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Monday, May 16th - Behavior

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

Friday, May 13th - Methods

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

Thursday, May 12 - Gonad

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

Wednesday, May 11th - Disease Models

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

Tuesday, May 10th - Evolution

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

Monday, May 9th - Larval and Adult Development

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

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

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

Thursday, May 5th - Disease Models

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

Wednesday, May 4th - Chromatin and DNA Repair

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

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

Tuesday, May 3rd - Chromatin and DNA Repair

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

Monday, May 2nd - RNA and Transposons

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

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