through the microbiome. This study developed a model for chronic, non-intoxicating ethanol ingestion in the adult fruit fly, Drosophila melanogaster, and paired this with the tractability of the fly gut microbiota, which can be experimentally removed. Numerous physiological, behavioral, and transcriptional variables were linked to fly fitness, including a combination of intestinal barrier integrity, stored triglyceride levels, feeding behavior, and the immunodeficiency pathway. These results reveal a complex tradeoff between lifespan and fecundity that is microbiome-dependent and modulated by dietary ethanol and feeding behavior.

Tuesday, May 3rd - Chromatin and DNA Repair

Stow, E. C., Simmons, J. R., An, R., Schoborg, T. A., Davenport, N. M. and Labrador, M. (2022). A Drosophila insulator interacting protein suppresses enhancer-blocking function and modulates replication timing. Gene 819: 146208. PubMed ID: 35092858
Insulators play important roles in genome structure and function in eukaryotes. Interactions between a DNA binding insulator protein and its interacting partner proteins define the properties of each insulator site. The different roles of insulator protein partners in the Drosophila genome and how they confer functional specificity remain poorly understood. The Suppressor of Hairy wing [Su(Hw)] insulator is targeted to the nuclear lamina, preferentially localizes at euchromatin/heterochromatin boundaries, and is associated with the gypsy retrotransposon. Insulator activity relies on the ability of the Su(Hw) protein to bind the DNA at specific sites and interact with Mod(mdg4)67.2 and CP190 partner proteins. HP1 and insulator partner protein 1 (HIPP1) is a partner of Su(Hw), but how HIPP1 contributes to the function of Su(Hw) insulator complexes is unclear. This study demonstrates that HIPP1 colocalizes with the Su(Hw) insulator complex in polytene chromatin and in stress-induced insulator bodies. The overexpression of either HIPP1 or Su(Hw) or mutation of the HIPP1 crotonase-like domain (CLD) causes defects in cell proliferation by limiting the progression of DNA replication. This study also showed that HIPP1 overexpression suppresses the Su(Hw) insulator enhancer-blocking function, while mutation of the HIPP1 CLD does not affect Su(Hw) enhancer blocking. These findings demonstrate a functional relationship between HIPP1 and the Su(Hw) insulator complex and suggest that the CLD, while not involved in enhancer blocking, influences cell cycle progression.
Crain, A. T., Klusza, S., Armstrong, R. L., Santa Rosa, P., Temple, B. R. S., Strahl, B. D., McKay, D. J., Matera, A. G. and Duronio, R. J. (2022). Distinct developmental phenotypes result from mutation of Set8/KMT5A and histone H4 lysine 20 in Drosophila melanogaster. Genetics. PubMed ID: 35404465
Mono-methylation of histone H4 lysine 20 (H4K20me1) is catalyzed by Set8/KMT5A and regulates numerous aspects of genome organization and function. Loss-of-function mutations in Drosophila melanogaster Set8 or mammalian KMT5A prevent H4K20me1 and disrupt development. Set8/KMT5A also has non-histone substrates, making it difficult to determine which developmental functions of Set8/KMT5A are attributable to H4K20me1 and which to other substrates or to non-catalytic roles. This study shows that human KMT5A can functionally substitute for Set8 during Drosophila development and that the catalytic SET domains of the two enzymes are fully interchangeable. A role in eye development was uncovered for the N-terminal domain of Set8 that cannot be complemented by human KMT5A. Whereas Set820/20 null mutants are inviable, this study found that an R634G mutation in Set8 predicted from in vitro experiments to ablate catalytic activity resulted in viable adults. Additionally, Set8(R634G) mutants retain significant, albeit reduced, H4K20me1, indicating that the R634G mutation does not eliminate catalytic activity in vivo and is functionally hypomorphic rather than null. Flies engineered to express only unmodifiable H4 histones (H4K20A) can also complete development, but are phenotypically distinct from H4K20R, Set820/20 null, and Set8R634G mutants. Taken together, these results demonstrate functional conservation of KMT5A and Set8 enzymes, as well as distinct roles for Set8 and H4K20me1 in Drosophila development.
Pahi, Z. G., Kovacs, L., Szucs, D., Borsos, B. N., Deak, P. and Pankotai, T. (2022). Usp5, Usp34, and Otu1 deubiquitylases mediate DNA repair in Drosophila melanogaster. Sci Rep 12(1): 5870. PubMed ID: 35393473
Ubiquitylation is critical for preventing aberrant DNA repair and for efficient maintenance of genome stability. As deubiquitylases (DUBs) counteract ubiquitylation, they must have a great influence on many biological processes, including DNA damage response. To elucidate the role of DUBs in DNA repair in Drosophila melanogaster, systematic siRNA screening was applied to identify DUBs with a reduced survival rate following exposure to ultraviolet and X-ray radiations. As a secondary validation, the direct repeat (DR)-white reporter system with which site-specific DSBs were induced was applied and the importance of the DUBs Ovarian tumor domain-containing deubiquitinating enzyme 1 (Otu1), Ubiquitin carboxyl-terminal hydrolase 5 (Usp5), and Ubiquitin carboxyl-terminal hydrolase 34 (Usp34) in DSB repair pathways were applied using Drosophila. The results indicate that the loss of Otu1 and Usp5 induces strong position effect variegation in Drosophila eye following I-SceI-induced DSB deployment. Otu1 and Usp5 are essential in DNA damage-induced cellular response, and both DUBs are required for the fine-tuned regulation of the non-homologous end joining pathway. Furthermore, the Drosophila DR-white assay demonstrated that homologous recombination does not occur in the absence of Usp34, indicating an indispensable role of Usp34 in this process.
Abraham, A., Villanyi, Z., Zsindely, N., Nagy, G., Szabo, A., Bodai, L., Henn, L. and Boros, I. M. (2022). Despite its sequence identity with canonical H4, Drosophila H4r product is enriched at specific chromatin regions. Sci Rep 12(1): 5007. PubMed ID: 35322122
Histone variants are different from their canonical counterparts in structure and are encoded by solitary genes with unique regulation to fulfill tissue or differentiation specific functions. A single H4 variant gene (His4r or H4r) that is located outside of the histone cluster and gives rise to a polyA tailed messenger RNA via replication-independent expression is preserved in Drosophila strains despite that its protein product is identical with canonical H4. In order to reveal information on the possible role of this alternative H4 endogenous H4r was epitope tagged, and its spatial and temporal expression was studied, and its genome-wide localization to chromatin was revealed at the nucleosomal level. RNA and immunohistochemistry analysis of H4r expressed under its cognate regulation indicate expression of the gene throughout zygotic and larval development and presence of the protein product is evident already in the pronuclei of fertilized eggs. In the developing nervous system a slight disequibrium in H4r distribution is observable, cholinergic neurons are the most abundant among H4r-expressing cells. ChIP-seq experiments revealed H4r association with regulatory regions of genes involved in cellular stress response. The data presented in this study indicate that H4r has a variant histone function.
Zhang, H., Eerland, J., Horn, V., Schellevis, R. and van Ingen, H. (2021). Mapping the electrostatic potential of the nucleosome acidic patch. Sci Rep 11(1): 23013. PubMed ID: 34837025
he nucleosome surface contains an area with negative electrostatic potential known as the acidic patch, which functions as a binding platform for various proteins to regulate chromatin biology. The dense clustering of acidic residues may impact their effective pKa and thus the electronegativity of the acidic patch, which in turn could influence nucleosome-protein interactions. This study set out to determine the pKa values of residues in and around the acidic patch in the free H2A-H2B dimer using NMR spectroscopy. A refined solution structure is presented of the H2A-H2B dimer based on intermolecular distance restraints, displaying a well-defined histone-fold core. The conserved histidines H2B H46 and H106 that line the acidic patch have pKa of 5.9 and 6.5, respectively, and most acidic patch carboxyl groups have pKa values well below 5.0. For H2A D89, strong evidence was found for an elevated pKa of 5.3. These data establish that the acidic patch is highly negatively charged at physiological pH, while protonation of H2B H106 and H2B H46 at slightly acidic pH will reduce electronegativity. These results will be valuable to understand the impact of pH changes on nucleosome-protein interactions in vitro, in silico or in vivo.
Chathoth, K. T., Mikheeva, L. A., Crevel, G., Wolfe, J. C., Hunter, I., Beckett-Doyle, S., Cotterill, S., Dai, H., Harrison, A. and Zabet, N. R. (2022). The role of insulators and transcription in 3D chromatin organization of flies. Genome Res 32(4): 682-698. PubMed ID: 35354608
The DNA in many organisms, including humans, is shown to be organized in topologically associating domains (TADs). In Drosophila, several architectural proteins are enriched at TAD borders, but it is still unclear whether these proteins play a functional role in the formation and maintenance of TADs. This study shows that depletion of BEAF-32, Cp190, Chro, and Dref leads to changes in TAD organization and chromatin loops. Their depletion predominantly affects TAD borders located in regions moderately enriched in repressive modifications and depleted in active ones, whereas TAD borders located in euchromatin are resilient to these knockdowns. Furthermore, transcriptomic data has revealed hundreds of genes displaying differential expression in these knockdowns and showed that the majority of differentially expressed genes are located within reorganized TADs. This work identifies a novel and functional role for architectural proteins at TAD borders in Drosophila and a link between TAD reorganization and subsequent changes in gene expression.

Monday, May 2nd - RNA and Transposons

Lewerentz, J., Johansson, A. M., Larsson, J. and Stenberg, P. (2022). Transposon activity, local duplications and propagation of structural variants across haplotypes drive the evolution of the Drosophila S2 cell line.. BMC Genomics 23(1): 276. PubMed ID: 35392795
Immortalized cell lines are widely used model systems whose genomes are often highly rearranged and polyploid. However, their genome structure is seldom deciphered and is thus not accounted for during analyses. This study therefore used linked short- and long-read sequencing to perform haplotype-level reconstruction of the genome of a Drosophila melanogaster cell line (S2-DRSC) with a complex genome structure. Using a custom implementation (that is designed to use ultra-long reads in complex genomes with nested rearrangements) to call structural variants (SVs), it was found that the most common SV was repetitive sequence insertion or deletion (> 80% of SVs), with Gypsy retrotransposon insertions dominating. The second most common SV was local sequence duplication. SNPs and other SVs were rarer, but several large chromosomal translocations and mitochondrial genome insertions were observed. Haplotypes were highly similar at the nucleotide level but structurally very different. Insertion SVs existed at various haplotype frequencies and were unlinked on chromosomes, demonstrating that haplotypes have different structures and suggesting the existence of a mechanism that allows SVs to propagate across haplotypes. Finally, using public short-read data, it was found that transposable element insertions and local duplications are common in other D. melanogaster cell lines. It is concluded that The S2-DRSC cell line evolved through retrotransposon activity and vast local sequence duplications, that are hypothesized to be the products of DNA re-replication events. Additionally, mutations can propagate across haplotypes (possibly explained by mitotic recombination), which enables fine-tuning of mutational impact and prevents accumulation of deleterious events, an inherent problem of clonal reproduction. It is concluded that traditional linear homozygous genome representation conceals the complexity when dealing with rearranged and heterozygous clonal cells.
Zipper, L., Batchu, S., Kaya, N. H., Antonello, Z. A. and Reiff, T. (2022). The MicroRNA miR-277 Controls Physiology and Pathology of the Adult Drosophila Midgut by Regulating the Expression of Fatty Acid beta-Oxidation-Related Genes in Intestinal Stem Cells. Metabolites 12(4). PubMed ID: 35448502
This study tested whether stem and progenitor cell types might have a distinctive metabolic profile in the intestinal lineage. This study tested that hypothesis and investigated the metabolism of the intestinal lineage from stem cell (ISC) to differentiated epithelial cell in their native context under homeostatic conditions. An initial in silico analysis of single cell RNAseq data and functional experiments identify the microRNA miR-277 as a posttranscriptional regulator of fatty acid beta-oxidation (FAO) in the intestinal lineage. Low levels of miR-277 are detected in ISC and progressively rising miR-277 levels are found in progenitors during their growth and differentiation. Supporting this, miR-277-regulated fatty acid beta-oxidation enzymes progressively declined from ISC towards more differentiated cells in pseudotime single-cell RNAseq analysis and in functional assays on RNA and protein level. In addition, in silico clustering of single-cell RNAseq data based on metabolic genes validates that stem cells and progenitors belong to two independent clusters with well-defined metabolic characteristics. Furthermore, studying FAO genes in silico indicates that two populations of ISC exist that can be categorized in mitotically active and quiescent ISC, of which the latter relies on FAO genes. In line with an FAO dependency of ISC, forced expression of miR-277 phenocopies RNAi knockdown of FAO genes by reducing ISC size and subsequently resulting in stem cell death. This study also investigated miR-277 effects on ISC in a benign and a newly developed CRISPR-Cas9-based colorectal cancer model and found effects on ISC survival, which as a consequence affects tumor growth, further underlining the importance of FAO in a pathological context. Taken together, this study provides new insights into the basal metabolic requirements of intestinal stem cell on beta-oxidation of fatty acids evolutionarily implemented by a sole microRNA. Gaining knowledge about the metabolic differences and dependencies affecting the survival of two central and cancer-relevant cell populations in the fly and human intestine might reveal starting points for targeted combinatorial therapy in the hope for better treatment of colorectal cancer in the future.
Rech, G. E., Radio, S., Guirao-Rico, S., Aguilera, L., Horvath, V., Green, L., Lindstadt, H., Jamilloux, V., Quesneville, H. and Gonzalez, J. (2022). Population-scale long-read sequencing uncovers transposable elements associated with gene expression variation and adaptive signatures in Drosophila. Nat Commun 13(1): 1948. PubMed ID: 35413957
High quality reference genomes are crucial to understanding genome function, structure and evolution. The availability of reference genomes has allowed inference of the role of genetic variation in biology, disease, and biodiversity conservation. However, analyses across organisms demonstrate that a single reference genome is not enough to capture the global genetic diversity present in populations. This work generated 32 high-quality reference genomes for the well-known model species D. melanogaster and focused on the identification and analysis of transposable element variation as they are the most common type of structural variant. Integrating the genetic variation across natural populations from five climatic regions increases the number of detected insertions by 58%. Moreover, 26% to 57% of the insertions identified using long-reads were missed by short-reads methods. Hundreds of transposable elements associated with gene expression variation and new TE variants likely to contribute to adaptive evolution in this species were identified. The results highlight the importance of incorporating the genetic variation present in natural populations to genomic studies, which is essential if how genomes function and evolve are to be understood.
Faucillion, M. L., Johansson, A. M. and Larsson, J. (2022). Modulation of RNA stability regulates gene expression in two opposite ways: through buffering of RNA levels upon global perturbations and by supporting adapted differential expression. Nucleic Acids Res. PubMed ID: 35390159
The steady state levels of RNAs, often referred to as expression levels, result from a well-balanced combination of RNA transcription and decay. Alterations in RNA levels will therefore result from tight regulation of transcription rates, decay rates or both. This study explored the role of RNA stability in achieving balanced gene expression and present genome-wide RNA stabilities in Drosophila melanogaster male and female cells as well as male cells depleted of proteins essential for dosage compensation. Two distinct RNA-stability mediated responses were found to be involved in regulation of gene expression. The first of these responds to acute and global changes in transcription and thus counteracts potentially harmful gene mis-expression by shifting the RNA stability in the direction opposite to the transcriptional change. The second response enhances inter-individual differential gene expression by adjusting the RNA stability in the same direction as a transcriptional change. Both mechanisms are global, act on housekeeping as well as non-housekeeping genes and were observed in both flies and mammals. Additionally, this study showed that, in contrast to mammals, modulation of RNA stability does not detectably contribute to dosage compensation of the sex-chromosomes in D. melanogaster.
Oshizuki, S., Matsumoto, E., Tanaka, S. and Kataoka, N. (2022). Mutations equivalent to Drosophila mago nashi mutants imply reduction of Magoh protein incorporation into exon junction complex. Genes Cells. PubMed ID: 35430764
Pre-mRNA splicing imprints mRNAs by depositing multi-protein complexes, termed exon junction complexes (EJCs). The EJC core consists of four proteins, eIF4AIII, MLN51, Y14 and Magoh. Magoh is a human homologue of Drosophila Mago nashi protein, which is involved in oskar mRNA localization in Drosophila oocytes. This study determined the effects of Magoh mutations equivalent to those of Drosophila mago nashi mutant proteins that cause mis-localization of oskar mRNA. It was found that Magoh I90T mutation caused mis-localization of Magoh protein in the cytoplasm by reducing its binding activity to Y14. On the other hand, G18R mutation did not affect its binding to Y14, but this mutation reduced its association with spliced mRNAs. These results strongly suggest that Magoh mutations equivalent to Drosophila mago nashi mutants cause improper EJC formation by reducing incorporation of Magoh into EJC.
Bose, M., Lampe, M., Mahamid, J. and Ephrussi, A. (2022). Liquid-to-solid phase transition of oskar ribonucleoprotein granules is essential for their function in Drosophila embryonic development. Cell 185(8): 1308-1324. PubMed ID: 35325593
Asymmetric localization of oskar ribonucleoprotein (RNP) granules to the oocyte posterior is crucial for abdominal patterning and germline formation in the Drosophila embryo. This study shows that oskar RNP granules in the oocyte are condensates with solid-like physical properties. Using purified oskar RNA and scaffold proteins Bruno and Hrp48, this study confirmed in vitro that oskar granules undergo a liquid-to-solid phase transition. Whereas the liquid phase allows RNA incorporation, the solid phase precludes incorporation of additional RNA while allowing RNA-dependent partitioning of client proteins. Genetic modification of scaffold granule proteins or tethering the intrinsically disordered region of human fused in sarcoma (FUS) to oskar mRNA allowed modulation of granule material properties in vivo. The resulting liquid-like properties impaired oskar localization and translation with severe consequences on embryonic development. This study reflects how physiological phase transitions shape RNA-protein condensates to regulate the localization and expression of a maternal RNA that instructs germline formation.

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