What's hot today: Current papers in developmental biology and gene function

What's hot today:
Current papers in developmental biology and gene function

Thursday April 26th, 2023 - Cell Cycle

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Taslim, T. H., Hussein, A. M., Keshri, R., Ishibashi, J. R., Chan, T. C., Nguyen, B. N., Liu, S., Brewer, D., Harper, S., Lyons, S., Garver, B., Dang, J., Balachandar, N., Jhajharia, S., Castillo, D. D., Mathieu, J. and Ruohola-Baker, H. (2023). Stress-induced reversible cell-cycle arrest requires PRC2/PRC1-mediated control of mitophagy in Drosophila germline stem cells and human iPSCs. Stem Cell Reports 18(1): 269-288. PubMed ID: 36493777
Summary:
Following acute genotoxic stress, both normal and tumorous stem cells can undergo cell-cycle arrest to avoid apoptosis and later re-enter the cell cycle to regenerate daughter cells. However, the mechanism of protective, reversible proliferative arrest, "quiescence," remains unresolved. This study shows that mitophagy is a prerequisite for reversible quiescence in both irradiated Drosophila germline stem cells (GSCs) and human induced pluripotent stem cells (hiPSCs). In GSCs, mitofission (Drp1) or mitophagy (Pink1/Parkin) genes are essential to enter quiescence, whereas mitochondrial biogenesis (PGC1α) or fusion (Mfn2) genes are crucial for exiting quiescence. Furthermore, mitophagy-dependent quiescence lies downstream of mTOR- and PRC2-mediated repression and relies on the mitochondrial pool of cyclin E. Mitophagy-dependent reduction of cyclin E in GSCs and in hiPSCs during mTOR inhibition prevents the usual G1/S transition, pushing the cells toward reversible quiescence (G0). This alternative method of G1/S control may present new opportunities for therapeutic purposes.

Nieken, K. J., O'Brien, K., McDonnell, A., Zhaunova, L. and Ohkura, H. (2023). A large-scale RNAi screen reveals that mitochondrial function is important for meiotic chromosome organization in oocytes. Chromosoma. PubMed ID: 36648541
Summary:
In prophase of the first meiotic division, chromatin forms a compact spherical cluster called the karyosome within the enlarged oocyte nucleus in Drosophila melanogaster. Similar clustering of chromatin has been widely observed in oocytes in many species including humans. To identify genes involved in karyosome formation, a large-scale cytological screen was carried out using Drosophila melanogaster oocytes. This screen comprised 3916 genes expressed in ovaries, of which 106 genes triggered reproducible karyosome defects upon knockdown. The karyosome defects in 24 out of these 106 genes resulted from activation of the meiotic recombination checkpoint, suggesting possible roles in DNA repair or piRNA processing. The other genes identified in this screen include genes with functions linked to chromatin, nuclear envelope, and actin. It was also found that silencing of genes with mitochondrial functions, including electron transport chain components, induced a distinct karyosome defect typically with de-clustered chromosomes located close to the nuclear envelope. Furthermore, mitochondrial dysfunction not only impairs karyosome formation and maintenance, but also delays synaptonemal complex disassembly in cells not destined to become the oocyte. These karyosome defects do not appear to be mediated by apoptosis. This large-scale unbiased study uncovered a set of genes required for karyosome formation and revealed a new link between mitochondrial dysfunction and chromatin organization in oocytes.

Strashnyuk, V. Y., Shakina, L. A. and Skorobagatko, D. A. (2023). Variability of polyteny of giant chromosomes in Drosophila melanogaster salivary glands. Genetica 151(1): 75-86. PubMed ID: 36163579
Summary:
Polyteny is an effective mechanism for accelerating growth and enhancing gene expression in eukaryotes. The purpose of investigation was to study the genetic variability of polyteny degree of giant chromosomes in the salivary glands of Drosophila. in relation to the differential fitness of different genotypes. 16 strains, lines and hybrids of fruit flies were studied. This study demonstrates the significant influence of hereditary factors on the level of polytenization of giant chromosomes in Drosophila. This is manifested in the differences between strains and lines, the effect of inbreeding, chromosome isogenization, hybridization, adaptively significant selection, sexual differences, and varying degrees of individual variability of a trait in different strains, lines, and hybrids. The genetic component in the variability of the degree of chromosome polyteny in Drosophila salivary glands was 45.3%, the effect of sex was 9.5%. It has been shown that genetic distances during inbreeding, outbreeding or hybridization, which largely determine the selective value of different genotypes, also affect polyteny patterns. Genetic, humoral, and epigenetic aspects of endocycle regulation, which may underlie the variations in the degree of chromosome polyteny, as well as the biological significance of the phenomenon of endopolyploidy, are discussed.

Corrales, G. M., Li, M., Svermova, T., Goncalves, A., Voicu, D., Dobson, A. J., Southall, T. D. and Alic, N. (2022). Transcriptional memory of dFOXO activation in youth curtails later-life mortality through chromatin remodelling and Xbp1. Nat Aging 2: 1176-1190. PubMed ID: 37064330
Summary:
A transient, homeostatic transcriptional response can result in transcriptional memory, programming subsequent transcriptional outputs. Transcriptional memory has great but unappreciated potential to alter animal ageing as animals encounter a multitude of diverse stimuli throughout their lifespan. This study shows that activating an evolutionarily conserved, longevity-promoting transcription factor, dFOXO, solely in early adulthood of female fruit flies is sufficient to improve their subsequent health and survival in mid- and late life. This youth-restricted dFOXO activation causes persistent changes to chromatin landscape in the fat body and requires chromatin remodellers such as the SWI/SNF and ISWI complexes to program health and longevity. Chromatin remodelling is accompanied by a long-lasting transcriptional programme that is distinct from that observed during acute dFOXO activation and includes induction of Xbp1. This later-life induction of Xbp1 is sufficient to curtail later-life mortality. This study demonstrates that transcriptional memory can profoundly alter how animals age.

Muhlen, D., Li, X., Dovgusha, O., Jackle, H. and Gunesdogan, U. (2023). Recycling of parental histones preserves the epigenetic landscape during embryonic development. Sci Adv 9(5): eadd6440. PubMed ID: 36724233
Summary:
Epigenetic inheritance during DNA replication requires an orchestrated assembly of nucleosomes from parental and newly synthesized histones. This study analyzed Drosophila His(C) mutant embryos harboring a deletion of all canonical histone genes, in which nucleosome assembly relies on parental histones from cell cycle 14 onward. Lack of new histone synthesis leads to more accessible chromatin and reduced nucleosome occupancy, since only parental histones are available. This leads to up-regulated and spurious transcription, whereas the control of the developmental transcriptional program is partially maintained. The genomic positions of modified parental histone H2A, H2B, and H3 are largely restored during DNA replication. However, parental histones with active marks become more dispersed within gene bodies, which is linked to transcription. Together, the results suggest that parental histones are recycled to preserve the epigenetic landscape during DNA replication in vivo.

Warsinger-Pepe, N., Chang, C., Desroberts, C. R. and Akbari, O. S. (2023). Polycomb response elements reduce leaky expression of Cas9 under temperature-inducible Hsp70Bb promoter in Drosophila melanogaster. G3 (Bethesda). PubMed ID: 36705519
Summary:
Heat shock inducible expression of genes through the use of heat inducible promoters is commonly used in research despite leaky expression of downstream genes of interest without targeted induction (i.e. heat shock). The development of non-leaky inducible expression systems are of broad interest for both basic and applied studies, to precisely control gene expression. This study characterizes the use of Polycomb response elements and the inducible Heat shock protein 70Bb promoter, previously described as a non-leaky inducible system, to regulate Cas9 endonuclease levels and function in Drosophila melanogaster after varying both heat shock durations and rearing temperatures. Polycomb response elements were shown to significantly reduce expression of Cas9 under Heat shock protein 70Bb promoter control using a range of conditions, corroborating previously published results. It was further demonstrated that this low transcript level of heat-induced Cas9 is sufficient to induce mutant mosaic phenotypes. Incomplete suppression of an inducible Cas9 system by Polycomb response elements with no heat shock suggests that further regulatory elements are required to precisely control Cas9 expression and abundance.

Wednesday April 26th - Behavior

Yadipour, M., Billah, M. A. and Faruque, I. A. (2023). Optic flow enrichment via Drosophila head and retina motions to support inflight position regulation. J Theor Biol 562: 111416. PubMed ID: 36681182
Summary:
Developing a functional description of the neural control circuits and visual feedback paths underlying insect flight behaviors is an active research area. Feedback controllers incorporating engineering models of the insect visual system outputs have described some flight behaviors, yet they do not explain how insects are able to stabilize their body position relative to nearby targets such as neighbors or forage sources, especially in challenging environments in which optic flow is poor. The insect experimental community is simultaneously recording a growing library of in-flight head and eye motions that may be linked to increased perception. This study develops a quantitative model of the optic flow experienced by a flying insect or robot during head yawing rotations (distinct from lateral peering motions in previous work) with a single other target in view. This study then applies a model of insect visuomotor feedback to show via analysis and simulation of five species that these head motions sufficiently enrich the optic flow and that the output feedback can provide relative position regulation relative to the single target (asymptotic stability). In the simplifying case of pure rotation relative to the body, theoretical analysis provides a stronger stability guarantee. The results are shown to be robust to anatomical neck angle limits and body vibrations, persist with more detailed Drosophila lateral-directional flight dynamics simulations, and generalize to recent retinal motion studies. Together, these results suggest that the optic flow enrichment provided by head or pseudopupil rotation could be used in an insect's neural processing circuit to enable position regulation.

Zhang, Q., Chen, J., Wang, Y., Lu, Y., Dong, Z., Shi, W., Pang, L., Ren, S., Chen, X. and Huang, J. (2023). The odorant receptor co-receptor gene contributes to mating and host-searching behaviors in parasitoid wasps. Pest Manag Sci 79(1): 454-463. PubMed ID: 36177949
Summary:
Biological control of pest insects by parasitoid wasps is an effective and environmentally friendly strategy compared with the use of synthetic pesticides. Successful courtship and host-search behaviors of parasitoid wasps are important for biological control efficiency and are often mediated by chemical odorant cues. The odorant receptor co-receptor (Orco) gene has an essential role in the perception of odors in insects. However, the function of Orco in the mating and host-searching behaviors of parasitoid wasps remains underexplored. This study identified the full-length Orco genes of four Drosophila parasitoid species in the genus Leptopilina, namely L. heterotoma, L. boulardi, L. syphax and L. drosophilae. Sequence alignment and membrane-topology analysis showed that Leptopilina Orcos had similar amino acid sequences and topology structures. Phylogenetic analysis revealed that Leptopilina Orcos were highly conserved. Furthermore, the results of quantitative real-time polymerase chain reactions showed that all four Orco genes had a typical antennae-biased tissue expression pattern. After knockdown of Orco in these different parasitoid species, it was found that Orco-deficient male parasitoid wasps, but not females, lost their courtship ability. Moreover, Orco-deficient female parasitoid wasps presented impaired host-searching performance and decreased oviposition rates. This study demonstrates that Orcos are essential in the mating and host-searching behaviors of parasitoid wasps. This is the first time that the functions of Orco genes have been characterized in parasitoid wasps, which broadens understanding of the chemoreception basis of parasitoid wasps and contributes to developing advanced pest management strategies.

Moulin, T. C., Stojanovic, T., Rajesh, R. P., Pareek, T., Donzelli, L., Williams, M. J. and Schioth, H. B. (2023). Effects of Transient Administration of the NMDA Receptor Antagonist MK-801 in Drosophila melanogaster Activity, Sleep, and Negative Geotaxis. Biomedicines 11(1). PubMed ID: 36672700
Summary:
MK-801, also called dizocilpine, is an N-methyl-D-aspartate (NMDA) receptor antagonist widely used in animal research to model schizophrenia-like phenotypes. Although its effects in rodents are well characterised, little is known about the outcomes of this drug in other organisms. This study characterise the effects of MK-801 on the locomotion, sleep, and negative geotaxis of the fruit fly Drosophila melanogaster. Acute (24 h) and chronic (7 days) administration of MK-801 enhanced negative geotaxis activity in the forced climbing assay for all tested concentrations (0.15 mM, 0.3 mM, and 0.6 mM). Moreover, acute administration, but not chronic, increased the flies' locomotion in a dose-dependent matter. Finally, average sleep duration was not affected by any concentration or administration protocol. These results indicate that acute MK-801 could be used to model hyperactivity phenotypes in Drosophila melanogaster. Overall, this study provides further evidence that the NMDA receptor system is functionally conserved in flies, suggesting the usefulness of this model to investigate several phenotypes as a complement and replacement of the rodent models within drug discovery.

Mishra, S., Sharma, N., Singh, S. K. and Lone, S. R. (2023). Peculiar sleep features in sympatric species may contribute to the temporal segregation. J Comp Physiol B 193(1): 57-70. PubMed ID: 36271924
Summary:
Sleep is conserved in the animal kingdom and plays a pivotal role in the adaptation of species. Sleep in Drosophila melanogaster is defined as any continuous 5 min of quiescence, shows a prominent siesta, and consolidated nighttime sleep. This study analyzed the sleep of two other species D. malerkotliana (DMK) and D. ananassae (DA), and compared it with D. melanogaster (DM). The DMK males and females have siesta like DM. However, unlike DM, flies continue to sleep beyond siesta till the evening. DA has a less prominent siesta compared to DM and DMK. In the morning, DA took a longer time to respond to the lights ON and continued to sleep for at least half an hour. The nighttime sleep of the DA flies is higher than the other two species. Average length of sleep episode is three times more than that of DM and DMK with few wake episodes. Thus, the nighttime sleep of DA males and females is deep and needs exposure to more potent stimuli to wake up relative to the other two species. DA males and females show higher sleep rebound than the other two species, suggesting the robustness of sleep homeostasis. Although total sleep of DMK and DA is similar, DA is a day-active species with highly consolidated night sleep. DMK, like DM, is a crepuscular species with a midday siesta. Thus, these results suggest that temporal partitioning of sleep, in sympatric species may contribute to temporal segregation.

Vernier, C. L., Leitner, N., Zelle, K. M., Foltz, M., Dutton, S., Liang, X., Halloran, S., Millar, J. G. and Ben-Shahar, Y. (2023). A pleiotropic chemoreceptor facilitates the production and perception of mating pheromones. iScience 26(1): 105882. PubMed ID: 36691619
Summary:
Optimal mating decisions depend on the robust coupling of signal production and perception because independent changes in either could carry a fitness cost. However, since the perception and production of mating signals are often mediated by different tissues and cell types, the mechanisms that drive and maintain their coupling remain unknown for most animal species.This study shows that in Drosophila, behavioral responses to, and the production of, a putative inhibitory mating pheromone are co-regulated by Gr8a, a member of the Gustatory receptor gene family. Specifically, through behavioral and pheromonal data, this study found that Gr8a independently regulates the behavioral responses of males and females to a putative inhibitory pheromone, as well as its production in the fat body and oenocytes of males. Overall, these findings provide a relatively simple molecular explanation for how pleiotropic receptors maintain robust mating signaling systems at the population and species levels.

Zhang, Y., Li, Y., Barber, A. F., Noya, S. B., Williams, J. A., Li, F., Daniel, S. G., Bittinger, K., Fang, J. and Sehgal, A. (2023). The microbiome stabilizes circadian rhythms in the gut. Proc Natl Acad Sci U S A 120(5): e2217532120. PubMed ID: 36689661
Summary:
The gut microbiome is well known to impact host physiology and health. Given widespread control of physiology by circadian clocks, it was asked how the microbiome interacts with circadian rhythms in the Drosophila gut. The microbiome did not cycle in flies fed ad libitum, and timed feeding (TF) drove limited cycling only in clockless per⁰¹ flies. However, TF and loss of the microbiome influenced the composition of the gut cycling transcriptome, independently and together. Moreover, both interventions increased the amplitude of rhythmic gene expression, with effects of TF at least partly due to changes in histone acetylation. Contrary to expectations, timed feeding rendered animals more sensitive to stress. Analysis of microbiome function in circadian physiology revealed that germ-free flies reset more rapidly with shifts in the light:dark cycle. We propose that the microbiome stabilizes cycling in the host gut to prevent rapid fluctuations with changing environmental conditions.

Tuesday April 25th - Larval and Adult Development

De Giorgio, E., Giannios, P., Espinas, M. L. and Llimargas, M. (2023). A dynamic interplay between chitin synthase and the proteins Expansion/Rebuf reveals that chitin polymerisation and translocation are uncoupled in Drosophila. PLoS Biol 21(1): e3001978. PubMed ID: 36689563
Summary:
Chitin is a highly abundant polymer in nature and a principal component of apical extracellular matrices in insects. In addition, chitin has proved to be an excellent biomaterial with multiple applications. In spite of its importance, the molecular mechanisms of chitin biosynthesis and chitin structural diversity are not fully elucidated yet. To investigate these issues, Drosophila was used as a model. Previous work showed that chitin deposition in ectodermal tissues requires the concomitant activities of the chitin synthase enzyme Kkv and the functionally interchangeable proteins Exp and Reb. Exp/Reb are conserved proteins, but their mechanism of activity during chitin deposition has not been elucidated yet. This study carried out a cellular and molecular analysis of chitin deposition, and it was shown that chitin polymerisation and chitin translocation to the extracellular space are uncoupled. Kkv activity was found in chitin translocation, but not in polymerisation, requires the activity of Exp/Reb, and in particular of its conserved Nα-MH2 domain. The activity of Kkv in chitin polymerisation and translocation correlate with Kkv subcellular localisation, and in absence of Kkv-mediated extracellular chitin deposition, chitin accumulates intracellularly as membrane-less punctae. Unexpectedly, this study found that although Kkv and Exp/Reb display largely complementary patterns at the apical domain, Exp/Reb activity nonetheless regulates the topological distribution of Kkv at the apical membrane. A model is proposed in which Exp/Reb regulate the organisation of Kkv complexes at the apical membrane, which, in turn, regulates the function of Kkv in extracellular chitin translocation.

Scholl, A., Ndoja, I., Dhakal, N., Morante, D., Ivan, A., Newman, D., Mossington, T., Clemans, C., Surapaneni, S., Powers, M. and Jiang, L. (2023). The Osiris family genes function as novel regulators of the tube maturation process in the Drosophila trachea. PLoS Genet 19(1): e1010571. PubMed ID: 36689473
Summary:
Tracheal tube maturation starts with an apical secretion pulse that deposits extracellular matrix components to form a chitin-based apical luminal matrix (aECM). This aECM is then cleared and followed by the maturation of taenidial folds. Finally, air fills the tubes. Meanwhile, the cellular junctions are maintained to ensure tube integrity. The Osiris (Osi) gene family is located at the Triplo-lethal (Tpl) locus on chromosome 3R 83D4-E3 and exhibits dosage sensitivity. This study shows that three Osi genes (Osi9, Osi15, Osi19), function redundantly to regulate adherens junction (AJ) maintenance, luminal clearance, taenidial fold formation, tube morphology, and air filling during tube maturation. The localization of Osi proteins in endosomes (Rab7-containing late endosomes, Rab11-containing recycling endosomes, Lamp-containing lysosomes) and the reduction of these endosomes in Osi mutants suggest the possible role of Osi genes in tube maturation through endosome-mediated trafficking. Tube maturation was examined in zygotic rab11 and rab7 mutants, respectively, to determine whether endosome-mediated trafficking is required. Interestingly, similar tube maturation defects were observed in rab11 but not in rab7 mutants, suggesting the involvement of Rab11-mediated trafficking, but not Rab7-mediated trafficking, in this process. To investigate whether Osi genes regulate tube maturation primarily through the maintenance of Rab11-containing endosomes, rab11 was overexpressed in Osi mutant trachea. Surprisingly, no obvious rescue was observed. Thus, increasing endosome numbers is not sufficient to rescue tube maturation defects in Osi mutants. These results suggest that Osi genes regulate other aspects of endosome-mediated trafficking, or regulate an unknown mechanism that converges or acts in parallel with Rab11-mediated trafficking during tube maturation.

Zaytseva, O., Mitchell, N. C., Muckle, D., Delandre, C., Nie, Z., Werner, J. K., Lis, J. T., Eyras, E., Hannan, R. D., Levens, D. L., Marshall, O. J. and Quinn, L. M. (2023). Psi promotes Drosophila wing growth via direct transcriptional activation of cell cycle targets and repression of growth inhibitors. Development 150(2). PubMed ID: 36692218
Summary:
The first characterised FUSE Binding Protein family member, FUBP1, binds single-stranded DNA to activate MYC transcription. Psi, the sole FUBP protein in Drosophila, binds RNA to regulate P-element and mRNA splicing. Previous work revealed pro-growth functions for Psi, which depend, in part, on transcriptional activation of Myc. Genome-wide functions for FUBP family proteins in transcriptional control remain obscure. Through the first genome-wide binding and expression profiles obtained for a FUBP family protein, this study demonstrates that, in addition to being required to activate Myc to promote cell growth, Psi also directly binds and activates stg to couple growth and cell division. Thus, Psi knockdown results in reduced cell division in the wing imaginal disc. In addition to activating these pro-proliferative targets, Psi directly represses transcription of the growth inhibitor tolkin (tok, a metallopeptidase implicated in TGFβ signalling). It was further demonstrated that tok overexpression inhibits proliferation, while tok loss of function increases mitosis alone and suppresses impaired cell division caused by Psi knockdown. Thus, Psi orchestrates growth through concurrent transcriptional activation of the pro-proliferative genes Myc and stg, in combination with repression of the growth inhibitor tok.

Scanlan, J. L., Robin, C. and Mirth, C. K. (2023). Rethinking the ecdysteroid source during Drosophila pupal-adult development. Insect Biochem Mol Biol 152: 103891. PubMed ID: 36481381
Summary:
Ecdysteroids, typified by 20-hydroxyecdysone (20E), are essential hormones for the development, reproduction and physiology of insects and other arthropods. For over half a century, Drosophila has been used as a model of ecdysteroid biology. Many aspects of the biosynthesis and regulation of ecdysteroids in this species are understood at the molecular level, particularly with respect to their secretion from the prothoracic gland (PG) cells of the ring gland, widely considered the dominant biosynthetic tissue during development. Discrete pulses of 20E orchestrate transitions during the D. melanogaster life cycle, the sources of which are generally well understood, apart from the large 20E pulse at the onset of pharate adult development, which has received little recent attention. As the source of this pharate adult pulse (PAP) is a curious blind spot in Drosophila endocrinology, this study evaluated published biochemical and genetic data as they pertain to three hypotheses for the source of PAP 20E: the PG; an alternative biosynthetic tissue; or the recycling of stored 20E. Based on multiple lines of evidence, it is contended the PAP cannot be derived from biosynthesis, with other data consistent with D. melanogaster able to recycle ecdysteroids before and during metamorphosis. Published data also suggest the PAP is conserved across Diptera, with evidence for pupal-adult ecdysteroid recycling occurring in other cyclorrhaphan flies. Further experimental work is required to test the ecdysteroid recycling hypothesis, which would establish fundamental knowledge of the function, regulation, and evolution of metamorphic hormones in dipterans and other insects.

Yeung, K., Bollepogu Raja, K. K., Shim, Y. K., Li, Y., Chen, R. and Mardon, G. (2022). Single cell RNA sequencing of the adult Drosophila eye reveals distinct clusters and novel marker genes for all major cell types. Commun Biol 5(1): 1370. PubMed ID: 36517671
Summary:
The adult Drosophila eye is a powerful model system for phototransduction and neurodegeneration research. However, single cell resolution transcriptomic data are lacking for this tissue. This study presents single cell RNA-seq data on 1-day male and female, 3-day and 7-day old male adult eyes, covering early to mature adult eyes. All major cell types, including photoreceptors, cone and pigment cells in the adult eye were captured and identified. The data sets identified novel cell type specific marker genes, some of which were validated in vivo. R7 and R8 photoreceptors form clusters that reflect their specific Rhodopsin expression and the specific Rhodopsin expression by each R7 and R8 cluster is the major determinant to their clustering. The transcriptomic data presented in this report will facilitate a deeper mechanistic understanding of the adult fly eye as a model system.

Urban, E. A., Chernoff, C., Layng, K. V., Han, J., Anderson, C., Konzman, D. and Johnston, R. J., Jr. (2023). Activating and repressing gene expression between chromosomes during stochastic fate specification. Cell Rep 42(1): 111910. PubMed ID: 36640351
Summary:
DNA elements act across long genomic distances to regulate gene expression. During transvection in Drosophila, DNA elements on one allele of a gene act between chromosomes to regulate expression of the other allele. Little is known about the biological roles and developmental regulation of transvection. The stochastic expression of spineless (ss) in photoreceptors in the fly eye was investigated to understand transvection. It was determine a biological role for transvection in regulating expression of naturally occurring ss alleles. DNA elements were identified required for activating and repressing transvection. Different enhancers participate in transvection at different times during development to promote gene expression and specify cell fates. Bringing a silencer element on a heterologous chromosome into proximity with the ss locus "reconstitutes" the gene, leading to repression. These studies show that transvection regulates gene expression via distinct DNA elements at specific timepoints in development, with implications for genome organization and architecture.

Monday, April 25th - Physiology and Metabolism

Lai, M. L., Li, A. Q., Senior, A. M., Neely, G. G., Simpson, S. J. and Wang, Q. P. (2023). Nutritional geometry framework of sleep. Life Sci 316: 121381. PubMed ID: 36640899:
Summary:
Sleep is a fundamental physiological function and is essential for all animals. Sleep is affected by diet compositions including protein (P) and carbohydrates (C), but there has not been a systematic investigation on the effect of dietary macronutrient balance on sleep. This study used the nutritional geometry framework (NGF) to explore the interactive effects on sleep of protein (P) and carbohydrates (C) in the model organism Drosophila. Both female and male flies were fed various diets containing seven ratios of protein-to-carbohydrates at different energetic levels for 5 days and sleep was monitored by the Drosophila Activity Monitor (DAM) system. The results showed that the combination of low protein and high carbohydrates (LPHC) prolonged sleep time and sleep quality, with fewer sleep episodes and longer sleep duration. We further found that the effects of macronutrients on sleep mirrored levels of hemolymph glucose and whole-body glycogen. Moreover, transcriptomic analyses revealed that a high-protein, low-carbohydrate (HPLC) diet significantly elevated the gene expression of metabolic pathways when compared to the LPHC diet, with the glycine, serine, and threonine metabolism pathway being most strongly elevated. Further studies confirmed that the contents of glycine, serine, and threonine affected sleep. These results demonstrate that sleep is affected by the dietary balance of protein and carbohydrates possibly mediated by the change in glucose, glycogen, glycine, serine, and threonine.

Li, Y., Wang, W. and Lim, H. Y. (2023). Drosophila transmembrane protein 214 (dTMEM214) regulates midgut glucose uptake and systemic glucose homeostasis. Dev Biol 495: 92-103. PubMed ID: 36657508Summary:
The availability of glucose transporter in the small intestine critically determines the capacity for glucose uptake and consequently systemic glucose homeostasis. Hence a better understanding of the physiological regulation of intestinal glucose transporter is pertinent. However, the molecular mechanisms that regulate sodium-glucose linked transporter 1 (SGLT1), the primary glucose transporter in the small intestine, remain incompletely understood. Recently, the Drosophila SLC5A5 (dSLC5A5) has been found to exhibit properties consistent with a dietary glucose transporter in the Drosophila midgut, the equivalence of the mammalian small intestine. Hence, the fly midgut could serve as a suitable model system for the study of the in vivo molecular underpinnings of SGLT1 function. This study reports the identification, through a genetic screen, of Drosophila transmembrane protein 214 (dTMEM214) that acts in the midgut enterocytes to regulate systemic glucose homeostasis and glucose uptake.dTMEM214 resides in the apical membrane and cytoplasm of the midgut enterocytes, and the proper subcellular distribution of dTMEM214 in the enterocytes is regulated by the Rab4 GTPase. As a corollary, Rab4 loss-of-function phenocopies dTMEM214 loss-of-function in the midgut as shown by a decrease in enterocyte glucose uptake and an alteration in systemic glucose homeostasis. It was further shown that dTMEM214 regulates the apical membrane localization of dSLC5A5 in the enterocytes, thereby revealing dTMEM214 as a molecular regulator of glucose transporter in the midgut.

Hwangbo, D. S., Kwon, Y. J., Iwanaszko, M., Jiang, P., Abbasi, L., Wright, N., Alli, S., Hutchison, A. L., Dinner, A. R., Braun, R. I. and Allada, R. (2023). Dietary Restriction Impacts Peripheral Circadian Clock Output Important for Longevity in Drosophila. bioRxiv. PubMed ID: 36711760
Summary:
Circadian clocks may mediate lifespan extension by caloric or dietary restriction (DR). The core clock transcription factor Clock is crucial for a robust longevity and fecundity response to DR in Drosophila. To identify clock-controlled mediators, RNA-sequencing was performed from abdominal fat bodies across the 24 h day after just 5 days under control or DR diets. In contrast to more chronic DR regimens, no significant changes were detected in the rhythmic expression of core clock genes. Yet it was discovered that DR induced de novo rhythmicity or increased expression of rhythmic clock output genes. Network analysis revealed that DR increased network connectivity in one module comprised of genes encoding proteasome subunits. Adult, fat body specific RNAi knockdown demonstrated that proteasome subunits contribute to DR-mediated lifespan extension. Thus, clock control of output links DR-mediated changes in rhythmic transcription to lifespan extension.

Lovegrove, M. R., Dearden, P. K. and Duncan, E. J. (2023). Honeybee queen mandibular pheromone induces a starvation response in Drosophila melanogaster. Insect Biochem Mol Biol 154: 103908. PubMed ID: 36657589
Summary:
Eusocial insect societies are defined by the reproductive division of labour, a social structure that is generally enforced by the reproductive dominant(s) or 'queen(s)'. Reproductive dominance is maintained through behavioural dominance or production of queen pheromones, or a mixture of both. Queen mandibular pheromone (QMP) is a queen pheromone produced by queen honeybees (Apis mellifera) which represses reproduction in worker honeybees. How QMP acts to repress worker reproduction, the mechanisms by which this repression is induced, and how it has evolved this activity, remain poorly understood. Surprisingly, QMP is capable of repressing reproduction in non-target arthropods. This study shows that in Drosophila melanogaster QMP treatment mimics the starvation response, disrupting reproduction. QMP exposure induces an increase in food consumption and activation of checkpoints in the ovary that reduce fecundity and depresses insulin signalling. The magnitude of these effects is indistinguishable between QMP-treated and starved individuals. As QMP triggers a starvation response in an insect diverged from honeybees, it is proposed that QMP originally evolved by co-opting nutrition signalling pathways to regulate reproduction.

Palermo, J., Chesi, A., Zimmerman, A., Sonti, S., Pahl, M. C., Lasconi, C., Brown, E. B., Pippin, J. A., Wells, A. D., Doldur-Balli, F., Mazzotti, D. R., Pack, A. I., Gehrman, P. R., Grant, S. F. A. and Keene, A. C. (2023). Variant-to-gene mapping followed by cross-species genetic screening identifies GPI-anchor biosynthesis as a regulator of sleep. Sci Adv 9(1): eabq0844. PubMed ID: 36608130
Summary:
Genome-wide association studies (GWAS) in humans have identified loci robustly associated with several heritable diseases or traits, yet little is known about the functional roles of the underlying causal variants in regulating sleep duration or quality. This study applied an ATAC-seq/promoter focused Capture C strategy in human iPSC-derived neural progenitors to carry out a "variant-to-gene" mapping campaign that identified 88 candidate sleep effector genes connected to relevant GWAS signals. To functionally validate the role of the implicated effector genes in sleep regulation, a neuron-specific RNA interference screen was performed in the fruit fly, Drosophila melanogaster, followed by validation in zebrafish. This approach identified a number of genes that regulate sleep including a critical role for glycosylphosphatidylinositol (GPI)-anchor biosynthesis. These results provide the first physical variant-to-gene mapping of human sleep genes followed by a model organism-based prioritization, revealing a conserved role for GPI-anchor biosynthesis in sleep regulation.

Zanco, B., Rapley, L., Johnstone, J. N., Dedman, A., Mirth, C. K., Sgro, C. M. and Piper, M. D. W. (2023). Drosophila melanogaster females prioritise dietary sterols for producing viable eggs. J Insect Physiol 144: 104472. PubMed ID: 36549582
Summary:
Limiting calories or specific nutrients without malnutrition, otherwise known as dietary restriction (DR), has been shown to extend lifespan and reduce reproduction across a broad range of taxa. Recent findings in Drosophila melanogaster show that supplementing flies on macronutrient-rich diets with additional cholesterol can extend lifespan to the same extent as DR, while also sustaining high egg production. Thus, DR may be beneficial for lifespan because it reduces egg production which in turn reduces the mother's demand for sterols, thus supporting longer lifespan. It is also possible that mothers live longer and lay more eggs on high sterol diets because the diet triggers enhanced somatic maintenance and promotes egg production, but at the cost of diminished egg quality. To test this, the viability of eggs was measure and development of offspring from mothers fed either cholesterol-sufficient or cholesterol-limiting diets. Even when the mother's diet was completely devoid of cholesterol, viable egg production persisted for ~10 days. Furthermore,it was shown that sterol-supplemented flies with long lives lay eggs that have high viability and the same developmental potential as those laid by shorter lived mothers on sterol limiting diets. These findings suggest that offspring viability is not a hidden cost of lifespan extension seen in response to dietary sterol supplementation.

Friday April 21st - Gonads

Monteiro, V. L., Safavian, D., Vasudevan, D. and Hurd, T. R. (2023). Mitochondrial remodelling is essential for female germ cell differentiation and survival. PLoS Genet 19(1): e1010610. PubMed ID: 36696418
Summary:
Stem cells often possess immature mitochondria with few inner membrane invaginations, which increase as stem cells differentiate. Despite this being a conserved feature across many stem cell types in numerous organisms, how and why mitochondria undergo such remodelling during stem cell differentiation has remained unclear. Using Drosophila germline stem cells (GSCs), this study shows that Complex V drives mitochondrial remodelling during the early stages of GSC differentiation, prior to terminal differentiation. This endows germline mitochondria with the capacity to generate large amounts of ATP required for later egg growth and development. Interestingly, impairing mitochondrial remodelling prior to terminal differentiation results in endoplasmic reticulum (ER) lipid bilayer stress, Protein kinase R-like ER kinase (PERK)-mediated activation of the Integrated Stress Response (ISR) and germ cell death. Taken together, these data suggest that mitochondrial remodelling is an essential and tightly integrated aspect of stem cell differentiation. This work sheds light on the potential impact of mitochondrial dysfunction on stem and germ cell function, highlighting ER lipid bilayer stress as a potential major driver of phenotypes caused by mitochondrial dysfunction.

Yang, X., Chen, D., Zheng, S., Yi, M., Wang, S., Liu, Y., Jing, L., Liu, Z., Yang, D., Liu, Y., Tang, L., Walters, J. R. and Huang, Y. (2023). The Prmt5-Vasa module is essential for spermatogenesis in Bombyx mori. PPLoS Genet 19(1): e1010600. PubMed ID: 36634107
Summary:
In lepidopteran insects, dichotomous spermatogenesis produces eupyrene spermatozoa, which are nucleated, and apyrene spermatozoa, which are anucleated. Both sperm morphs are essential for fertilization, as eupyrene sperm fertilize the egg, and apyrene sperm is necessary for the migration of eupyrene sperm. In Drosophila, Prmt5 acts as a type II arginine methyltransferase that catalyzes the symmetrical dimethylation of arginine residues in the RNA helicase Vasa. Prmt5 is critical for the regulation of spermatogenesis, but Vasa is not. To date, functional genetic studies of spermatogenesis in the lepidopteran model Bombyx mori has been limited. In this study, mutations were engineered in BmPrmt5 and BmVasa through CRISPR/Cas9-based gene editing. Both BmPrmt5 and BmVasa loss-of-function mutants had similar male and female sterility phenotypes. Through immunofluorescence staining analysis, it was found that the morphs of sperm from both BmPrmt5 and BmVasa mutants have severe defects, indicating essential roles for both BmPrmt5 and BmVasa in the regulation of spermatogenesis. Mass spectrometry results identified that R35, R54, and R56 of BmVasa were dimethylated in WT while unmethylated in BmPrmt5 mutants. RNA-seq analyses indicate that the defects in spermatogenesis in mutants resulted from reduced expression of the spermatogenesis-related genes, including BmSxl (see Drosophila Sxl), implying that BmSxl acts downstream of BmPrmt5 and BmVasa to regulate apyrene sperm development. These findings indicate that BmPrmt5 and BmVasa constitute an integral regulatory module essential for spermatogenesis in B. mori.

Hetie, P., de Cuevas, M. and Matunis, E. L. (2023). The adult Drosophila testis lacks a mechanism to replenish missing niche cells. Development 150(2). PubMed ID: 36503989
Summary:
The adult Drosophila testis contains a well-defined niche created by a cluster of hub cells, which secrete signals that maintain adjacent germline stem cells and somatic cyst stem cells (CySCs). Hub cells are normally quiescent in adult flies but can exit quiescence, delaminate from the hub and convert into CySCs after ablation of all CySCs. The opposite event, CySC conversion into hub cells, was proposed to occur under physiological conditions, but the frequency of this event is debated. To probe further the question of whether or not hub cells can be regenerated, methods were developed to genetically ablate some or all hub cells. Surprisingly, when flies were allowed to recover from ablation, the missing hub cells were not replaced. Hub cells did not exit quiescence after partial ablation of hub cells, and labeled cells from outside the hub did not enter the hub during or after ablation. Despite its ability to exit quiescence in response to CySC ablation, it is concluded that the hub in the adult Drosophila testis does not have a mechanism to replenish missing hub cells.

Shao, L., Fingerhut, J. M., Falk, B. L., Han, H., Maldonado, G., Qiao, Y., Lee, V., Hall, E., Chen, L., Polevoy, G., Hernandez, G., Lasko, P. and Brill, J. A. (2023). Eukaryotic translation initiation factor eIF4E-5 is required for spermiogenesis in Drosophila melanogaster. Development. PubMed ID: 36695474
Summary:
Drosophila sperm development is characterized by extensive post-transcriptional regulation whereby thousands of transcripts are preserved for translation during later stages. A key step in translation initiation is the binding of eukaryotic initiation factor 4E (eIF4E) to the 5' mRNA cap. In addition to canonical eIF4E-1, Drosophila has multiple eIF4E paralogs, including four (eIF4E-3, -4, -5, and -7) that are highly expressed in the testis. Among these, only eIF4E-3 has been characterized genetically. Using CRISPR/Cas9 mutagenesis, this study determined that eIF4E-5 is essential for male fertility. eIF4E-5 protein localizes to the distal ends of elongated spermatid cysts, and eIF4E-5 mutants exhibit defects during post-meiotic stages, including a mild defect in spermatid cyst polarization. eIF4E-5 mutants also have a fully penetrant defect in individualization, resulting in failure to produce mature sperm. Indeed, the data indicate that eIF4E-5 regulates non-apoptotic caspase activity during individualization by promoting local accumulation of the E3 ubiquitin ligase inhibitor Soti. These results further extend the diversity of non-canonical eIF4Es that carry out distinct spatiotemporal roles during spermatogenesis.

Lee, E. H., Zinshteyn, D., Miglo, F., Wang, M. Q., Reinach, J., Chau, C. M., Grosstephan, J. M., Correa, I., Costa, K., Vargas, A., Johnson, A., Longo, S. M., Alexander, J. I. and O'Reilly, A. M. (2023). Sequential events during the quiescence to proliferation transition establish patterns of follicle cell differentiation in the Drosophila ovary. Biol Open 12(1). PubMed ID: 36524613
Summary:
Stem cells cycle between periods of quiescence and proliferation to promote tissue health. In Drosophila ovaries, quiescence to proliferation transitions of follicle stem cells (FSCs) are exquisitely feeding-dependent. This study demonstrate sfeeding-dependent induction of follicle cell differentiation markers, Eyes absent (Eya) and Castor (Cas) in FSCs, a patterning process that does not depend on proliferation induction. Instead, FSCs extend micron-scale cytoplasmic projections that dictate Eya-Cas patterning. still life and sickie were identified as necessary and sufficient for FSC projection growth and Eya-Cas induction. These results suggest that sequential, interdependent events establish long-term differentiation patterns in follicle cell precursors, independently of FSC proliferation induction.

Ku, H. Y., Harris, L. K. and Bilder, D. (2023). Specialized cells that sense tissue mechanics to regulate Drosophila morphogenesis. Dev Cell. PubMed ID: 36708706
Summary:
Shaping of developing organs requires dynamic regulation of force and resistance to achieve precise outcomes, but how organs monitor tissue mechanical properties is poorly understood. This study shows that in developing Drosophila follicles (egg chambers), a single pair of cells performs such monitoring to drive organ shaping. These anterior polar cells secrete a matrix metalloproteinase (MMP) that specifies the appropriate degree of tissue elongation, rather than hyper- or hypo-elongated organs. MMP production is negatively regulated by basement membrane (BM) mechanical properties, which are sensed through focal adhesion signaling and autonomous contractile activity; MMP then reciprocally regulates BM remodeling, particularly at the anterior region. Changing BM properties at remote locations alone is sufficient to induce a remodeling response in polar cells. It is proposed that this small group of cells senses both local and distant stiffness cues to produce factors that pattern the organ's BM mechanics, ensuring proper tissue shape and reproductive success.

Thursday April 20th - Adult Neural Development and Function

Yan, W., Lin, H., Yu, J., Wiggin, T. D., Wu, L., Meng, Z., Liu, C. and Griffith, L. C. (2023). Subtype-Specific Roles of Ellipsoid Body Ring Neurons in Sleep Regulation in Drosophila. J Neurosci 43(5): 764-786. PubMed ID: 36535771
Summary:
The ellipsoid body (EB) is a major structure of the central complex of the Drosophila melanogaster brain. Twenty-two subtypes of EB ring neurons have been identified based on anatomic and morphologic characteristics by light-level microscopy and EM connectomics. A few studies have associated ring neurons with the regulation of sleep homeostasis and structure. However, cell type-specific and population interactions in the regulation of sleep remain unclear. Using an unbiased thermogenetic screen of EB drivers using female flies, the following was found the: (1) multiple ring neurons are involved in the modulation of amount of sleep and structure in a synergistic manner; (2) analysis of data for &deltaP^doze/&Delta:P^wake using a mixed Gaussian model detected 5 clusters of GAL4 drivers which had similar effects on sleep pressure and/or depth: lines driving arousal contained R4m neurons, whereas lines that increased sleep pressure had R3m cells; (3) a GLM analysis correlating ring cell subtype and activity-dependent changes in sleep parameters across all lines identified several cell types significantly associated with specific sleep effects: R3p was daytime sleep-promoting, and R4m was nighttime wake-promoting; and (4) R3d cells present in 5HT7-GAL4 and in GAL4 lines, which exclusively affect sleep structure, were found to contribute to fragmentation of sleep during both day and night. Thus, multiple subtypes of ring neurons distinctively control sleep amount and/or structure. The unique highly interconnected structure of the EB suggests a local-network model worth future investigation; understanding EB subtype interactions may provide insight how sleep circuits in general are structured.

Truman, J. W., Price, J., Miyares, R. L. and Lee, T. (2023). Metamorphosis of memory circuits in Drosophila reveals a strategy for evolving a larval brain. Elife 12. PubMed ID: 36695420
Summary:
This study has focused on the mushroom bodies (MB) of Drosophila to determine how the larval circuits are formed and then transformed into those of the adult at metamorphosis. The adult MB has a core of thousands of Kenyon neurons; axons of the early-born g class form a medial lobe and those from later-born a'b' and ab classes form both medial and vertical lobes. The larva, however, hatches with only g neurons and forms a vertical lobe 'facsimile' using larval-specific axon branches from its g neurons. Computations by the MB involves MB input (MBINs) and output (MBONs) neurons that divide the lobes into discrete compartments. The larva has 10 such compartments while the adult MB has 16. This study determined the fates of 28 of the 32 types of MBONs and MBINs that define the 10 larval compartments. Seven larval compartments are eventually incorporated into the adult MB; four of their larval MBINs die, while 12 MBINs/MBONs continue into the adult MB although with some compartment shifting. The remaining three larval compartments are larval specific, and their MBIN/MBONs trans-differentiate at metamorphosis, leaving the MB and joining other adult brain circuits. With the loss of the larval vertical lobe facsimile, the adult vertical lobes, are made de novo at metamorphosis, and their MBONs/MBINs are recruited from the pool of adult-specific cells. The combination of cell death, compartment shifting, trans-differentiation, and recruitment of new neurons result in no larval MBIN-MBON connections persisting through metamorphosis. At this simple level, then, no anatomical substrate was found for a memory trace persisting from larva to adult. For the neurons that trans-differentiate, the data suggest that their adult phenotypes are in line with their evolutionarily ancestral roles while their larval phenotypes are derived adaptations for the larval stage. These cells arise primarily within lineages that also produce permanent MBINs and MBONs, suggesting that larval specifying factors may allow information related to birth-order or sibling identity to be interpreted in a modified manner in these neurons to cause them to adopt a modified, larval phenotype. The loss of such factors at metamorphosis, though, would then allow these cells to adopt their ancestral phenotype in the adult system.

Tanaka, R., Zhou, B., Agrochao, M., Badwan, B. A., Au, B., Matos, N. C. B. and Clark, D. A. (2023). Drosophila integrates visual evidence and counterevidence in self motion estimation. bioRxiv. PubMed ID: 36711843
Summary:
In selecting a behavior, animals should weigh sensory evidence both for and against their beliefs about the world. For instance, animals use optic flow to estimate and control their own rotation. However, existing models of flow detection can confuse the movement of external objects with genuine self motion. This study shows that stationary patterns on the retina, which constitute negative evidence against self rotation, are used by the fruit fly Drosophila to suppress inappropriate stabilizing rotational behavior. In parallel in silico experiments, it was shown that artificial neural networks trained to distinguish self and world motion incorporate similar negative evidence. Neural measurements and genetic manipulations were used to identify components of the circuitry for stationary pattern detection, which is parallel to the fly's motion- and optic flow-detectors. These results exemplify how the compact brain of the fly incorporates negative evidence to improve heading stability, exploiting geometrical constraints of the visual world.

Yang, Q., Zhou, J., Wang, L., Hu, W., Zhong, Y. and Li, Q. (2023). Spontaneous recovery of reward memory through active forgetting of extinction memory. Curr Biol. PubMed ID: 36731465
Summary:
Learned behavior can be suppressed by the extinction procedure. Such extinguished memory often returns spontaneously over time, making it difficult to treat diseases such as addiction. However, the biological mechanisms underlying such spontaneous recovery remain unclear. This study reports that the extinguished reward memory in Drosophila recovers spontaneously because extinction training forms an aversive memory that can be actively forgotten via the Dia pathway. Manipulating Rac1 activity does not affect sugar-reward memory and its immediate extinction effect but bidirectionally regulates spontaneous recovery-the decay process of extinction. Experiments using thermogenetic inhibition and functional imaging support that such extinction appears to be coded as an aversive experience. Genetic and pharmacological inhibition of formin Dia, a downstream effector of Rac1, specifically prevents spontaneous recovery after extinction in both behavioral performance and corresponding physiological traces. Together, the data suggest that spontaneous recovery is caused by active forgetting of the opposing extinction memory.

Spalthoff, C., Salgado, V. L., Balu, N., David, M. D., Hehlert, P., Huang, H., Jones, J. E., Kandasamy, R., Knudsen, G. A., Lelito, K. R., Machamer, J. B., Nesterov, A., Tomalski, M., Wahl, G. D., Wedel, B. J. and Gopfert, M. C. (2023). The Novel Pyridazine Pyrazolecarboxamide Insecticide Dimpropyridaz Inhibits Chordotonal Organ Function Upstream of Trpv Channels. Pest Manag Sci. PubMed ID: 36622360
Summary:
Pyridazine pyrazolecarboxamides (PPCs) are a novel insecticide class discovered and optimized at BASF. Dimpropyridaz is the first PPC to be submitted for registration and controls many aphid species as well as whiteflies and other piercing-sucking insects. Dimpropyridaz and other tertiary amide PPCs are proinsecticides that are converted in vivo into secondary amide active forms by N-dealkylation. Active secondary amide metabolites of PPCs potently inhibit the function of insect chordotonal neurons. Unlike Groups 9 and 29 insecticides, which hyper-activate chordotonal neurons and increase Ca(2+) levels, active metabolites of PPCs silence chordotonal neurons and decrease intracellular Ca(2+) levels. Whereas the effects of Groups 9 and 29 insecticides require TRPV channels, PPCs act in a TRPV-independent fashion, without compromising cellular responses to Group 9 and 29 insecticides, placing the molecular PPC target upstream of TRPVs. It is concluded that PPCs are a new class of chordotonal organ modulator insecticides for control of piercing-sucking pests. Dimpropyridaz is a PPC proinsecticide that is activated in target insects to secondary amide forms that inhibit the firing of chordotonal organs. The inhibition occurs at a site upstream of TRPVs and is TRPV-independent, providing a novel mode of action for resistance management. This article is protected by copyright.

Suver, M. P., Medina, A. M. and Nagel, K. I. (2023). Active antennal movements in Drosophila can tune wind encoding. Curr Biol. PubMed ID: 36731464
Summary:
Insects use their antennae to smell odors, detect auditory cues, and sense mechanosensory stimuli such as wind and objects, frequently by combining sensory processing with active movements. Genetic access to antennal motor systems would therefore provide a powerful tool for dissecting the circuit mechanisms underlying active sensing, but little is known about how the most genetically tractable insect, Drosophila melanogaster, moves its antennae. This study used deep learning to measure how tethered Drosophila move their antennae in the presence of sensory stimuli and identified genetic reagents for controlling antennal movement. Flies perform both slow adaptive movements and fast flicking movements in response to wind-induced deflections, but not the attractive odor apple cider vinegar. Next, four muscles are described in the first antennal segment that control antennal movements and identify genetic driver lines that provide access to two groups of antennal motor neurons and an antennal muscle. Through optogenetic inactivation, evidence is provided that antennal motor neurons contribute to active movements with different time courses. Finally, it was shown that activation of antennal motor neurons and muscles can adjust the gain and acuity of wind direction encoding by antennal displacement. Together, these experiments provide insight into the neural control of antennal movement and suggest that active antennal positioning in Drosophila may tune the precision of wind encoding.

Wednesday, April 19th - Enhancers and transcriptional regulation

Klaus, L., de Almeida, B. P., Vlasova, A., Nemcko, F., Schleiffer, A., Bergauer, K., Hofbauer, L., Rath, M. and Stark, A. (2023). Systematic identification and characterization of repressive domains in Drosophila transcription factors. Embo j 42(3): e112100. PubMed ID: 36545802
Summary:
All multicellular life relies on differential gene expression, determined by regulatory DNA elements and DNA-binding transcription factors that mediate activation and repression via cofactor recruitment. While activators have been extensively characterized, repressors are less well studied: the identities and properties of their repressive domains (RDs) are typically unknown and the specific co-repressors (CoRs) they recruit have not been determined. This study developed a high-throughput, next-generation sequencing-based screening method, repressive-domain (RD)-seq, to systematically identify RDs in complex DNA-fragment libraries. Screening more than 200,000 fragments covering the coding sequences of all transcription-related proteins in Drosophila melanogaster, this study identified 195 RDs in known repressors and in proteins not previously associated with repression. Many RDs contain recurrent short peptide motifs, which are conserved between fly and human and are required for RD function, as demonstrated by motif mutagenesis. Moreover, it was shown that RDs that contain one of five distinct repressive motifs interact with and depend on different CoRs, such as Groucho, CtBP, Sin3A, or Smrter. These findings advance understanding of repressors, their sequences, and the functional impact of sequence-altering mutations and should provide a valuable resource for further studies.

Hodkinson, L. J., Smith, C., Comstra, H. S., Albanese, E. H., Ajani, B. A., Arsalan, K., Daisson, A. P., Forrest, K. B., Fox, E. H., Guerette, M. R., Khan, S., Koenig, M. P., Lam, S., Lewandowski, A. S., Mahoney, L. J., Manai, N., Miglay, J., Miller, B. A., Milloway, O., Ngo, V. D., Oey, N. F., Punjani, T. A., SiMa, H., Zeng, H., Schmidt, C. A. and Rieder, L. E. (2023). A bioinformatics screen reveals Hox and chromatin remodeling factors at the Drosophila histone locus. bioRxiv. PubMed ID: 36711759
Summary:
Cells orchestrate histone biogenesis with strict temporal and quantitative control. To efficiently regulate histone biogenesis, the repetitive Drosophila melanogaster replication-dependent histone genes are arrayed and clustered at a single locus. Regulatory factors concentrate in a nuclear body known as the histone locus body (HLB), which forms around the locus. Historically, HLB factors are largely discovered by chance, and few are known to interact directly with DNA. It is therefore unclear how the histone genes are specifically targeted for unique and coordinated regulation. To expand the list of known HLB factors, a candidate-based screen was performed by mapping 30 publicly available ChIP datasets and 27 factors to the Drosophila histone gene array. Novel transcription factor candidates were identified, including the Drosophila Hox proteins Ultrabithorax, Abdominal-A and Abdominal-B, suggesting a new pathway for these factors in influencing body plan morphogenesis. Additionally, six other transcription factors were identified that target the histone gene array: JIL-1, Hr78, the long isoform of fs(1)h as well as the generalized transcription factors TAF-1, TFIIB, and TFIIF. This foundational screen provides several candidates for future studies into factors that may influence histone biogenesis. Further, this study emphasizes the powerful reservoir of publicly available datasets, which can be mined as a primary screening technique.

Hoedjes, K. M., Kostic, H., Flatt, T. and Keller, L. (2023). A single nucleotide variant in the PPARgamma-homolog Eip75B affects fecundity in Drosophila. Mol Biol Evol. PubMed ID: 36703226
Summary:
Single nucleotide polymorphisms are the most common type of genetic variation, but how these variants contribute to the adaptation of complex phenotypes is largely unknown. Experimental evolution and genome-wide association studies have demonstrated that variation in the PPARγ-homolog Eip75B is associated with longevity and life-history differences in the fruit fly Drosophila melanogaster. Using RNAi knockdown, this study first demonstrate that reduced expression of Eip75B in adult flies affects lifespan, egg-laying rate and egg volume. The effects of a naturally occurring SNP within a cis-regulatory domain of Eip75B were tested by applying two complementary approaches: a Mendelian randomization approach using lines of the Drosophila Genetic Reference Panel, and allelic replacement using precise CRISPR/Cas9-induced genome editing. These experiments reveal that this natural polymorphism has a significant pleiotropic effect on fecundity and egg-to-adult viability, but not on longevity or other life-history traits. These results provide a rare functional validation at the nucleotide level and identify a natural allelic variant affecting fitness and life-history adaptation.

Syed, S., Duan, Y. and Lim, B. (2023). Modulation of protein-DNA binding reveals mechanisms of spatiotemporal gene control in early Drosophila embryos. bioRxiv. PubMed ID: 36711729
Summary:
It is well known that enhancers regulate the spatiotemporal expression of their target genes by recruiting transcription factors (TFs) to the cognate binding sites in the region. However, the role of multiple binding sites for the same TFs and their specific spatial arrangement in determining the overall competency of the enhancer has yet to be fully understood. This study utilized the MS2-MCP live imaging technique to quantitatively analyze the regulatory logic of the snail distal enhancer in early Drosophila embryos. Through systematic modulation of Dorsal and Twist binding motifs in this enhancer, it was found that a mutation in any one of these binding sites causes a drastic reduction in transcriptional amplitude, resulting in a reduction in total mRNA production of the target gene. Evidence is provided of synergy, such that multiple binding sites with moderate affinities cooperatively recruit more TFs to drive stronger transcriptional activity than a single site. Moreover, a Hidden Markov-based stochastic model of transcription reveals that embryos with mutated binding sites have a higher probability of returning to the inactive promoter state. It is proposed that TF-DNA binding regulates spatial and temporal gene expression and drives robust pattern formation by modulating transcriptional kinetics and tuning bursting rates.

Wooten, M., Takushi, B., Ahmad, K. and Henikoff, S. (2023). Aclarubicin stimulates RNA polymerase II elongation at closely spaced divergent promoters. bioRxiv. PubMed ID: 36712130
Summary:
Anthracyclines are a class of widely prescribed anti-cancer drugs that disrupt chromatin by intercalating into DNA and enhancing nucleosome turnover. To understand the molecular consequences of anthracycline-mediated chromatin disruption, this study utilized CUT&Tag to profile RNA polymerase II during anthracycline treatment in Drosophila cells. It was observed that treatment with the anthracycline aclarubicin leads to elevated levels of elongating RNA polymerase II and changes in chromatin accessibility. Promoter proximity and orientation impacts chromatin changes during aclarubicin treatment, as closely spaced divergent promoter pairs show greater chromatin changes when compared to codirectionally-oriented tandem promoters. This study also found that aclarubicin treatment changes the distribution of non-canonical DNA G-quadruplex structures both at promoters and at G-rich pericentromeric repeats. This work suggests that the anti-cancer activity of aclarubicin is driven by the effects of nucleosome disruption on RNA polymerase II, chromatin accessibility and DNA structures.

Tuesday April 18th - Enzymes and Protein Expression, Evolution, Structure and Function

Fedir, B., Yannick, M., Marco, M., Patrizia, F., Catherine, Z., Frederic, V., Dirk, E., Joerg, K., Clemens, S., Camilo, V. V. and Patrick, C. (2023). N-terminal β-strand in YAP is critical for stronger binding to scalloped relative to TEAD transcription factor. Protein Sci 32(1):e4545. PubMed ID: 36522189
Summary:
The yes-associated protein (Yap; see Drosophila Yorkie) regulates the transcriptional activity of the TEAD transcrip../tion factors that are key in the control of organ morphogenesis. YAP interacts with TEAD via three secondary structure elements: a β-strand, an α-helix, and an Ω-loop. Earlier results have shown that the β-strand has only a marginal contribution in the YAP:TEAD interaction, but this study shows that it significantly enhances the affinity of YAP for the Drosophila homolog of TEAD, scalloped (Sd). Nuclear magnetic resonance shows that the β-strand adopts a more rigid conformation once bound to Sd; pre-steady state kinetic measurements show that the YAP:Sd complex is more stable. Although the crystal structures of the YAP:TEAD and YAP:Sd complexes reveal no differences at the binding interface that could explain these results. Molecular Dynamics simulations are in line with these experimental findings regarding β-strand stability and overall binding affinity of YAP to TEAD and Sd. In particular, RMSF, correlated motion and MMGBSA analyses suggest that β-sheet fluctuations play a relevant role in YAP(53-57) β-strand dissociation from TEAD4 and contribute to the lower affinity of YAP for TEAD4. Identifying a clear mechanism leading to the difference in YAP's β-strand stability proved to be challenging, pointing to the potential relevance of multiple modest structural changes or fluctuations for regulation of binding affinity.

Wishard, R., Jayaram, M., Ramesh, S. R. and Nongthomba, U. (2023). Spatial and temporal requirement of Mlp60A isoforms during muscle development and function in Drosophila melanogaster. Cells 12(2). Exp Cell Res 422(1): 113430. PubMed ID: 36423661
Summary:
Many myofibrillar proteins undergo isoform switching in a spatio-temporal manner during muscle development. The biological significance of the variants of several of these myofibrillar proteins remains elusive. One such myofibrillar protein, the Muscle LIM Protein (MLP), is a vital component of the Z-discs. This paper shows that one of the Drosophila MLP encoding genes, Mlp60A, gives rise to two isoforms: a short (279 bp, 10 kDa) and a long (1461 bp, 54 kDa) one. The short isoform is expressed throughout development, but the long isoform is adult-specific, being the dominant of the two isoforms in the indirect flight muscles (IFMs). A concomitant, muscle-specific knockdown of both isoforms leads to partial developmental lethality, with most of the surviving flies being flight defective. A global loss of both isoforms in a Mlp60A-null background also leads to developmental lethality, with muscle defects in the individuals that survive to the third instar larval stage. This lethality could be rescued partially by a muscle-specific overexpression of the short isoform. Genetic perturbation of only the long isoform, through a P-element insertion in the long isoform-specific coding sequence, leads to defective flight, in around 90% of the flies. This phenotype was completely rescued when the P-element insertion was precisely excised from the locus. Hence, these data show that the two Mlp60A isoforms are functionally specialized: the short isoform being essential for normal embryonic muscle development and the long isoform being necessary for normal adult flight muscle function.

Naz, R., Saeed, A., Tirth, V., Shukla, N. K., Mayet, A. M., Khan, A., Vrinceanu, N., Racheriu, M., Amir, T. and Iqbal, A. (2023). Structural and Functional Characterization of Novel Phosphotyrosine Phosphatase Protein from Drosophila melanogaster (Pupal Retina).ACS Omega 8(2): 1937-1945. PubMed ID: 36687094
Summary:
A novel pair of protein tyrosine phosphatases in Drosophila melanogaster (pupal retina) has been identified. Phosphotyrosyl protein phosphatases (PTPs) are structurally diverse enzymes increasingly recognized as having a fundamental role in cellular processes including effects on metabolism, cell proliferation, and differentiation. This study presents identification of novel sequences of PTPs and their comparative homology modeling from Drosophila melanogaster (Dr-PTPs) and complexation with the potent inhibitor HEPES. The 3D structure was predicted based on sequence homology with bovine heart low molecular weight PTPs (Bh-PTPs). The sequence homologies are approximately 50% identical to each other and to low molecular weight protein tyrosine phosphatases (PTPs) in other species. Comparison of the 3D structures of Bh-PTPs and Dr-PTPs (primo-2) reveals a remarkable similarity having a four stranded central parallel β sheet with flanking α helices on both sides, showing two right handed β-α-β motifs. The inhibitor shows similar binding features as seen in other PTPs. The study also highlights the key catalytic residues important for target recognition and PTPs' activation. The structure guided studies of both proteins clearly reveal a common mechanism of action and inhibitor binding at the active site and will be expected to contribute toward the basic understanding of functional association of this enzyme with other molecules.

Lee, S. A., Kim, V., Choi, B., Lee, H., Chun, Y. J., Cho, K. S. and Kim, D. (2023). Functional Characterization of Drosophila melanogaster CYP6A8 Fatty Acid Hydroxylase. Biomol Ther (Seoul) 31(1): 82-88. PubMed ID: 35934685
Summary:
Genomic analysis indicated that the genome of Drosophila melanogaster contains more than 80 cytochrome P450 genes. To date, the enzymatic activity of these P450s has not been extensively studied. In this study, the biochemical properties of CYP6A8 were characterized. CYP6A8 was cloned into the pCW vector, and its recombinant enzyme was expressed in Escherichia coli and purified using Ni(2+)-nitrilotriacetate affinity chromatography. Its expression level was approximately 130 nmol per liter of culture. Purified CYP6A8 exhibited a low-spin state in the absolute spectra of the ferric forms. Binding titration analysis indicated that lauric acid and capric acid produced type І spectral changes, with K(d) values 28 ± 4 and 144 ± 20 &mi;M, respectively. Ultra-performance liquid chromatography-mass spectrometry analysis showed that the oxidation reaction of lauric acid produced (ω-1)-hydroxylated lauric acid as a major product and ω-hydroxy-lauric acid as a minor product. Steady-state kinetic analysis of lauric acid hydroxylation yielded a k(cat) value of 0.038 ± 0.002 min(-1) and a K(m) value of 10 ± 2 &mi;M. In addition, capric acid hydroxylation of CYP6A8 yielded kinetic parameters with a k(cat) value of 0.135 ± 0.007 min(-1) and a K(m) value of 21 ± 4 &mi;M. Because of the importance of various lipids as carbon sources, the metabolic analysis of fatty acids using CYP6A8 in this study can provide an understanding of the biochemical roles of P450 enzymes in many insects, including Drosophila melanogaster.

Tang, X., Liu, N., Qi, H. and Lin, H. (2023). Piwi maintains homeostasis in the Drosophila adult intestine. Stem Cell Reports. PubMed ID: 36736325
Summary:
PIWI genes are well known for their germline but not somatic functions. Here, we report the function of the Drosophila piwi gene in the adult gut, where intestinal stem cells (ISCs) produce enteroendocrine cells and enteroblasts that generate enterocytes. piwi is expressed in ISCs and enteroblasts. Piwi deficiency reduced ISC number, compromised enteroblasts maintenance, and induced apoptosis in enterocytes, but did not affect ISC proliferation and its differentiation to enteroendocrine cells. In addition, deficiency of zygotic but not maternal piwi mildly de-silenced several retrotransposons in the adult gut. Importantly, either piwi mutations or piwi knockdown specifically in ISCs and enteroblasts shortened the Drosophila lifespan, indicating that intestinal piwi contributes to longevity. Finally, mRNA sequencing data implied that Piwi may achieve its intestinal function by regulating diverse molecular processes involved in metabolism and oxidation-reduction reaction.

Buhr, A., Schiemann, R. and Meyer, H. (2023). Neprilysin 4: an essential peptidase with multifaceted physiological relevance. Biol Chem. PubMed ID: 36653344
Summary:
Neprilysins are highly conserved ectoenzymes that hydrolyze and thus inactivate signaling peptides in the extracellular space. Herein, we focus on Neprilysin 4 from Drosophila melanogaster and evaluate the existing knowledge on the physiological relevance of the peptidase. Particular attention is paid to the role of the neprilysin in regulating feeding behavior and the expression of insulin-like peptides in the central nervous system. In addition, this study assessed the function of the peptidase in controlling the activity of the sarcoplasmic and endoplasmic reticulum Ca(2+) ATPase in myocytes, as well as the underlying molecular mechanism in detail expression evolution structure and function

Monday April 14th - Synapse and Vesicles

Vacassenno, R. M., Haddad, C. N. and Cooper, R. L. (2023). The effects of doxapram (blocker of K2p channels) on resting membrane potential and synaptic transmission at the Drosophila neuromuscular junction. Comp Biochem Physiol C Toxicol Pharmacol 263: 109497. PubMed ID: 36306997
Summary:
The resting membrane potential of most cells is maintained by potassium K2p channels. The pharmacological profile and distribution of various K2p channel subtypes in organisms are still being investigated. The Drosophila genome contains 11 subtypes; however, their function and expression profiles have not yet been determined. Doxapram is clinically used to enhance respiration in humans and blocks the acid-sensitive K2p TASK subtype in mammals. The resting membrane potential of larval Drosophila muscle and synaptic transmission at the neuromuscular junction are pH sensitive. The present study investigated the effects of doxapram on membrane potential and synaptic transmission using intracellular recordings of larval Drosophila muscles. Doxapram (1 mM and 10 mM) depolarizes the muscle and appears to depolarize motor neurons, causing an increase in the frequency of spontaneous quantal events and evoked excitatory junction potentials. Verapamil (1 and 10 mM) paralleled the action of doxapram. These changes were matched by an extracellular increase in KCl (50 mM) and blocked by Cd(2+). It is assumed that the motor nerve depolarizes to open voltage-gated Ca(2+) channels in presynaptic nerve terminals because of exposure to doxapram. These findings are significant for building models to better understand the function of pharmacological agents that affect K2p channels and how K2p channels contribute to the physiology of tissues. Drosophila offers a genetically amenable model that can alter the tissue-specific expression of K2p channel subtypes to simulate known human diseases related to this family of channels.

Walsh, E. N., Shetty, M. S., Diba, K. and Abel, T. (2023). Chemogenetic Enhancement of cAMP Signaling Renders Hippocampal Synaptic Plasticity Resilient to the Impact of Acute Sleep Deprivation. eNeuro 10(1). PubMed ID: 36635248
Summary:
Sleep facilitates memory storage and even brief periods of sleep loss lead to impairments in memory, particularly memories that are hippocampus dependent. Previous studies have shown that the deficit in memory seen after sleep loss is accompanied by deficits in synaptic plasticity. Previous work has also found that sleep deprivation (SD) is associated with reduced levels of cyclic adenosine monophosphate (cAMP) in the hippocampus and that the reduction of cAMP mediates the diminished memory observed in sleep-deprived animals. Based on these findings, it was hypothesized that cAMP acts as a mediator for not only the cognitive deficits caused by sleep deprivation, but also the observed deficits in synaptic plasticity. This study expressed the heterologous Drosophila melanogaster Gαs-protein-coupled octopamine receptor (DmOctβ1R) in mouse hippocampal neurons. This receptor is selectively activated by the systemically injected ligand (octopamine), thus leading to increased cAMP levels in hippocampal neurons during a 5-h sleep deprivation period. The results show that chemogenetic enhancement of cAMP during the period of sleep deprivation prevents deficits in a persistent form of long-term potentiation (LTP) that is induced at the Schaffer collateral synapses in the hippocampal CA1 region. It was found that elevating cAMP levels in either the first or second half of sleep deprivation successfully prevented LTP deficits. These findings reveal that cAMP-dependent signaling pathways are key mediators of sleep deprivation at the synaptic level. Targeting these pathways could be useful in designing strategies to prevent the impact of sleep loss.

Lehne, F. and Bogdan, S. (2023). EFhD2/Swip-1 promotes exocytosis of glue granules in the exocrine Drosophila salivary gland. J Cell Sci. PubMed ID: 36727484
Summary:
Exocytosis is a fundamental cellular process by which cells secret cargos from their apical membrane into the extracellular lumen. Cargo release proceeds in sequential steps that depend on a coordinated assembly and organization of an actin cytoskeletal network. This study identified the conserved actin-crosslinking protein EFhD2/Swip-1 as a novel regulator controlling exocytosis of glue granules in the Drosophila salivary gland. Real-time imaging revealed that EFhD2/Swip-1 is simultaneously recruited with F-actin onto secreting granules in proximity to the apical membrane. EFhD2/Swip-1 is rapidly cleared at the point of secretory vesicle fusion and colocalizes with actomyosin network around the fused vesicles. Loss of EFhD2/Swip-1 function impairs secretory cargo expulsion resulting in strongly delayed secretion. Thus, these results uncover a novel role of EFhD2/Swip-1 in secretory vesicle compression and expulsion of cargo during regulated exocytosis. Remarkably, this function neither require calcium-binding nor dimerization of EFhD2/Swip-1. These data rather suggest that EFhD2/Swip-1 regulates actomyosin activity upstream of Rho-GTPase signaling to drive proper vesicle membrane crumpling and expulsion of cargo.

Leahy, S. N., Song, C., Vita, D. J. and Broadie, K. (2023). FMRP activity and control of Csw/SHP2 translation regulate MAPK-dependent synaptic transmission. PLoS Biol 21(1): e3001969. PubMed ID: 36701299
Summary:
Noonan syndrome (NS) and NS with multiple lentigines (NSML) cognitive dysfunction are linked to SH2 domain-containing protein tyrosine phosphatase-2 (SHP2) gain-of-function (GoF) and loss-of-function (LoF), respectively. In Drosophila disease models, this study found both SHP2 mutations from human patients and corkscrew (csw) homolog LoF/GoF elevate glutamatergic transmission. Cell-targeted RNAi and neurotransmitter release analyses reveal a presynaptic requirement. Consistently, all mutants exhibit reduced synaptic depression during high-frequency stimulation. Both LoF and GoF mutants also show impaired synaptic plasticity, including reduced facilitation, augmentation, and post-tetanic potentiation. NS/NSML diseases are characterized by elevated MAPK/ERK signaling, and drugs suppressing this signaling restore normal neurotransmission in mutants. Fragile X syndrome (FXS) is likewise characterized by elevated MAPK/ERK signaling. Fragile X Mental Retardation Protein (FMRP) binds csw mRNA and neuronal Csw protein is elevated in Drosophila fragile X mental retardation 1 (dfmr1) nulls. Moreover, phosphorylated ERK (pERK) is increased in dfmr1 and csw null presynaptic boutons. Presynaptic pERK activation was found in response to stimulation is reduced in dfmr1 and csw nulls. Trans-heterozygous csw/+; dfmr1/+ recapitulate elevated presynaptic pERK activation and function, showing FMRP and Csw/SHP2 act within the same signaling pathway. Thus, a FMRP and SHP2 MAPK/ERK regulative mechanism controls basal and activity-dependent neurotransmission strength.

Jetti, S. K., Crane, A. S. B., Akbergenova, Y., Aponte-Santiago, N. A., Cunningham, K. L., Whittaker, C. A. and Littleton, J. T. (2023). Molecular Logic of Synaptic Diversity Between Drosophila Tonic and Phasic Motoneurons. bioRxiv. PubMed ID: 36711745
Summary:
Although neuronal subtypes display unique synaptic organization and function, the underlying transcriptional differences that establish these features is poorly understood. To identify molecular pathways that contribute to synaptic diversity, single neuron PatchSeq RNA profiling was performed on Drosophila tonic and phasic glutamatergic motoneurons. Tonic motoneurons form weaker facilitating synapses onto single muscles, while phasic motoneurons form stronger depressing synapses onto multiple muscles. Super-resolution microscopy and in vivo imaging demonstrated synaptic active zones in phasic motoneurons are more compact and display enhanced Ca (2+) influx compared to their tonic counterparts. Genetic analysis identified unique synaptic properties that mapped onto gene expression differences for several cellular pathways, including distinct signaling ligands, post-translational modifications and intracellular Ca (2+) buffers. These findings provide insights into how unique transcriptomes drive functional and morphological differences between neuronal subtypes.

Kozlov, E. N., Tokmatcheva, E. V., Khrustaleva, A. M., Grebenshchikov, E. S., Deev, R. V., Gilmutdinov, R. A., Lebedeva, L. A., Zhukova, M., Savvateeva-Popova, E. V., Schedl, P. and Shidlovskii, Y. V. (2023). Long-Term Memory Formation in Drosophila Depends on the 3'UTR of CPEB Gene orb2. Cells 12(2). PubMed ID: 36672258
Summary:
Activation of local translation in neurites in response to stimulation is an important step in the formation of long-term memory (LTM). CPEB proteins are a family of translation factors involved in LTM formation. The Drosophila CPEB protein Orb2 plays an important role in the development and function of the nervous system. Mutations of the coding region of the orb2 gene have previously been shown to impair LTM formation. This study found that a deletion of the 3'UTR of the orb2 gene similarly results in loss of LTM in Drosophila. As a result of the deletion, the content of the Orb2 protein remained the same in the neuron soma, but significantly decreased in synapses. Using RNA immunoprecipitation followed by high-throughput sequencing, more than 6000 potential Orb2 mRNA targets expressed in the Drosophila brain were detected. Importantly, deletion of the 3'UTR of orb2 mRNA also affected the localization of the Csp, Pyd, and Eya proteins, which are encoded by putative mRNA targets of Orb2. Therefore, the 3'UTR of the orb2 mRNA is important for the proper localization of Orb2 and other proteins in synapses of neurons and the brain as a whole, providing a molecular basis for LTM formation.

Friday April 14th - Larval and Adult Development

Scholl, A., Ndoja, I., Dhakal, N., Morante, D., Ivan, A., Newman, D., Mossington, T., Clemans, C., Surapaneni, S., Powers, M. and Jiang, L. (2023). The Osiris family genes function as novel regulators of the tube maturation process in the Drosophila trachea. PLoS Genet 19(1): e1010571. PubMed ID: 36689473
Summary:
Drosophila trachea is a premier model to study tube morphogenesis. After the formation of continuous tubes, tube maturation follows. Tracheal tube maturation starts with an apical secretion pulse that deposits extracellular matrix components to form a chitin-based apical luminal matrix (aECM). This aECM is then cleared and followed by the maturation of taenidial folds. Finally, air fills the tubes. Meanwhile, the cellular junctions are maintained to ensure tube integrity. Previous research has identified several key components (ER, Golgi, several endosomes) of protein trafficking pathways that regulate the secretion and clearance of aECM, and the maintenance of cellular junctions. The Osiris (Osi) gene family is located at the Triplo-lethal (Tpl) locus on chromosome 3R 83D4-E3 and exhibits dosage sensitivity. This study shows that three Osi genes (Osi9, Osi15, Osi19), function redundantly to regulate adherens junction (AJ) maintenance, luminal clearance, taenidial fold formation, tube morphology, and air filling during tube maturation. The localization of Osi proteins in endosomes (Rab7-containing late endosomes, Rab11-containing recycling endosomes, Lamp-containing lysosomes) and the reduction of these endosomes in Osi mutants suggest the possible role of Osi genes in tube maturation through endosome-mediated trafficking. This study analyzed tube maturation in zygotic rab11 and rab7 mutants, respectively. Interestingly, similar tube maturation defects were observed in rab11 but not in rab7 mutant. To investigate whether Osi genes regulate tube maturation primarily through the maintenance of Rab11-containing endosomes, rab11 was overexpressed in Osi mutant trachea. Surprisingly, no obvious rescue was observed. Thus, increasing endosome numbers is not sufficient to rescue tube maturation defects in Osi mutants. These results suggest that Osi genes regulate other aspects of endosome-mediated trafficking, or regulate an unknown mechanism that converges or acts in parallel with Rab11-mediated trafficking during tube maturation.

De Giorgio, E., Giannios, P., Espinas, M. L. and Llimargas, M. (2023). A dynamic interplay between chitin synthase and the proteins Expansion/Rebuf reveals that chitin polymerisation and translocation are uncoupled in Drosophila.. PLoS Biol 21(1): e3001978. PubMed ID: 36689563
Summary:
Chitin is a highly abundant polymer in nature and a principal component of apical extracellular matrices in insects. In addition, chitin has proved to be an excellent biomaterial with multiple applications. In spite of its importance, the molecular mechanisms of chitin biosynthesis and chitin structural diversity are not fully elucidated yet. To investigate these issues, Drosophila was used as a model. Previously work showed that chitin deposition in ectodermal tissues requires the concomitant activities of the chitin synthase enzyme Kkv and the functionally interchangeable proteins Exp and Reb. Exp/Reb are conserved proteins, but their mechanism of activity during chitin deposition has not been elucidated yet. This study carry out a cellular and molecular analysis of chitin deposition, and it was shown that chitin polymerisation and chitin translocation to the extracellular space are uncoupled. We find that Kkv activity in chitin translocation, but not in polymerisation, requires the activity of Exp/Reb, and in particular of its conserved Nα-MH2 domain. The activity of Kkv in chitin polymerisation and translocation correlate with Kkv subcellular localisation, and in absence of Kkv-mediated extracellular chitin deposition, chitin accumulates intracellularly as membrane-less punctae. Unexpectedly, it was found that although Kkv and Exp/Reb display largely complementary patterns at the apical domain, Exp/Reb activity nonetheless regulates the topological distribution of Kkv at the apical membrane. A model is proposed in which Exp/Reb regulate the organisation of Kkv complexes at the apical membrane, which, in turn, regulates the function of Kkv in extracellular chitin translocation.

Scanlan, J. L., Robin, C. and Mirth, C. K. (2023). Rethinking the ecdysteroid source during Drosophila pupal-adult development. Insect Biochem Mol Biol 152: 103891. PubMed ID: 36481381
Summary:
Ecdysteroids, typified by 20-hydroxyecdysone (20E), are essential hormones for the development, reproduction and physiology of insects and other arthropods. For over half a century, the vinegar fly Drosophila melanogaster (Ephydroidea: Diptera) has been used as a model of ecdysteroid biology. Many aspects of the biosynthesis and regulation of ecdysteroids in this species are understood at the molecular level, particularly with respect to their secretion from the prothoracic gland (PG) cells of the ring gland, widely considered the dominant biosynthetic tissue during development. Discrete pulses of 20E orchestrate transitions during the D. melanogaster life cycle, the sources of which are generally well understood, apart from the large 20E pulse at the onset of pharate adult development, which has received little recent attention. As the source of this pharate adult pulse (PAP) is a curious blind spot in Drosophila endocrinology, published biochemical and genetic data were evaluated as they pertain to three hypotheses for the source of PAP 20E: the PG; an alternative biosynthetic tissue; or the recycling of stored 20E. Based on multiple lines of evidence, it is contended that the PAP cannot be derived from biosynthesis, with other data consistent with D. melanogaster able to recycle ecdysteroids before and during metamorphosis. Published data also suggest the PAP is conserved across Diptera, with evidence for pupal-adult ecdysteroid recycling occurring in other cyclorrhaphan flies. Further experimental work is required to test the ecdysteroid recycling hypothesis, which would establish fundamental knowledge of the function, regulation, and evolution of metamorphic hormones in dipterans and other insects.

Ramakrishnan, P., Joshi, A., Tulasi, M. and Yadav, P. (2022). Monochromatic visible lights modulate the timing of pre-adult developmental traits in Drosophila melanogaster. Photochem Photobiol Sci. PubMed ID: 36583814
Summary:
Light exposure impacts several aspects of Drosophila development including the establishment of circadian rhythms, neuroendocrine regulation, life-history traits, etc. Introduction of artificial lights in the environment has caused almost all animals to develop ecological and physiological adaptations. White light which comprises different lights of differing wavelengths shortens the lifespan in fruit flies Drosophila melanogaster. The wavelength-specific effects of white light on Drosophila development remains poorly understood. This study shows that different wavelengths of white light differentially modulate Drosophila development in all its concomitant stages when maintained in a 12-h light: 12-h dark photoperiod. It was observed that exposure to different monochromatic lights significantly alters larval behaviours such as feeding rate and phototaxis that influence pre-adult development. Larvae grown under shorter wavelengths of light experienced an altered feedingrate. Similarly, larvae were found to avoid shorter wavelengths of light but were highly attracted to the longer wavelengths of light. Most of the developmental processes were greatly accelerated under the green light regime while in other light regimes, the effects were highly varied. Interestingly, pre-adult survivorship remained unaltered across all light regimes but light exposure was found to show its impact on sex determination. This study for the first time reveals how different wavelengths of white light modulate Drosophila development which in the future might help in developing non-invasive therapies and effective pest measures.

April 13th - Larval and Adult Development

Nandy, N. and Roy, J. K. (2023). Rab11 negatively regulates wingless preventing JNK-mediated apoptosis in Drosophila epithelium during embryonic dorsal closure. Cell Tissue Res. PubMed ID: 36705747
Summary:
Rab11, a small Ras like GTPase marking the recycling endosomes, plays instrumental roles in Drosophila embryonic epithelial morphogenesis where an array of reports testify its importance in the maintenance of cyto-architectural as well as functional attributes of the concerned cells. Proper Rab11 functions ensure a precise regulation of developmentally active cell signaling pathways which in turn promote the expression of morphogens and other physico-chemical cues which finally forge an embryo out of a single layer of cells. Earlier reports have established that Rab11 functions are vital for fly embryonic development where amorphic mutants such as EP3017 homozygotes show a fair degree of epithelial defects along with incomplete dorsal closure. This study presents a detailed account of the effects of Rab11 loss of function in the dorso-lateral epithelium which resulted in severe dorsal closure defects along with an elevated JNK-Dpp expression. It was further observed that the dorso-lateral epithelial cells undergo epithelial to mesenchymal transition as well as apoptosis in Rab11 mutants with elevated expression levels of MMP1 and Caspase-3, where Caspase-3 contributes to the Rab11 knockout phenotype contrary to the knockdown mutants or hypomorphs. Interestingly, the elevated expressions of the core JNK-Dpp signaling could be rescued with a simultaneous knockdown of wingless in the Rab11 knockout mutants suggesting a genetic interaction of Rab11 with the Wingless pathway during dorsal closure, an ideal model of epithelial wound healing.

Velagala, V., Soundarrajan, D. K., Unger, M. F., Gazzo, D., Kumar, N., Li, J. and Zartman, J. (2023). The multimodal action of G alpha q in coordinating growth and homeostasis in the Drosophila wing imaginal disc. bioRxiv. PubMed ID: 36711848
Summary:
G proteins mediate cell responses to various ligands and play key roles in organ development. This study employed the Gal4/UAS binary system to inhibit or overexpress Gαq in the wing disc, followed by phenotypic analysis. This study characterized how the G protein subunit Gαq tunes the size and shape of the wing in the larval and adult stages of development. Downregulation of Gαq in the wing disc reduced wing growth and delayed larval development. Gαq overexpression is sufficient to promote global Ca (2+) waves in the wing disc with a concomitant reduction in the Drosophila final wing size and a delay in pupariation. The reduced wing size phenotype is further enhanced when downregulating downstream components of the core Ca (2+) signaling toolkit, suggesting that downstream Ca (2+) signaling partially ameliorates the reduction in wing size. In contrast, Gαq -mediated pupariation delay is rescued by inhibition of IP (3) R, a key regulator of Ca (2+) signaling. This suggests that Gαq regulates developmental phenotypes through both Ca (2+) -dependent and Ca (2+) -independent mechanisms. RNA seq analysis shows that disruption of Gαq homeostasis affects nuclear hormone receptors, JAK/STAT pathway, and immune response genes. Notably, disruption of Gαq homeostasis increases expression levels of Dilp8, a key regulator of growth and pupariation timing. It is concluded that Gαq activity contributes to cell size regulation and wing metamorphosis. Disruption to Gαq homeostasis in the peripheral wing disc organ delays larval development through ecdysone signaling inhibition. Overall, Gαq signaling mediates key modules of organ size regulation and epithelial homeostasis through the dual action of Ca (2+) -dependent and independent mechanisms.

Trammell, C. E., Rowe, E. H., Jones, B. J., Char, A. B., Fawcett, S., Ahlers, L. R. H. and Goodman, A. G. (2023). Insulin-mediated endothelin signaling is antiviral during West Nile virus infection. bioRxiv. PubMed ID: 36712090
Summary:
West Nile virus (WNV) is the most prevalent mosquito-borne virus in the United States with approximately 2,000 cases each year. There are currently no approved human vaccines and a lack of prophylactic and therapeutic treatments. Understanding host responses to infection may reveal potential intervention targets to reduce virus replication and disease progression. The use of Drosophila melanogaster as a model organism to understand innate immunity and host antiviral responses is well established. Previous studies revealed that insulin-mediated signaling regulates WNV infection in invertebrates by regulating canonical antiviral pathways. Because insulin signaling is well-conserved across insect and mammalian species,tthis study sought to determine if results using D. melanogaster can be extrapolated for the analysis of orthologous pathways in humans. This study identified insulin-mediated endothelin signaling using the D. melanogaster model and evaluated an orthologous pathway in human cells during WNV infection. It was demonstrated that endothelin signaling reduces WNV replication through the activation of canonical antiviral signaling. Taken together, these findings show that endothelin-mediated antiviral immunity is broadly conserved across species and reduces replication of viruses that can cause severe human disease.

Marmion, R. A., Simpkins, A. G., Barrett, L. A., Denberg, D. W., Zusman, S., Schottenfeld-Roames, J., Schupbach, T. and Shvartsman, S. Y. (2023). Stochastic phenotypes in RAS-dependent developmental diseases. Curr Biol. PubMed ID: 36706752
Summary:
Germline mutations upregulating RAS signaling are associated with multiple developmental disorders. A hallmark of these conditions is that the same mutation may present vastly different phenotypes in different individuals, even in monozygotic twins. This study demonstrates how the origins of such largely unexplained phenotypic variations may be dissected using highly controlled studies in Drosophila that have been gene edited to carry activating variants of MEK, a core enzyme in the RAS pathway. This allowed measurement of the small but consistent increase in signaling output of such alleles in vivo. The fraction of mutation carriers reaching adulthood was strongly reduced, but most surviving animals had normal RAS-dependent structures. These results were rationalized using a stochastic signaling model and support it by quantifying cell fate specification errors in bilaterally symmetric larval trachea, a RAS-dependent structure that allows isolation the effects of mutations from potential contributions of genetic modifiers and environmental differences. It is proposed that the small increase in signaling output shifts the distribution of phenotypes into a regime, where stochastic variation causes defects in some individuals, but not in others. These findings shed light on phenotypic heterogeneity of developmental disorders caused by deregulated RAS signaling and offer a framework for investigating causal effects of other pathogenic alleles and mild mutations in general.

Sakakibara, Y., Yamashiro, R., Chikamatsu, S., Hirota, Y., Tsubokawa, Y., Nishijima, R., Takei, K., Sekiya, M. and Iijima, K. M. (2023). Drosophila Toll-9 is induced by aging and neurodegeneration to modulate stress signaling and its deficiency exacerbates tau-mediated neurodegeneration. iScience 26(2): 105968. PubMed ID: 36718365
Summary:
Drosophila Toll-9 is most closely related to mammalian Toll-like receptors; however, physiological functions of Toll-9 remain elusive. This study examined the roles of Toll-9 in fly brains in aging and neurodegeneration. Toll-9 mRNA levels were increased in aged fly heads accompanied by activation of nuclear factor-kappa B (NF-kB) and stress-activated protein kinase (SAPK; Jun-N-terminal Kinase pathway) signaling, and many of these changes were modulated by Toll-9 in glial cells. The loss of Toll-9 did not affect lifespan or brain integrity, whereas it exacerbated hydrogen peroxide-induced lethality. Toll-9 expression was also induced by nerve injury but did not affect acute stress response or glial engulfment activity, suggesting Toll-9 may modulate subsequent neurodegeneration. In a fly tauopathy model, Toll-9 deficiency enhanced neurodegeneration and disease-related tau phosphorylation with reduced SAPK activity, and blocking SAPK enhanced tau phosphorylation and neurodegeneration. In sum, Toll-9 is induced upon aging and nerve injury and affects neurodegeneration by modulating stress kinase signaling.

Moreno, M. R., Hunton, R., Strutt, D. and Bulgakova, N. A. (2023). Deciphering the roles of cell shape and Fat and Dachsous planar polarity in arranging the Drosophila apical microtubule network through quantitative image analysis. Mol Biol Cell: mbcE22090442. PubMed ID: 36735484
Summary:
In epithelial cells, planar polarisation of subapical microtubule networks is thought to be important for both breaking cellular symmetry and maintaining the resulting cellular polarity. Studies in the Drosophila pupal wing and other tissues have suggested two alternative mechanisms for specifying network polarity. On one hand mechanical strain and/or cell shape have been implicated as key determinants, on the other the Fat-Dachsous planar polarity pathway has been suggested to be the primary polarising cue. Using quantitative image analysis in the pupal wing, this study reassessed these models. It was found that cell shape was a strong predictor of microtubule organisation in the developing wing epithelium. Conversely Fat-Dachsous polarity cues do not play any direct role in the organisation of the subapical microtubule network, despite being able to weakly recruit the microtubule minus-end capping protein Patronin to cell boundaries. It is concluded that any effect of Fat-Dachsous on microtubule polarity is likely to be indirect, via their known ability to regulate cell shape.

Wednesday April 12th - Larval and Adult Physiology and Metabolism

Lai, M. L., Li, A. Q., Senior, A. M., Neely, G. G., Simpson, S. J. and Wang, Q. P. (2023). Nutritional geometry framework of sleep. Life Sci 316: 121381. PubMed ID: 36640899
Summary:
Sleep is a fundamental physiological function and is essential for all animals. Sleep is affected by diet compositions including protein (P) and carbohydrates (C), but there has not been a systematic investigation on the effect of dietary macronutrient balance on sleep. This study used the nutritional geometry framework (NGF) to explore the interactive effects on sleep of protein (P) and carbohydrates (C) in the model organism Drosophila. Both female and male flies were fed various diets containing seven ratios of protein-to-carbohydrates at different energetic levels for 5 days and sleep was monitored by the Drosophila Activity Monitor (DAM) system. The results showed that the combination of low protein and high carbohydrates (LPHC) prolonged sleep time and sleep quality, with fewer sleep episodes and longer sleep duration. It was further found that the effects of macronutrients on sleep mirrored levels of hemolymph glucose and whole-body glycogen. Moreover, transcriptomic analyses revealed that a high-protein, low-carbohydrate (HPLC) diet significantly elevated the gene expression of metabolic pathways when compared to the LPHC diet, with the glycine, serine, and threonine metabolism pathway being most strongly elevated. Further studies confirmed that the contents of glycine, serine, and threonine affected sleep. These results demonstrate that sleep is affected by the dietary balance of protein and carbohydrates possibly mediated by the change in glucose, glycogen, glycine, serine, and threonine.

Li, Y., Wang, W. and Lim, H. Y. (2023). Drosophila transmembrane protein 214 (dTMEM214) regulates midgut glucose uptake and systemic glucose homeostasis. Dev Biol 495: 92-103. PubMed ID: 36657508
Summary:
The availability of glucose transporter in the small intestine critically determines the capacity for glucose uptake and consequently systemic glucose homeostasis. Hence a better understanding of the physiological regulation of intestinal glucose transporter is pertinent. However, the molecular mechanisms that regulate sodium-glucose linked transporter 1 (SGLT1), the primary glucose transporter in the small intestine, remain incompletely understood. Recently, the Drosophila SLC5A5 (dSLC5A5) has been found to exhibit properties consistent with a dietary glucose transporter in the Drosophila midgut, the equivalence of the mammalian small intestine. Hence, the fly midgut could serve as a suitable model system for the study of the in vivo molecular underpinnings of SGLT1 function. This study reports the identification, through a genetic screen, of Drosophila transmembrane protein 214 (dTMEM214) that acts in the midgut enterocytes to regulate systemic glucose homeostasis and glucose uptake. dTMEM214 resides in the apical membrane and cytoplasm of the midgut enterocytes, and that the proper subcellular distribution of dTMEM214 in the enterocytes is regulated by the Rab4 GTPase. As a corollary, Rab4 loss-of-function phenocopies dTMEM214 loss-of-function in the midgut as shown by a decrease in enterocyte glucose uptake and an alteration in systemic glucose homeostasis. This study further showed that dTMEM214 regulates the apical membrane localization of dSLC5A5 in the enterocytes, thereby revealing dTMEM214 as a molecular regulator of glucose transporter in the midgut (Li, 2023).

Tsao, D. D., Chang, K. R., Kockel, L., Park, S. and Kim, S. K. (2023). A genetic strategy to measure insulin signaling regulation and physiology in Drosophila. PLoS Genet 19(2): e1010619. PubMed ID: 36730473
Summary:
Insulin regulation is a hallmark of health, and impaired insulin signaling promotes metabolic diseases like diabetes mellitus. However, current assays for measuring insulin signaling in all animals remain semi-quantitative and lack the sensitivity, tissue-specificity or temporal resolution needed to quantify in vivo physiological signaling dynamics. Insulin signal transduction is remarkably conserved across metazoans, including insulin-dependent phosphorylation and regulation of Akt/Protein kinase B. This study generated transgenic fruit flies permitting tissue-specific expression of an immunoepitope-labelled Akt (AktHF). Enzyme-linked immunosorption assays (ELISA) were developed to quantify picomolar levels of phosphorylated (pAktHF) and total AktHF in single flies, revealing dynamic tissue-specific physiological regulation of pAktHF in response to fasting and re-feeding, exogenous insulin, or targeted genetic suppression of established insulin signaling regulators. Genetic screening revealed Pp1-87B as an unrecognized regulator of Akt and insulin signaling. Tools and concepts here provide opportunities to discover tissue-specific regulators of in vivo insulin signaling responses.

Lovegrove, M. R., Dearden, P. K. and Duncan, E. J. (2023). Honeybee queen mandibular pheromone induces a starvation response in Drosophila melanogaster. Insect Biochem Mol Biol 154: 103908. PubMed ID: 36657589
Summary:
Eusocial insect societies are defined by the reproductive division of labour, a social structure that is generally enforced by the reproductive dominant(s) or 'queen(s)'. Reproductive dominance is maintained through behavioural dominance or production of queen pheromones, or a mixture of both. Queen mandibular pheromone (QMP) is a queen pheromone produced by queen honeybees (Apis mellifera) which represses reproduction in worker honeybees. How QMP acts to repress worker reproduction, the mechanisms by which this repression is induced, and how it has evolved this activity, remain poorly understood. Surprisingly, QMP is capable of repressing reproduction in non-target arthropods. This study showed that in Drosophila melanogaster QMP treatment mimics the starvation response, disrupting reproduction. QMP exposure induces an increase in food consumption and activation of checkpoints in the ovary that reduce fecundity and depresses insulin signalling. The magnitude of these effects is indistinguishable between QMP-treated and starved individuals. As QMP triggers a starvation response in an insect diverged from honeybees, it is proposed that QMP originally evolved by co-opting nutrition signalling pathways to regulate reproduction.

Hwangbo, D. S., Kwon, Y. J., Iwanaszko, M., Jiang, P., Abbasi, L., Wright, N., Alli, S., Hutchison, A. L., Dinner, A. R., Braun, R. I. and Allada, R. (2023). Dietary Restriction Impacts Peripheral Circadian Clock Output Important for Longevity in Drosophila. bioRxiv. PubMed ID: 36711760
Summary:
Circadian clocks may mediate lifespan extension by caloric or dietary restriction (DR). This study found that the core clock transcription factor Clock is crucial for a robust longevity and fecundity response to DR in Drosophila. To identify clock-controlled mediators, RNA-sequencing was performed from abdominal fat bodies across the 24 h day after just 5 days under control or DR diets. In contrast to more chronic DR regimens, no significant changes were detected in the rhythmic expression of core clock genes. Yet it was discovered that DR induced de novo rhythmicity or increased expression of rhythmic clock output genes. Network analysis revealed that DR increased network connectivity in one module comprised of genes encoding proteasome subunits. Adult, fat body specific RNAi knockdown demonstrated that proteasome subunits contribute to DR-mediated lifespan extension. Thus, clock control of output links DR-mediated changes in rhythmic transcription to lifespan extension.

Tuesday April 11th - Disease Models

Paul, M. S., Duncan, A. R., Genetti, C. A., Pan, H., Jackson, A., Grant, P. E., Shi, J., Pinelli, M., Brunetti-Pierri, N., Garza-Flores, A., Shahani, D., Saneto, R. P., Zampino, G., Leoni, C., Agolini, E., Novelli, A., Blumlein, U., Haack, T. B., Heinritz, W., Matzker, E., Alhaddad, B., Abou Jamra, R., Bartolomaeus, T., AlHamdan, S., Carapito, R., Isidor, B., Bahram, S., Ritter, A., Izumi, K., Shakked, B. P., Barel, O., Ben Zeev, B., Begtrup, A., Carere, D. A., Mullegama, S. V., Palculict, T. B., Calame, D. G., Schwan, K., Aycinena, A. R. P., Traberg, R., Douzgou, S., Pirt, H., Ismayilova, N., Banka, S., Chao, H. T. and Agrawal, P. B. (2023). Rare EIF4A2 variants are associated with a neurodevelopmental disorder characterized by intellectual disability, hypotonia, and epilepsy. Am J Hum Genet 110(1): 120-145. PubMed ID: 36528028
Summary:
Eukaryotic initiation factor-4A2 (EIF4A2) is an ATP-dependent RNA helicase and a member of the DEAD-box protein family that recognizes the 5' cap structure of mRNAs, allows mRNA to bind to the ribosome, and plays an important role in microRNA-regulated gene repression. This study reports on 15 individuals from 14 families presenting with global developmental delay, intellectual disability, hypotonia, epilepsy, and structural brain anomalies, all of whom have extremely rare de novo mono-allelic or inherited bi-allelic variants in EIF4A2. Neurodegeneration was predominantly reported in individuals with bi-allelic variants. Molecular modeling predicts these variants would perturb structural interactions in key protein domains. To determine the pathogenicity of the EIF4A2 variants in vivo, the mono-allelic variants were studied in Drosophila melanogaster and identified variant-specific behavioral and developmental defects. The fruit fly homolog of EIF4A2 is eIF4A, a negative regulator of decapentaplegic (dpp) signaling that regulates embryo patterning, eye and wing morphogenesis, and stem cell identity determination. A loss-of-function (LOF) rescue assay demonstrated a pupal lethality phenotype induced by loss of eIF4A, which was fully rescued with human EIF4A2 wild-type (WT) cDNA expression. In comparison, the EIF4A2 variant cDNAs failed or incompletely rescued the lethality. Overall, our findings reveal that EIF4A2 variants cause a genetic neurodevelopmental syndrome with both LOF and gain of function as underlying mechanisms.

Srivastava, S., Shaked, H. M., Gable, K., Gupta, S. D., Pan, X., Somashekarappa, N., Han, G., Mohassel, P., Gotkine, M., Doney, E., Goldenberg, P., Tan, Q. K. G., Gong, Y., Kleinstiver, B., Wishart, B., Cope, H., Pires, C. B., Stutzman, H., Spillmann, R. C., Sadjadi, R., Elpeleg, O., Lee, C. H., Bellen, H. J., Edvardson, S., Eichler, F. and Dunn, T. M. (2023). SPTSSA variants alter sphingolipid synthesis and cause a complex hereditary spastic paraplegia. Brain. PubMed ID: 36718090
Summary:
Sphingolipids are a diverse family of lipids with critical structural and signalling functions in the mammalian nervous system, where they are abundant in myelin membranes. Serine palmitoyltransferase, the enzyme that catalyses the rate-limiting reaction of sphingolipid synthesis, is comprised of multiple subunits including an activating subunit, SPTSSA. Sphingolipids are both essential and cytotoxic and their synthesis must therefore be tightly regulated. Key to the homeostatic regulation are the ORMDL proteins that are bound to serine palmitoyltransferase and mediate feedback inhibition of enzymatic activity when sphingolipid levels become excessive. Exome sequencing identified potential disease-causing variants in SPTSSA in three children presenting with a complex form of hereditary spastic paraplegia. The effect of these variants on the catalytic activity and homeostatic regulation of serine palmitoyltransferase was investigated in human embryonic kidney cells, patient fibroblasts and Drosophila. The results showed that two different pathogenic variants in SPTSSA caused a hereditary spastic paraplegia resulting in progressive motor disturbance with variable sensorineural hearing loss and language/cognitive dysfunction in three individuals. The variants in SPTSSA impaired the negative regulation of serine palmitoyltransferase by ORMDLs leading to excessive sphingolipid synthesis based on biochemical studies and in vivo studies in Drosophila. These findings support the pathogenicity of the SPTSSA variants and point to excessive sphingolipid synthesis due to impaired homeostatic regulation of serine palmitoyltransferase as responsible for defects in early brain development and function.

Reyes-Ortiz, A. M., Abud, E. M., Burns, M. S., Wu, J., Hernandez, S. J., McClure, N., Wang, K. Q., Schulz, C. J., Miramontes, R., Lau, A., Michael, N., Miyoshi, E., Van Vactor, D., Reidling, J. C., Blurton-Jones, M., Swarup, V., Poon, W. W., Lim, R. G. and Thompson, L. M. (2023). Single-nuclei transcriptome analysis of Huntington disease iPSC and mouse astrocytes implicates maturation and functional deficits. iScience 26(1): 105732. PubMed ID: 36590162
Summary:
Huntington disease (HD) is a neurodegenerative disorder caused by expanded CAG repeats in the huntingtin gene that alters cellular homeostasis, particularly in the striatum and cortex. Astrocyte signaling that establishes and maintains neuronal functions are often altered under pathological conditions. In this study single-nuclei RNA-sequencing was performed on human HD patient-induced pluripotent stem cell (iPSC)-derived astrocytes and on striatal and cortical tissue from R6/2 HD mice to investigate high-resolution HD astrocyte cell state transitions. Altered maturation and glutamate signaling were observed in HD human and mouse astrocytes. Human HD astrocytes also showed upregulated actin-mediated signaling, suggesting that some states may be cell-autonomous and human specific. In both species, astrogliogenesis transcription factors may drive HD astrocyte maturation deficits, which are supported by rescued climbing deficits in HD Drosophila with NFIA knockdown. Thus, dysregulated HD astrocyte states may induce dysfunctional astrocytic properties, in part due to maturation deficits influenced by astrogliogenesis transcription factor dysregulation.

Taminato, T., Takeuchi, T., Ueyama, M., Mori, K., Ikeda, M., Mochizuki, H. and Nagai, Y. (2023). Therapeutic reduction of GGGGCC repeat RNA levels by hnRNPA3 suppresses neurodegeneration in drosophila models of C9orf72-linked ALS/FTD. Hum Mol Genet. PubMed ID: 36611007
Summary:
The abnormal expansion of GGGGCC hexanucleotide repeats within the C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The accumulation of GGGGCC repeat-containing RNAs as RNA foci, and the deposition of dipeptide repeat proteins (DPR) produced from these repeat RNAs by unconventional translation are major pathological hallmarks of C9orf72-linked ALS/FTD (C9-ALS/FTD), and are both thought to play a crucial role in the pathogenesis of these diseases. Because GGGGCC repeat RNA is likely to be the most upstream therapeutic target in the pathogenic cascade of C9-ALS/FTD, lowering the cellular level of GGGGCC repeat RNA is expected to mitigate repeat RNA toxicity, and will therefore be a disease-modifying therapeutic strategy for the treatment of C9-ALS/FTD. This study demonstrated using a Drosophila model of C9-ALS/FTD that elevated expression of a subset of human RNA-binding proteins that bind to GGGGCC repeat RNA, including hnRNPA3, IGF2BP1, hnRNPA2B1, hnRNPR, and SF3B3, reduces the level of GGGGCC repeat RNA, resulting in the suppression of neurodegeneration. It was further shown that hnRNPA3-mediated reduction of GGGGCC repeat RNA suppresses disease pathology, such as RNA foci and DPR accumulation. These results demonstrate that hnRNPA3 and other RNA-binding proteins negatively regulate the level of GGGGCC repeat RNA, and mitigate repeat RNA toxicity in vivo, indicating the therapeutic potential of the repeat RNA-lowering approach mediated by endogenous RNA-binding proteins for the treatment of C9-ALS/FTD.

Shaposhnikov, M. V., Gorbunova, A. A., Zemskaya, N. V., Ulyasheva, N. S., Pakshina, N. R., Yakovleva, D. V. and Moskalev, A. (2023). Simultaneous activation of the hydrogen sulfide biosynthesis genes (CBS and CSE) induces sex-specific geroprotective effects in Drosophila melanogaster. Biogerontology. PubMed ID: 36662374
Summary:
Hydrogen sulfide (H(2)S) is one of the most important gasotransmitters that affect lifespan and provide resistance to adverse environmental conditions. This study investigated geroprotective effects of the individual and simultaneous overexpression of genes encoding key enzymes of H(2)S biosynthesis - cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE) on D. melanogaster model. Simultaneous overexpression of CBS and CSE resulted in additive (in males) and synergistic (in females) beneficial effects on median lifespan. Individual overexpression of CBS was associated with increased thermotolerance and decreased transcription level of genes encoding stress-responsive transcription factors HIF1 and Hsf, while individual overexpression of CSE was associated with increased resistance to paraquat. Simultaneous overexpression of both genes increased resistance to hyperthermia in old females or paraquat in old males. The obtained results suggest sex-specific epistatic interaction of CBS and CSE overexpression effects on longevity and stress resistance.

Tandon, S. and Sarkar, S. (2023). Glipizide ameliorates human poly(Q) mediated neurotoxicity by upregulating insulin signalling in Drosophila disease models. Biochem Biophys Res Commun 645: 88-96. PubMed ID: 36680941
Summary:
ncreasing reports suggest insulin signalling pathway as a putative drug target against polyglutamine [poly(Q)] disorders, such as Huntington's disease (HD), Spinocerebellar ataxias (SCA) 1, 2, 3 etc. However, studies on drug-based stimulation of insulin signalling cascade to mitigate poly(Q) pathogenesis are lacking. This study adopted an evidence-based approach to examine if some established insulin stimulating drug can be utilized to restrict poly(Q) aetiology in Drosophila disease models. For the first time, this study reports that glipizide, an FDA approved anti-diabetic drug upregulates insulin signalling in poly(Q) expressing tissues and restricts formation of inclusion bodies and neurodegeneration. Moreover, it reinstates the chromatin architecture by improving histone acetylation, which is otherwise abrogated due to poly(Q) toxicity. In view of the functional conservation of insulin signalling pathway in Drosophila and humans, this finding strongly suggests that glipizide can be repurposed as an effective treatment strategy against the neurodegenerative poly(Q) disorders. Also, with appropriate validation studies in mammalian disease models, glipizide could be subsequently considered for the clinical trials in human patients.

Monday April 10th - Neural Development

Nguyen, C. T., Nguyen, V. M. and Jeong, S. (2022). Regulation of Off-track bidirectional signaling by Semaphorin-1a and Wnt signaling in the Drosophila motor axon guidance. Insect Biochem Mol Biol 150: 103857. PubMed ID: 36244650
Summary:
Off-track receptor tyrosine kinase (OTK) has been shown to play an important role in the Drosophila motor axon pathfinding. The results of biochemical and genetic interactions previously suggested that OTK acts as a component of Semaphorin-1a/Plexin A (Sema-1a/PlexA) signaling during embryonic motor axon guidance and further showed that OTK binds to Wnt family members Wnt2 and Wnt4 and their common receptor Frizzled (Fz). However, the molecular mechanisms underlying the motor axon guidance function of OTK remain elusive. This study concludes that OTK mediates the forward and reverse signaling required for intersegmental nerve b (ISNb) motor axon pathfinding and it was also demonstrated that the loss of two copies of Sema-1a synergistically enhances the bypass phenotype observed in otk mutants. Furthermore, the amorphic wnt2 mutation resulted in increased premature branching phenotypes, and the loss of fz function caused a frequent inability of ISNb motor axons to defasciculate at specific choice points. Consistent with a previous study, wnt4 mutant axons were often defective in recognizing target muscles. Interestingly, the bypass phenotype of otk mutants was robustly suppressed by loss of function mutations in wnt2, wnt4, or fz. In contrast, total ISNb defects of otk were increased by the loss-of-function alleles in wnt2 and wnt4, but not fz. These findings indicate that OTK may participate in the crosstalk between the Sema-1a/PlexA and Wnt signaling pathways, thereby contributing to ISNb motor axon pathfinding and target recognition.

Xu, W., Kong, W., Gao, Z., Huang, E., Xie, W., Wang, S. and Rui, M. (2023). Establishment of a novel axon pruning model of Drosophila motor neuron. Biol Open 12(1). PubMed ID: 36606515
Summary:
Developmental neuronal pruning is a process by which neurons selectively remove excessive or unnecessary neurite without causing neuronal death. Importantly, this process is widely used for the refinement of neural circuits in both vertebrates and invertebrates, and may also contribute to the pathogenesis of neuropsychiatric disorders, such as autism and schizophrenia. In the peripheral nervous system (PNS), class IV dendritic arborization (da) sensory neurons of Drosophila, selectively remove the dendrites without losing their somas and axons, while the dendrites and axons of mushroom body (MB) γ neuron in the central nervous system (CNS) are eliminated by localized fragmentation during metamorphosis. Alternatively, dendrite pruning of ddaC neurons is usually investigated via live-cell imaging, while dissection and fixation are currently used for evaluating MB γ neuron axon pruning. Thus, an excellent model system to assess axon specific pruning directly via live-cell imaging remains elusive. This study reports that the Drosophila motor neuron offers a unique advantage for studying axon pruning. Interestingly, a long-range projecting axon bundle from soma at ventral nerve cord (VNC) undergoes degeneration rather than retraction during metamorphosis. Strikingly, the pruning process of the motor axon bundle is straightforward to investigate via live imaging and it occurs approximately at 22 h after pupal formation (APF), when axon bundles are completely cleared. Consistently, the classical axon pruning regulators in the Drosophila MB γ neuron, including TGF-β signaling, ecdysone signaling, JNK signaling, and the ubiquitin-proteasome system are also involved in governing motor axon pruning. Finally, these findings establish an unprecedented axon pruning mode that will serve to systematically screen and identify undiscovered axon pruning regulators.

Rubio-Ferrera, I., Clarembaux-Badell, L., Baladron-de-Juan, P., Berrocal-Rubio, M., Thor, S., Cobeta, I. M. and Benito-Sipos, J. (2023). Specification of the Drosophila Orcokinin A neurons by combinatorial coding. Cell Tissue Res 391(2): 269-286. PubMed ID: 36512054
Summary:
The central nervous system contains a daunting number of different cell types. Understanding how each cell acquires its fate remains a major challenge for neurobiology. The developing embryonic ventral nerve cord (VNC) of Drosophila melanogaster has been a powerful model system for unraveling the basic principles of cell fate specification. This pertains specifically to neuropeptide neurons, which typically are stereotypically generated in discrete subsets, allowing for unambiguous single-cell resolution in different genetic contexts. The specification of the OrcoA-LA neurons, characterized by the expression of the neuropeptide Orcokinin A and located laterally in the A1-A5 abdominal segments of the VNC, was studied. The progenitor neuroblast (NB; NB5-3) and the temporal window (castor/grainyhead) that generate the OrcoA-LA neurons were identified. The role of the Ubx, abd-A, and Abd-B Hox genes in the segment-specific generation of these neurons was studied. Additionally, these results indicate that the OrcoA-LA neurons are "Notch Off" cells, and neither programmed cell death nor the BMP pathway appears to be involved in their specification. Finally, a targeted genetic screen was performed of 485 genes known to be expressed in the CNS and nab, vg, and tsh were identified as crucial determinists for OrcoA-LA neurons. This work provides a new neuropeptidergic model that will allow for addressing new questions related to neuronal specification mechanisms in the future.

Singh, B. N., Tran, H., Kramer, J., Kirishenko, E., Changela, N., Wang, F., Feng, Y., Kumar, D., Tu, M., Liang, S., Lan, J., Bizet, M., Fuks, F. and Steward, R. (2023). Tet-dependent 5-hydroxymethyl-Cytosine modification of mRNA regulates the axon guidance genes robo2 and slit in Drosophila. bioRxiv. PubMed ID: 36711932
Summary:
Modifications of mRNA, especially methylation of adenosine, have recently drawn much attention. The much rarer modification, 5-hydroxymethylation of cytosine (5hmC), is not well understood and is the subject of this study. Vertebrate Tet proteins are 5-methylcytosine (5mC) hydroxylases enzymes catalyzing the transition of 5mC to 5hmC in DNA and have recently been shown to have the same function in messenger RNAs in both vertebrates and in Drosophila. The Tet gene is essential in Drosophila because Tet knock-out animals do not reach adulthood. This study describes the identification of Tet-target genes in the embryo and larval brain by determining Tet DNA-binding sites throughout the genome and by mapping the Tet-dependent 5hmrC modifications transcriptome-wide. 5hmrC-modified sites can be found along the entire transcript and are preferentially located at the promoter where they overlap with histone H3K4me3 peaks. The identified mRNAs are frequently involved in neuron and axon development and Tet knock-out led to a reduction of 5hmrC marks on specific mRNAs. Among the Tet-target genes were the robo2 receptor and its slit ligand that function in axon guidance in Drosophila and in vertebrates. Tet knock-out embryos show overlapping phenotypes with robo2 and are sensitized to reduced levels of slit. Both Robo2 and Slit protein levels were markedly reduced in Tet KO larval brains. These results establish a role for Tet-dependent 5hmrC in facilitating the translation of modified mRNAs, primarily in developing nerve cells.

Zang, Y., Chaudhari, K. and Bashaw, G. J. (2022). Tace/ADAM17 is a bi-directional regulator of axon guidance that coordinates distinct Frazzled and Dcc receptor signaling outputs. Cell Rep 41(10): 111785. PubMed ID: 36476876
Summary:
Frazzled (Fra) and deleted in colorectal cancer (Dcc) are homologous receptors that promote axon attraction in response to netrin. In Drosophila, Fra also acts independently of netrin by releasing an intracellular domain (ICD) that activates gene transcription. How neurons coordinate these pathways to make accurate guidance decisions is unclear. This study showed that the ADAM metalloprotease Tace cleaves Fra, and this instructs the switch between the two pathways. Genetic manipulations that either increase or decrease Tace levels disrupt midline crossing of commissural axons. These conflicting phenotypes reflect Tace's function as a bi-directional regulator of axon guidance, a function conserved in its vertebrate homolog ADAM17: while Tace induces the formation of the Fra ICD to activate transcription, excessive Tace cleavage of Fra and Dcc suppresses the response to netrin. It is proposed that Tace and ADAM17 are key regulators of midline axon guidance by establishing the balance between netrin-dependent and netrin-independent signaling.

Linskens, A., Doe, C. and Lee, K. (2022). Developmental origin of the Pair1 descending interneuron. MicroPubl Biol 2022. PubMed ID: 36606082
Summary:
Pair1 is part of a Drosophila larval locomotor circuit that promotes backward locomotion by inhibiting forward locomotion. It was hypothesized that lineage related neurons may function in neuronal circuits together. Testing this hypothesis requires knowing the progenitor of each neuron within this locomotor circuit, and this study focussed exclusively on Pair1. During Drosophila melanogaster embryogenesis, unique neuroblasts form by inheriting the spatial transcription factors (TFs) expressed in their birth location within the neuroectoderm. The Pair1 neurons were examined using immunofluorescence to determine which neuroblast the Pair1s derive from. The results show that Pair1 is derived from gnathal neuroblast 5-3 which expresses Gooseberry (Gsb) and Intermediate neuroblasts defective (Ind). When Gsb or Ind were overexpressed in the Pair1 lineage, extra neurons formed with similar Pair1 morphology.

Friday April 7th - Cytoskeleton and Junctions

Fang, C. T., Kuo, H. H., Amartuvshin, O., Hsu, H. J., Liu, S. L., Yao, J. S. and Yih, L. H. (2023). Inhibition of acetyl-CoA carboxylase impaired tubulin palmitoylation and induced spindle abnormalities. Cell Death Discov 9(1): 4. PubMed ID: 36617578
Summary:
Tubulin s-palmitoylation involves the thioesterification of a cysteine residue in tubulin with palmitate. The palmitate moiety is produced by the fatty acid synthesis pathway, which is rate-limited by acetyl-CoA carboxylase (ACC). While it is known that ACC is phosphorylated at serine 79 (pSer(79)) by AMPK and accumulates at the spindle pole (SP) during mitosis, a functional role for tubulin palmitoylation during mitosis has not been identified. This study found that modulating pSer(79)-ACC level at the SP using AMPK agonist and inhibitor induced spindle defects. Loss of ACC function induced spindle abnormalities in cell lines and in germ cells of the Drosophila germarium, and palmitic acid (PA) rescued the spindle defects in the cell line treated transiently with the ACC inhibitor, TOFA. Furthermore, inhibition of protein palmitoylating or depalmitoylating enzymes also induced spindle defects. Together, these data suggested that precisely regulated cellular palmitate level and protein palmitoylation may be required for accurate spindle assembly. It was then shown that tubulin was largely palmitoylated in interphase cells but less palmitoylated in mitotic cells. TOFA treatment diminished tubulin palmitoylation at doses that disrupt microtubule (MT) instability and cause spindle defects. Moreover, spindle MTs comprised of α-tubulins mutated at the reported palmitoylation site exhibited disrupted dynamic instability. It was also found that TOFA enhanced the MT-targeting drug-induced spindle abnormalities and cytotoxicity. Thus, this study reveals that precise regulation of ACC during mitosis impacts tubulin palmitoylation to delicately control MT dynamic instability and spindle assembly, thereby safeguarding nuclear and cell division.

Hou, Y., Zheng, S., Wu, Z., Augière, C., Morel, V., Cortier, E., Duteyrat, J. L., Zhang, Y., Chen, H., Peng, Y., Durand, B. and Wei, Q. (2023). Drosophila transition fibers are essential for IFT-dependent ciliary elongation but not basal body docking and ciliary budding. Curr Biol. PubMed ID: 36669498
Summary:
Cilia are highly conserved organelles critical for animal development and perception. Dysfunction of cilia has been linked to a wide spectrum of human genetic diseases, termed ciliopathies. Transition fibers (TFs) are striking ciliary base structures essential for cilia assembly. Vertebrates' TFs that originate from centriole distal appendages (DAs) mediate basal body docking to ciliary vesicles to initiate ciliogenesis and regulate the entry of ciliary proteins for axoneme assembly via intraflagellar transport (IFT) machinery. Although no distal appendages can be observed on Drosophila centrioles, three key TF proteins, FBF1, CEP164, and CEP89, have obvious homologs in Drosophila. This study aimed to compare their functions with their mammalian counterparts in Drosophila ciliogenesis. All three proteins were shown to be localized like TF proteins at the ciliary base in both sensory neurons and spermatocytes, the only two types of ciliated cells in flies. Fbf1 and Cep89 are essential for the formation of IFT-dependent neuronal cilia, but Cep164 is dispensable for ciliogenesis in flies. Strikingly, none are required for basal body docking and transition zone (TZ) assembly in IFT-dependent neuronal cilia or IFT-independent spermatocyte cilia. Furthermore, it was demonstrate that Unc is essential to recruit all three TF proteins and establish a hierarchical order, with Cep89 acting on Fbf1. Collectively, these results not only demonstrate that TF proteins are required for IFT-dependent ciliogenesis in Drosophila, in agreement with an evolutionarily conserved function of these proteins in regulating ciliary protein entry, but also that the basal body docking function of TFs has diverged during evolution.

Ikawa, K., Ishihara, S., Tamori, Y. and Sugimura, K. (2023). Attachment and detachment of cortical myosin regulates cell junction exchange during cell rearrangement in the Drosophila wing epithelium. Curr Biol 33(2): 263-275. PubMed ID: 36543168
Summary:
Epithelial cells remodel cell adhesion and change their neighbors to shape a tissue. This cellular rearrangement proceeds in three steps: the shrinkage of a junction, exchange of junctions, and elongation of the newly generated junction. By combining live imaging and physical modeling, this study showed that the formation of myosin-II (myo-II) cables around the cell vertices underlies the exchange of junctions in the Drosophila wing epithelium. The local and transient detachment of myo-II from the cell cortex is regulated by the LIM domain-containing protein Jub and the tricellular septate junction protein M6. Moreover, M6 shifts to the adherens junction plane on jub RNAi and that Jub is persistently retained at reconnecting junctions in m6 RNAi cells. This interplay between Jub and M6 can depend on the junction length and thereby couples the detachment of cortical myo-II cables and the shrinkage/elongation of the junction during cell rearrangement. Furthermore, this study developed a mechanical model based on the wetting theory and clarified how the physical properties of myo-II cables are integrated with the junction geometry to induce the transition between the attached and detached states and support the unidirectionality of cell rearrangement. Collectively, this study elucidates the orchestration of geometry, mechanics, and signaling for exchanging junctions.

Perez-Vale, K. Z., Yow, K. D., Gurley, N. J., Greene, M. and Peifer, M. (2023). Rap1 regulates apical contractility to allow embryonic morphogenesis without tissue disruption and acts in part via Canoe-independent mechanisms. Mol Biol Cell 34(1): ar7. PubMed ID: 36287827
Summary:
Embryonic morphogenesis is powered by dramatic changes in cell shape and arrangement driven by the cytoskeleton and its connections to adherens junctions. This requires robust linkage allowing morphogenesis without disrupting tissue integrity. The small GTPase Rap1 is a key regulator of cell adhesion, controlling both cadherin-mediated and integrin-mediated processes.This study has defined multiple roles in morphogenesis for one Rap1 effector, Canoe/Afadin, which ensures robust junction-cytoskeletal linkage. It is now asked what mechanisms regulate Canoe and other junction-cytoskeletal linkers during Drosophila morphogenesis, defining roles for Rap1 and one of its guanine nucleotide exchange factor (GEF) regulators, Dizzy. Rap1 uses Canoe as one effector, regulating junctional planar polarity. However, Rap1 has additional roles in junctional protein localization and balanced apical constriction-in its absence, Bazooka/Par3 localization is fragmented, and cells next to mitotic cells apically constrict and invaginate, disrupting epidermal integrity. In contrast, the GEF Dizzy has phenotypes similar to but slightly less severe than Canoe loss, suggesting that this GEF regulates Rap1 action via Canoe. Taken together, these data reveal that Rap1 is a crucial regulator of morphogenesis, likely acting in parallel via Canoe and other effectors, and that different Rap1 GEFs regulate distinct functions of Rap1.

Sheppard, L., Green, D. G., Lerchbaumer, G., Rothenberg, K. E., Fernandez-Gonzalez, R. and Tepass, U. (2023). The alpha-Catenin mechanosensing M region is required for cell adhesion during tissue morphogenesis. J Cell Biol 222(2). PubMed ID: 36520419
Summary:
α-Catenin couples the cadherin-catenin complex to the actin cytoskeleton. The mechanosensitive α-Catenin M region undergoes conformational changes upon application of force to recruit interaction partners. This study took advantage of the tension landscape in the Drosophila embryo to define three different states of α-Catenin mechanosensing in support of cell adhesion. Low-, medium-, and high-tension contacts showed a corresponding recruitment of Vinculin and Ajuba, which was dependent on the α-Catenin M region. In contrast, the Afadin homolog Canoe acts in parallel to α-Catenin at bicellular low- and medium-tension junctions but requires an interaction with α-Catenin for its tension-sensitive enrichment at high-tension tricellular junctions. Individual M region domains make complex contributions to cell adhesion through their impact on interaction partner recruitment, and redundancies with the function of Canoe. These data argue that α-Catenin and its interaction partners are part of a cooperative and partially redundant mechanoresponsive network that supports AJs remodeling during morphogenesis.

Schueder, F., Mangeol, P., Chan, E. H., Rees, R., Schunemann, J., Jungmann, R., Gorlich, D. and Schnorrer, F. (2023). Nanobodies combined with DNA-PAINT super-resolution reveal a staggered titin nanoarchitecture in flight muscles. Elife 12. PubMed ID: 36645127
Summary:
Sarcomeres are the force-producing units of all striated muscles. Their nanoarchitecture critically depends on the large titinprotein, which in vertebrates spans from the sarcomeric Z-disc to the M-band and hence links actin and myosin filaments stably together. This ensures sarcomeric integrity and determines the length of vertebrate sarcomeres. However, the instructive role of titins for sarcomeric architecture outside of vertebrates is not as well understood. This study used a series of nanobodies, the Drosophila titin nanobody toolbox, recognising specific domains of the two Drosophila titin homologs Sallimus and Projectin to determine their precise location in intact flight muscles. By combining nanobodies with DNA-PAINT super-resolution microscopy, this study found that, similar to vertebrate titin, Sallimus bridges across the flight muscle I-band, whereas Projectin is located at the beginning of the A-band. Interestingly, the ends of both proteins overlap at the I-band/A-band border, revealing a staggered organisation of the two Drosophila titin homologs. This architecture may help to stably anchor Sallimus at the myosin filament and hence ensure efficient force transduction during flight.

Thursday April 6th - Adult Neural Development and Function

α-Catenin couples the cadherin-catenin complex to the actin cytoskeleton. The mechanosensitive α-Catenin M region undergoes conformational changes upon application of force to recruit interaction partners. This study took advantage of the tension landscape in the Drosophila embryo to define three different states of α-Catenin mechanosensing in support of cell adhesion. Low-, medium-, and high-tension contacts showed a corresponding recruitment of Vinculin and Ajuba, which was dependent on the α-Catenin M region. In contrast, the Afadin homolog Canoe acts in parallel to α-Catenin at bicellular low- and medium-tension junctions but requires an interaction with α-Catenin for its tension-sensitive enrichment at high-tension tricellular junctions. Individual M region domains make complex contributions to cell adhesion through their impact on interaction partner recruitment, and redundancies with the function of Canoe. These data argue that α-Catenin and its interaction partners are part of a cooperative and partially redundant mechanoresponsive network that supports AJs remodeling during morphogenesis.

Perez-Vale, K. Z., Yow, K. D., Gurley, N. J., Greene, M. and Peifer, M. (2023). Rap1 regulates apical contractility to allow embryonic morphogenesis without tissue disruption and acts in part via Canoe-independent mechanisms. Curr Biol 33(2): 263-275. Mol Biol Cell 34(1): ar7. PubMed ID: 36287827
Summary:
Embryonic morphogenesis is powered by dramatic changes in cell shape and arrangement driven by the cytoskeleton and its connections to adherens junctions. This requires robust linkage allowing morphogenesis without disrupting tissue integrity. The small GTPase Rap1 is a key regulator of cell adhesion, controlling both cadherin-mediated and integrin-mediated processes. We have defined multiple roles in morphogenesis for one Rap1 effector, Canoe/Afadin, which ensures robust junction-cytoskeletal linkage. We now ask what mechanisms regulate Canoe and other junction-cytoskeletal linkers during Drosophila morphogenesis, defining roles for Rap1 and one of its guanine nucleotide exchange factor (GEF) regulators, Dizzy. Rap1 uses Canoe as one effector, regulating junctional planar polarity. However, Rap1 has additional roles in junctional protein localization and balanced apical constriction-in its absence, Bazooka/Par3 localization is fragmented, and cells next to mitotic cells apically constrict and invaginate, disrupting epidermal integrity. In contrast, the GEF Dizzy has phenotypes similar to but slightly less severe than Canoe loss, suggesting that this GEF regulates Rap1 action via Canoe. Taken together, these data reveal that Rap1 is a crucial regulator of morphogenesis, likely acting in parallel via Canoe and other effectors, and that different Rap1 GEFs regulate distinct functions of Rap1.

Schueder, F., Mangeol, P., Chan, E. H., Rees, R., Schunemann, J., Jungmann, R., Gorlich, D. and Schnorrer, F. (2023). Nanobodies combined with DNA-PAINT super-resolution reveal a staggered titin nanoarchitecture in flight muscles. Curr Biol 33(2): 263-275. Elife 12. PubMed ID: 36645127
Summary:
Sarcomeres are the force-producing units of all striated muscles. Their nanoarchitecture critically depends on the large titinprotein, which in vertebrates spans from the sarcomeric Z-disc to the M-band and hence links actin and myosin filaments stably together. This ensures sarcomeric integrity and determines the length of vertebrate sarcomeres. However, the instructive role of titins for sarcomeric architecture outside of vertebrates is not as well understood. This study used a series of nanobodies, the Drosophila titin nanobody toolbox, recognising specific domains of the two Drosophila titin homologs Sallimus and Projectin to determine their precise location in intact flight muscles. By combining nanobodies with DNA-PAINT super-resolution microscopy, this study found that, similar to vertebrate titin, Sallimus bridges across the flight muscle I-band, whereas Projectin is located at the beginning of the A-band. Interestingly, the ends of both proteins overlap at the I-band/A-band border, revealing a staggered organisation of the two Drosophila titin homologs. This architecture may help to stably anchor Sallimus at the myosin filament and hence ensure efficient force transduction during flight.

Pasnuri, N., Jaiswal, M., Ray, K. and Mazumder, A. (2023). Buffered EGFR signaling regulated by spitz-to-argos expression ratio is a critical factor for patterning the Drosophila eye. PLoS Genet 19(2): e1010622. PubMed ID: 36730442
Summary:
The Epidermal Growth Factor Receptor (EGFR) signaling pathway plays a critical role in regulating tissue patterning. Drosophila EGFR signaling achieves specificity through multiple ligands and feedback loops to finetune signaling outcomes spatiotemporally. The principal Drosophila EGF ligand, cleaved Spitz, and the negative feedback regulator, Argos are diffusible and can act both in a cell autonomous and non-autonomous manner. The expression dose of Spitz and Argos early in photoreceptor cell fate determination has been shown to be critical in patterning the Drosophila eye, but the exact identity of the cells expressing these genes in the larval eye disc has been elusive. Using single molecule RNA Fluorescence in situ Hybridization (smFISH), this study revealed an intriguing differential expression of spitz and argos mRNA in the Drosophila third instar eye imaginal disc indicative of directional non-autonomous EGFR signaling. By genetically tuning EGFR signaling, it was shown that rather than absolute levels of expression, the ratio of expression of spitz-to-argos to be a critical determinant of the final adult eye phenotype. Proximate effects on EGFR signaling in terms of cell cycle and differentiation markers are affected differently in the different perturbations. Proper ommatidial patterning is robust to thresholds around a tightly maintained wildtype spitz-to-argos ratio, and breaks down beyond. This provides a powerful instance of developmental buffering against gene expression fluctuations.

Schenk, J. E. and Gaudry, Q. (2023). Nonspiking Interneurons in the Drosophila Antennal Lobe Exhibit Spatially Restricted Activity. eNeuro 10(1). PubMed ID: 36650069
Summary:
Inhibitory interneurons are important for neuronal circuit function. They regulate sensory inputs and enhance output discriminability. Often, the identities of interneurons can be determined by location and morphology, which can have implications for their functions. While most interneurons fire traditional action potentials, many are nonspiking. These can be seen in insect olfaction and the vertebrate retina. This study presents the novel observation of nonspiking inhibitory interneurons in the antennal lobe (AL) of the adult fruit fly, Drosophila melanogaster These neurons have a morphology where they innervate a patchwork of glomeruli. Electrophysiology was used to determine whether their nonspiking characteristic is because of a lack of sodium current. Immunohistochemsitry and in situ hybridization were used to show this is likely achieved through translational regulation of the voltage-gated sodium channel gene, para Using in vivo calcium imaging, this study explored how these cells respond to odors, finding regional isolation in their responses' spatial patterns. Further, their response patterns were dependent on both odor identity and concentration. Thus, we surmise these neurons are electrotonically compartmentalized such that activation of the neurites in one region does not propagate across the whole antennal lobe. It is proposed that these neurons may be the source of intraglomerular inhibition in the AL and may contribute to regulation of spontaneous activity within glomeruli.

Palmateer, C. M., Artikis, C., Brovero, S. G., Friedman, B., Gresham, A. and Arbeitman, M. N. (2023). Single-cell transcriptome profiles of Drosophila fruitless-expressing neurons from both sexes. Elife 12. PubMed ID: 36724009
Summary:
Drosophila melanogaster reproductive behaviors are orchestrated by fruitless neurons. Single-cell RNA-sequencing was performed on pupal neurons that produce sex-specifically spliced fru transcripts, the fru P1-expressing neurons. Uniform Manifold Approximation and Projection (UMAP) with clustering generates an atlas containing 113 clusters. While the male and female neurons overlap in UMAP space, more than half the clusters have sex differences in neuron number, and nearly all clusters display sex-differential expression. Based on an examination of enriched marker genes, clusters were annotated as circadian clock neurons, mushroom body Kenyon cell neurons, neurotransmitter- and/or neuropeptide-producing, and those that express doublesex. Marker gene analyses also show that genes that encode members of the immunoglobulin superfamily of cell adhesion molecules, transcription factors, neuropeptides, neuropeptide receptors, and Wnts have unique patterns of enriched expression across the clusters. In vivo spatial gene expression links to the clusters are examined. A functional analysis of fru P1 circadian neurons shows they have dimorphic roles in activity and period length. Given that most clusters are comprised of male and female neurons indicates that the sexes have fru P1 neurons with common gene expression programs. Sex-specific expression is overlaid on this program, to build the potential for vastly different sex-specific behaviors.

Shang, J., Tang, G., Yang, J., Lu, M., Wang, C. Z. and Wang, C. (2023). Sensing of a spore surface protein by a Drosophila chemosensory protein induces behavioral defense against fungal parasitic infections. Curr Biol 33(2): 276-286.e275. PubMed ID: 36423638
Summary:
In addition to innate immunity in a physiological context, insects have evolved behavioral defenses against parasite attacks. This study reports that Drosophila can sense the CFEM (common in fungal extracellular membrane) protein Mcdc9, which acts as a negative virulence factor of the entomopathogenic fungus Metarhizium robertsii. The individual deletions of 18 CFEM genes in Metarhizium followed by fly infection identified three null mutants that could kill the flies more quickly than the wild-type strain, among which Mcdc9 can coat fungal spores and interact with the fly chemosensory protein CheA75a. The deletion of Mcdc9 in the fungus or the knockdown of CheA75a in flies had a similar effect, in which a greater number of fungal spores were left on flies than on the respective controls after topical infection. Thus, similar to the accelerated death of the wild-type flies treated with ΔMcdc9, the CheA75a(RNAi) flies succumbed more quickly than the control insects topically challenged with the wild-type strain. The CheA75a gene is highly transcribed in fly legs and wings, and positive electrophysiological responses were evidenced in tarsal sensilla after stimulation with the Mcdc9 protein. The results imply that this CFEM protein could be sensed as a contact elicitor inducing the hygienic behavior of flies against fungal parasitic infection, which reveals a previously unsuspected mechanism of fungus-insect interactions.

Shekar, A., Mabry, S. J., Cheng, M. H., Aguilar, J. I., Patel, S., Zanella, D., Saleeby, D. P., Zhu, Y., Romanazzi, T., Ulery-Reynolds, P., Bahar, I., Carter, A. M., Matthies, H. J. G. and Galli, A. (2023). Syntaxin 1 Ser(14) phosphorylation is required for nonvesicular dopamine release. Sci Adv 9(2): eadd8417. PubMed ID: 36630507
Summary:
Amphetamine (AMPH) is a psychostimulant that is commonly abused. The stimulant properties of AMPH are associated with its ability to increase dopamine (DA) neurotransmission. This increase is promoted by nonvesicular DA release mediated by reversal of DA transporter (DAT) function. Syntaxin 1 (Stx1) is a SNARE protein that is phosphorylated at Ser(14) by casein kinase II. This study shows that Stx1 phosphorylation is critical for AMPH-induced nonvesicular DA release and, in Drosophila melanogaster, regulates the expression of AMPH-induced preference and sexual motivation. Molecular dynamics simulations of the DAT/Stx1 complex demonstrate that phosphorylation of these proteins is pivotal for DAT to dwell in a DA releasing state. This state is characterized by the breakdown of two key salt bridges within the DAT intracellular gate, causing the opening and hydration of the DAT intracellular vestibule, allowing DA to bind from the cytosol, a mechanism that is hypothesized to underlie nonvesicular DA release.

Pardo-Garcia, T. R., Gu, K., Woerner, R. K. R. and Dus, M. (2023). Food memory circuits regulate eating and energy balance. Curr Biol 33(2): 215-227. PubMed ID: 36528025
Summary:
In mammals, learning circuits play an essential role in energy balance by creating associations between sensory cues and the rewarding qualities of food. This process is altered by diet-induced obesity, but the causes and mechanisms are poorly understood. This study exploited the relative simplicity and wealth of knowledge about the D. melanogaster reinforcement learning network, the mushroom body, in order to study the relationship between the dietary environment, dopamine-induced plasticity, and food associations. Flies that are fed a high-sugar diet were shown to be unable make associations between sensory cues and the rewarding properties of sugar. This deficit was caused by diet exposure, not fat accumulation, and specifically by lower dopamine-induced plasticity onto mushroom body output neurons (MBONs) during learning. Importantly, food memories dynamically tune the output of MBONs during eating, which instead remains fixed in sugar-diet animals. Interestingly, manipulating the activity of MBONs influenced eating and fat mass, depending on the diet. Altogether, this work advances fundamental understanding of the mechanisms, causes, and consequences of the dietary environment on reinforcement learning and ingestive behavior.

Wednesday April 5th - Disease Models

Ochoa, E., Ramirez, P., Gonzalez, E., De Mange, J., Ray, W. J., Bieniek, K. F. and Frost, B. (2023). Pathogenic tau-induced transposable element-derived dsRNA drives neuroinflammation. Sci Adv 9(1): eabq5423. PubMed ID: 36608133
Summary:
Deposition of tau protein aggregates in the brain of affected individuals is a defining feature of "tauopathies," including Alzheimer's disease. Studies of human brain tissue and various model systems of tauopathy report that toxic forms of tau negatively affect nuclear and genomic architecture, identifying pathogenic tau-induced heterochromatin decondensation and consequent retrotransposon activation as a causal mediator of neurodegeneration. On the basis of their similarity to retroviruses, retrotransposons drive neuroinflammation via toxic intermediates, including double-stranded RNA (dsRNA). dsRNA and dsRNA sensing machinery were found to be elevated in astrocytes of postmortem brain tissue from patients with Alzheimer's disease and progressive supranuclear palsy and in brains of tau transgenic mice. Using a Drosophila model of tauopathy, this study identified specific tau-induced retrotransposons that form dsRNA and find that pathogenic tau and heterochromatin decondensation causally drive dsRNA-mediated neurodegeneration and neuroinflammation. This study suggests that pathogenic tau-induced heterochromatin decondensation and retrotransposon activation cause elevation of inflammatory, transposable element-derived dsRNA in the adult brain.

Mo, L., Li, R., He, C., Chen, Q., Xu, C., Shen, L., Chen, K. and Wu, Y. (2023). Hedgehog pathway is negatively regulated during the development of Drosophila melanogaster PheRS-m (Drosophila homologs gene of human FARS2) mutants. Hum Cell 36(1): 121-131. PubMed ID: 36205831
Summary:
Hereditary spastic paraplegia (HSP) is a neurodegeneration disease, one of the reasons is caused by autosomal recessive missense mutation of the karyogene that encodes phenylalanyl-tRNA synthetase 2, mitochondrial (FARS2). However, the molecular mechanism underlying FARS2-mediated HSP progression is unknown. Mitochondrial phenylalanyl-tRNA synthetase gene (PheRS-m) is the Drosophila melanogaster homolog gene of human FARS2. This study constructed a Drosophila HSP missense mutation model and a PheRS-m knockout model. Some of the mutant fly phenotypes included developmental delay, shortened lifespan, wing-structure abnormalities and decreased mobility. RNA-sequencing results revealed a relationship between abnormal phenotypes and the hedgehog (Hh) pathway. A qRT-PCR assay was used to determine the key genes (ptc, hib, and slmb) of the Hh pathway that exhibited increased expression during different developmental stages. It was demonstrated that Hh signaling transduction is negatively regulated during the developmental stages of PheRS-m mutants but positively regulated during adulthood. By inducing the agonist and inhibitor of Hh pathway in PheRS-m larvae, the developmental delay in mutants can be partly salvaged or postponed. Collectively, these findings indicate that Hh signaling negatively regulates the development of PheRS-m mutants, subsequently leading to developmental delay.

Oliveira, A. C., Santos, M., Pinho, M. and Lopes, C. S. (2023). String/Cdc25 phosphatase is a suppressor of Tau-associated neurodegeneration. Dis Model Mech 16(1). PubMed ID: 36601903
Summary:
Tau pathology is defined by the intracellular accumulation of abnormally phosphorylated Tau (MAPT) and is prevalent in several neurodegenerative disorders. The identification of modulators of Tau abnormal phosphorylation and aggregation is key to understanding disease progression and developing targeted therapeutic approaches. This study identified String (Stg)/Cdc25 phosphatase as a suppressor of abnormal Tau phosphorylation and associated toxicity. Using a Drosophila model of tauopathy, it was shown that Tau dephosphorylation by Stg/Cdc25 correlates with reduced Tau oligomerization, brain vacuolization and locomotor deficits in flies. Moreover, using a disease mimetic model, evidence is provided that Stg/Cdc25 reduces Tau phosphorylation levels independently of Tau aggregation status and delays neurodegeneration progression in the fly. These findings uncover a role for Stg/Cdc25 phosphatases as regulators of Tau biology that extends beyond their well-characterized function as cell-cycle regulators during cell proliferation, and indicate Stg/Cdc25-based approaches as promising entry points to target abnormal Tau phosphorylation.

Merino, M. M. (2023). Azot expression in the Drosophila gut modulates organismal lifespan. Commun Integr Biol 16(1): 2156735. PubMed ID: 36606245
Summary:
Cell Competition emerged in Drosophila as an unexpected phenomenon, when confronted clones of fit vs unfit cells genetically induced. During the last decade, it has been shown that this mechanism is physiologically active in Drosophila and higher organisms. In Drosophila, Flower (Fwe) eliminates unfit cells during development, regeneration and disease states. Furthermore, studies suggest that Fwe signaling is required to eliminate accumulated unfit cells during adulthood extending Drosophila lifespan. Indeed, ahuizotl (azot) mutants accumulate unfit cells during adulthood and after physical insults in the brain and other epithelial tissues, showing a decrease in organismal lifespan. On the contrary, flies carrying three functional copies of the gene, unfit cell culling seems to be more efficient and show an increase in lifespan. During aging, Azot is required for the elimination of unfit cells, however, the specific organs modulating organismal lifespan by Azot remain unknown. This study found a potential connection between gut-specific Azot expression and lifespan which may uncover a more widespread organ-specific mechanism modulating organismal survival.

Megat, S., Mora, N., Sanogo, J., Roman, O., Catanese, A., Alami, N. O., Freischmidt, A., Mingaj, X., De Calbiac, H., Muratet, F., Dirrig-Grosch, S., Dieterle, S., Van Bakel, N., Müller, K., Sieverding, K., Weishaupt, J., Andersen, P. M., Weber, M., Neuwirth, C., Margelisch, M., Sommacal, A., Van Eijk, K. R., Veldink, J. H., Lautrette, G., Couratier, P., Camuzat, A., Le Ber, I., Grassano, M., Chio, A., Boeckers, T., Ludolph, A. C., Roselli, F., Yilmazer-Hanke, D., Millecamps, S., Kabashi, E., Storkebaum, E., Sellier, C. and Dupuis, L. (2023). Integrative genetic analysis illuminates ALS heritability and identifies risk genes. Nat Commun 14(1): 342. PubMed ID: 36670122
Summary:
Amyotrophic lateral sclerosis (ALS) has substantial heritability, in part shared with fronto-temporal dementia (FTD). This study shows that ALS heritability is enriched in splicing variants and in binding sites of 6 RNA-binding proteins including TDP-43 and FUS. A transcriptome wide association study (TWAS) identified 6 loci associated with ALS, including in NUP50 encoding for the nucleopore basket protein NUP50. Independently, rare variants in NUP50 were associated with ALS risk in a cohort of 9,390 ALS/FTD patients and 4,594 controls. Cells from one patient carrying a NUP50 frameshift mutation displayed a decreased level of NUP50. Loss of NUP50 leads to death of cultured neurons, and motor defects in Drosophila and zebrafish. Thus, this study identifies alterations in splicing in neurons as critical in ALS and provides genetic evidence linking nuclear pore defects to ALS.

Pallares, L. F., Lea, A. J., Han, C., Filippova, E. V., Andolfatto, P. and Ayroles, J. F. (2023). Dietary stress remodels the genetic architecture of lifespan variation in outbred Drosophila. Nat Genet 55(1): 123-129. PubMed ID: 36550361
Summary:
Evolutionary theory suggests that lifespan-reducing alleles should be purged from the gene pool, and yet decades of genome-wide association and model organism studies have shown that they persist. One potential explanation is that alleles that regulate lifespan do so only in certain environmental contexts. This study exposed outbred Drosophila to control and high-sugar diets and genotyped more than 10,000 adult flies to track allele frequency changes over the course of a single adult lifespan. Thousands of lifespan-associated alleles associated with early versus late-life trade-offs, late-onset effects and genotype-by-environment interactions. Remarkably, a third of lifespan-associated genetic variation had environmentally dependent effects on lifespan. Lifespan-reducing alleles are often recently derived, have stronger effects on a high-sugar diet and show signatures of selection in wild Drosophila populations, consistent with the evolutionary mismatch hypothesis. These results provide insight into the highly polygenic and context-dependent genetic architecture of lifespan variation and the evolutionary processes that shape this key trait.

Tuesday April 4th - Adult Neural Development and Function

Lones, L. and DiAntonio, A. (2023). SIK3 and Wnk converge on Fray to regulate glial K+ buffering and seizure susceptibility. PLoS Genet 19(1): e1010581. PubMed ID: 36626385
Summary:
Glial cells play a critical role in maintaining homeostatic ion concentration gradients. Salt-inducible kinase 3 (SIK3) regulates a gene expression program that controls K+ buffering in glia, and upregulation of this pathway suppresses seizure behavior in the eag, Shaker hyperexcitability mutant. This study show that boosting the glial SIK3 K+ buffering pathway suppresses seizures in three additional molecularly diverse hyperexcitable mutants, highlighting the therapeutic potential of upregulating glial K+ buffering. Additional mechanisms regulating glial K+ buffering were explored. Fray, a transcriptional target of the SIK3 K+ buffering program, is a kinase that promotes K+ uptake by activating the Na+/K+/Cl- co-transporter, Ncc69. The href="http://flybase.org/reports/FBgn0037098">Wnk kinase phosphorylates Fray in Drosophila glia and that this activity is required to promote K+ buffering. This identifies Fray as a convergence point between the SIK3-dependent transcriptional program and Wnk-dependent post-translational regulation. Bypassing both regulatory mechanisms via overexpression of a constitutively active Fray in glia is sufficient to robustly suppress seizure behavior in multiple Drosophila models of hyperexcitability. Finally, cortex glia were identified as a critical cell type for regulation of seizure susceptibility, as boosting K+ buffering via expression of activated Fray exclusively in these cells is sufficient to suppress seizure behavior. These findings highlight Fray as a key convergence point for distinct K+ buffering regulatory mechanisms and cortex glia as an important locus for control of neuronal excitability.

Maiya, R., Dey, S., Ray, K. and Menon, G. I. (2023). The interplay of active and passive mechanisms in slow axonal transport. Biophys J 122(2): 333-345. PubMed ID: 36502274
Summary:
A combination of intermittent active movement of transient aggregates and a paused state that intervenes between periods of active transport has been proposed to underlie the slow, directed transport of soluble proteins in axons. A component of passive diffusion in the axoplasm may also contribute to slow axonal transport, although quantitative estimates of the relative contributions of diffusive and active movement in the slow transport of a soluble protein, and in particular how they might vary across developmental stages, are lacking. This work proposes and studies a model for slow axonal transport, addressing data from bleach recovery measurements on a small, soluble, protein, choline acetyltransferase, in thin axons of the lateral chordotonal (lch5) sensory neurons of Drosophila. Choline acetyltransferase is mainly present in soluble form in the axon and catalyzes the acetylation of choline at the synapse. It does not form particulate structures in axons and moves at rates characteristic of slow component b (≈ 1-10 mm/day or 0.01-0.1 &mi;m/s). Using this model, which incorporates active transport with paused and/or diffusive states, bleach recovery, transport rates, and cargo trajectories obtained through kymographs were predicted, comparing these with experimental observations at different developmental stages. Changes were shown in the diffusive fraction of cargo during these developmental stages dominate bleach recovery, and a combination of active motion with a paused state alone could reproduce the data. Predictions of the model were compared with results from photoactivation experiments. The importance of the diffusive state in reproducing the bleach recovery signal in the slow axonal transport of small soluble proteins is our central result.

Mannino, M. C., Cassidy, M. B., Florez, S., Rusan, Z., Chakraborty, S. and Schoborg, T. (2023). The neurodevelopmental transcriptome of the Drosophila melanogaster microcephaly gene abnormal spindle reveals a role for temporal transcription factors and the immune system in regulating brain size. bioRxiv. PubMed ID: 36711768
Summary:
The coordination of cellular behaviors during neurodevelopment is critical for determining the form, function, and size of the central nervous system. Mutations in the vertebrate Abnormal Spindle-Like, Microcephaly Associated (ASPM) gene and its Drosophila melanogaster ortholog abnormal spindle (asp) lead to microcephaly, a reduction in overall brain size whose etiology remains poorly defined. This study provides the neurodevelopmental transcriptional landscape for a Drosophila model for autosomal recessive primary microcephaly (MCPH) and extend the findings into the functional realm in an attempt to identify the key cellular mechanisms responsible for Asp-dependent brain growth and development. Multiple transcriptomic signatures were identified, including new patterns of co-expressed genes in the developing CNS. Defects in optic lobe neurogenesis were detected in larval brains through downregulation of temporal transcription factors (tTFs) and Notch signaling targets, which correlated with a significant reduction in brain size and total cell numbers during the neurogenic window of development. Inflammation was found as a hallmark of asp MCPH brains, detectable throughout every stage of CNS development, which also contributes to the brain size phenotype. Finally, we show that apoptosis is not a primary driver of the asp MCPH phenotype, further highlighting an intrinsic Asp-dependent neurogenesis promotion mechanism that is independent of cell death. Collectively, these results suggest that the etiology of asp MCPH is complex and that a comprehensive view of the cellular basis of the disorder requires an understanding of how multiple pathway inputs collectively determine the microcephaly phenotype.

Monagas-Valentin, P., Bridger, R., Chandel, I., Koff, M., Novikov, B., Schroeder, P., Wells, L. and Panin, V. (2023). Protein Tyrosine Phosphatase 69D is a substrate of Protein O-Mannosyltransferases 1-2 that is required for the wiring of sensory axons in Drosophila. J Biol Chem: 102890. PubMed ID: 36634851
Summary:
Mutations in Protein O-Mannosyltransferases (POMTs) result in severe brain defects and congenital muscular dystrophies characterized by abnormal glycosylation of α-Dystroglycan (α-Dg). However, neurological phenotypes of POMT mutants are not well understood, and the functional substrates of POMTs other than α-Dg remain unknown. Using a Drosophila model, this study revealed that Dg alone cannot account for the phenotypes of POMT mutants, and Receptor Protein Tyrosine Phosphatase 69D (PTP69D) was identified as a gene interacting with POMT genes in producing the abdomen rotation phenotype. Using RNAi-mediated knockdown, mutant alleles, and a dominant-negative form of PTP69D, this study reveal that PTP69D is required for the wiring of larval sensory axons. It was also found that PTP69D and POMT genes interact in this process, and that their interactions lead to complex synergistic or antagonistic effects on axon wiring phenotypes, depending on the mode of genetic manipulation. Using glycoproteomic approaches, the glycosylation was further characterized of the PTP69D transgenic construct expressed in genetic strains with different levels of POMT activity. The PTP69D construct carries many O-linked hexose modifications when expressed in Drosophila with wild-type or ectopically upregulated expression of POMTs, but these modifications were absent in POMT mutants, suggesting that PTP69D is a substrate of POMT-mediated O-mannosylation. Taken together, our results indicate that PTP69D is a novel functional substrate of POMTs that is required for axon connectivity. This mechanism of POMT-mediated regulation of RPTP functions could potentially be conserved in mammals and may shed new light on the etiology of neurological defects in muscular dystrophies.

Noyes, N. C. and Davis, R. L. (2023). Innate and learned odor-guided behaviors utilize distinct molecular signaling pathways in a shared dopaminergic circuit. Cell Rep 42(2): 112026. PubMed ID: 36701232
Summary:
Odor-based learning and innate odor-driven behavior have been hypothesized to require separate neuronal circuitry. Contrary to this notion, innate behavior and olfactory learning were recently shown to share circuitry that includes the Drosophila mushroom body (MB). But how a single circuit drives two discrete behaviors remains unknown. This study defines an MB circuit responsible for both olfactory learning and innate odor avoidance and the distinct dDA1 dopamine receptor-dependent signaling pathways that mediate these behaviors. Associative learning and learning-induced MB plasticity require rutabaga-encoded adenylyl cyclase activity in the MB. In contrast, innate odor preferences driven by naive MB neurotransmission are rutabaga independent, requiring the adenylyl cyclase ACXD. Both learning and innate odor preferences converge on PKA and the downstream MBON-γ2α'1. Importantly, the utilization of this shared circuitry for innate behavior only becomes apparent with hunger, indicating that hardwired innate behavior becomes more flexible during states of stress.

Mano, O., Choi, M., Tanaka, R., Creamer, M. S., Matos, N. C. B., Shomar, J., Badwan, B. A., Clandinin, T. R. and Clark, D. A. (2023). Long timescale anti-directional rotation in Drosophila optomotor behavior. bioRxiv. PubMed ID: 36711627
Summary:
Locomotor movements cause visual images to be displaced across the eye, a retinal slip that is counteracted by stabilizing reflexes in many animals. In insects, optomotor turning causes the animal to turn in the direction of rotating visual stimuli, thereby reducing retinal slip and stabilizing trajectories through the world. This behavior has formed the basis for extensive dissections of motion vision. This study reports that under certain stimulus conditions, two Drosophila species, including the widely studied D. melanogaster, can suppress and even reverse the optomotor turning response over several seconds. Such 'anti-directional turning' is most strongly evoked by long-lasting, high-contrast, slow-moving visual stimuli that are distinct from those that promote syn-directional optomotor turning. Anti-directional turning, like the syndirectional optomotor response, requires the local motion detecting neurons T4 and T5; a subset of lobula plate tangential cells, CH cells, show involvement in these responses. Imaging from a variety of direction-selective cells in the lobula plate shows no evidence of dynamics that match the behavior, suggesting that the observed inversion in turning direction emerges downstream of the lobula plate. Further, anti-directional turning declines with age and exposure to light. These results show that Drosophila optomotor turning behaviors contain rich, stimulus-dependent dynamics that are inconsistent with simple reflexive stabilization responses.

Monday April 3rd - Cell Cycle

Kabakci, Z., Yamada, H., Vernizzi, L., Gupta, S., Weber, J., Sun, M. S. and Lehner, C. F. (2022). Teflon promotes chromosomal recruitment of homolog conjunction proteins during Drosophila male meiosis. PLoS Genet 18(10): e1010469. PubMed ID: 36251690
Summary:
Meiosis in males of higher dipterans is achiasmate. In their spermatocytes, pairing of homologs into bivalent chromosomes does not include synaptonemal complex and crossover formation. While crossovers preserve homolog conjunction until anaphase I during canonical meiosis, an alternative system is used in dipteran males. Mutant screening in Drosophila melanogaster has identified teflon (tef) as being required specifically for alternative homolog conjunction (AHC) of autosomal bivalents. The additional known AHC genes, snm, uno and mnm, are needed for the conjunction of autosomal homologs and of sex chromosomes. This study has analyzed the pattern of TEF protein expression. TEF is present in early spermatocytes but cannot be detected on bivalents at the onset of the first meiotic division, in contrast to SNM, UNO and MNM (SUM). TEF binds to polytene chromosomes in larval salivary glands, recruits MNM by direct interaction and thereby, indirectly, also SNM and UNO. However, chromosomal SUM association is not entirely dependent on TEF, and residual autosome conjunction occurs in tef null mutant spermatocytes. The higher tef requirement for autosomal conjunction is likely linked to the quantitative difference in the amount of SUM protein that provides conjunction of autosomes and sex chromosomes, respectively. During normal meiosis, SUM proteins are far more abundant on sex chromosomes compared to autosomes. Beyond promoting SUM recruitment, TEF has a stabilizing effect on SUM proteins. Increased SUM causes excess conjunction and consequential chromosome missegregation during meiosis I after co-overexpression. Similarly, expression of SUM without TEF, and even more potently with TEF, interferes with chromosome segregation during anaphase of mitotic divisions in somatic cells, suggesting that the known AHC proteins are sufficient for establishment of ectopic chromosome conjunction. Overall, these findings suggest that TEF promotes alternative homolog conjunction during male meiosis without being part of the final physical linkage between chromosomes.

Kabakci, Z., Reichle, H. E., Lemke, B., Rousova, D., Gupta, S., Weber, J., Schleiffer, A., Weir, J. R. and Lehner, C. F. (2022). Homologous chromosomes are stably conjoined for Drosophila male meiosis I by SUM, a multimerized protein assembly with modules for DNA-binding and for separase-mediated dissociation co-opted from cohesin. PLoS Genet 18(12): e1010547. PubMed ID: 36480577
Summary:
For meiosis I, homologous chromosomes must be paired into bivalents. Maintenance of homolog conjunction in bivalents until anaphase I depends on crossovers in canonical meiosis. However, instead of crossovers, an alternative system achieves homolog conjunction during the achiasmate male meiosis of Drosophila melanogaster. The proteins SNM, UNO and MNM are likely constituents of a physical linkage that conjoins homologs in D. melanogaster spermatocytes. This study reports that SNM binds tightly to the C-terminal region of UNO. This interaction is homologous to that of the cohesin subunits stromalin/Scc3/STAG and α-kleisin, as revealed by sequence similarities, structure modeling and cross-link mass spectrometry. Importantly, purified SU_C, the heterodimeric complex of SNM and the C-terminal region of UNO, displayed DNA-binding in vitro. DNA-binding was severely impaired by mutational elimination of positively charged residues from the C-terminal helix of UNO. Phenotypic analyses in flies fully confirmed the physiological relevance of this basic helix for chromosome-binding and homolog conjunction during male meiosis. Beyond DNA, SU_C also bound MNM, one of many isoforms expressed from the complex mod(mdg4) locus. This binding of MNM to SU_C was mediated by the MNM-specific C-terminal region, while the purified N-terminal part common to all Mod(mdg4) isoforms multimerized into hexamers in vitro. Similarly, the UNO N-terminal domain formed tetramers in vitro. Thus, it is suggested that multimerization confers to SUM, the assemblies composed of SNM, UNO and MNM, the capacity to conjoin homologous chromosomes stably by the resultant multivalent DNA-binding. Moreover, to permit homolog separation during anaphase I, SUM is dissociated by separase, since UNO, the α-kleisin-related protein, includes a separase cleavage site. In support of this proposal, this study demonstrates that UNO cleavage by tobacco etch virus protease is sufficient to release homolog conjunction in vivo after mutational exchange of the separase cleavage site with that of the bio-orthogonal protease.

Zhang, J., Zhang, S., Sun, Z., Cai, Y., Zhong, G. and Yi, X. (2023). Camptothecin Effectively Regulates Germline Differentiation through Bam-Cyclin A Axis in Drosophila melanogaster. Int J Mol Sci 24(2). PubMed ID: 36675143
Summary:
Camptothecin (CPT), first isolated from Chinese tree Camptotheca acuminate, produces rapid and prolonged inhibition of DNA synthesis and induction of DNA damage by targeting topoisomerase I (top1), which is highly activated in cancer cells. CPT thus exhibits remarkable anticancer activities in various cancer types, and is a promising therapeutic agent for the treatment of cancers. However, it remains to be uncovered underlying its cytotoxicity toward germ cells. This study found that CPT, a cell cycle-specific anticancer agent, reduced fecundity and exhibited significant cytotoxicity toward GSCs and two-cell cysts. CPT was shown to induce GSC loss and retarded two-cell cysts differentiation in a niche- or apoptosis-independent manner. Instead, CPT induced ectopic expression of a differentiation factor, bag of marbles (Bam), and regulated the expression of cyclin A, which contributed to GSC loss. In addition, CPT compromised two-cell cysts differentiation by decreasing the expression of Bam and inducing cell arrest at G1/S phase via cyclin A, eventually resulting in two-cell accumulation. Collectively, this study demonstrates, for the first time in vivo, that the Bam-cyclin A axis is involved in CPT-mediated germline stem cell loss and two-cell cysts differentiation defects via inducing cell cycle arrest, which could provide information underlying toxicological effects of CPT in the productive system, and feature its potential to develop as a pharmacology-based germline stem cell regulation agent.

Hayden, L., Hur, W., Vergassola, M. and Di Talia, S. (2022). Manipulating the nature of embryonic mitotic waves. Curr Biol 32(22): 4989-4996.e4983. PubMed ID: 36332617
Summary:
Early embryogenesis is characterized by rapid and synchronous cleavage divisions, which are often controlled by wave-like patterns of Cdk1 activity. Two mechanisms have been proposed for mitotic waves: sweep and trigger waves. The two mechanisms give rise to different wave speeds, dependencies on physical and molecular parameters, and spatial profiles of Cdk1 activity: upward sweeping gradients versus traveling wavefronts. Both mechanisms hinge on the transient bistability governing the cell cycle and are differentiated by the speed of the cell-cycle progression: sweep and trigger waves arise for rapid and slow drives, respectively. This study, using quantitative imaging of Cdk1 activity and theory, illustrates that sweep waves are the dominant mechanism in Drosophila embryos and test two fundamental predictions on the transition from sweep to trigger waves. Sweep waves can be turned into trigger waves if the cell cycle is slowed down genetically or if significant delays in the cell-cycle progression are introduced across the embryo by altering nuclear density. Genetic experiments demonstrate that Polo kinase is a major rate-limiting regulator of the blastoderm divisions, and genetic perturbations reducing its activity can induce the transition from sweep to trigger waves. Furthermore, it was shown that changes in temperature cause an essentially uniform slowdown of interphase and mitosis. That results in sweep waves being observed across a wide temperature range despite the cell-cycle durations being significantly different. Collectively, our combination of theory and experiments elucidates the nature of mitotic waves in Drosophila embryogenesis, their control mechanisms, and their mutual transitions.

Neville, K. E., Finegan, T. M., Lowe, N., Bellomio, P. M., Na, D. and Bergstralh, D. T. (2023). The Drosophila mitotic spindle orientation machinery requires activation, not just localization. EMBO Rep: e56074. PubMed ID: 36629398
Summary:
The orientation of the mitotic spindle at metaphase determines the placement of the daughter cells. Spindle orientation in animals typically relies on an evolutionarily conserved biological machine comprised of at least four proteins - called Pins, Gαi, Mud, and Dynein in flies - that exerts a pulling force on astral microtubules and reels the spindle into alignment. The canonical model for spindle orientation holds that the direction of pulling is determined by asymmetric placement of this machinery at the cell cortex. In most cell types, this placement is thought to be mediated by Pins, and a substantial body of literature is therefore devoted to identifying polarized cues that govern localized cortical enrichment of Pins. This study revisits the canonical model and finds that it is incomplete. Spindle orientation in the Drosophila follicular epithelium and embryonic ectoderm requires not only Pins localization but also direct interaction between Pins and the multifunctional protein Discs large. This requirement can be over-ridden by interaction with another Pins interacting protein, Inscuteable.

Crucianelli, C., Jaiswal, J., Vijayakumar Maya, A., Nogay, L., Cosolo, A., Grass, I. and Classen, A. K. (2022). Distinct signaling signatures drive compensatory proliferation via S-phase acceleration. PLoS Genet 18(12): e1010516. PubMed ID: 36520882
Summary:
Regeneration relies on cell proliferation to restore damaged tissues. Multiple signaling pathways activated by local or paracrine cues have been identified to promote regenerative proliferation. How different types of tissue damage may activate distinct signaling pathways and how these differences converge on regenerative proliferation is less well defined. To better understand how tissue damage and proliferative signals are integrated during regeneration, this study investigated models of compensatory proliferation in Drosophila imaginal discs. Compensatory proliferation was found to be associated with a unique cell cycle profile, which is characterized by short G1 and G2 phases and, surprisingly, by acceleration of the S-phase. S-phase acceleration can be induced by two distinct signaling signatures, aligning with inflammatory and non-inflammatory tissue damage. Specifically, non-autonomous activation of JAK/STAT and Myc in response to inflammatory damage, or local activation of Ras/ERK and Hippo/Yki in response to elevated cell death, promote accelerated nucleotide incorporation during S-phase. This previously unappreciated convergence of different damaging insults on the same regenerative cell cycle program reconciles previous conflicting observations on proliferative signaling in different tissue regeneration and tumor models.

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