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Current papers in developmental biology and gene function


Friday, June 30th, 2023 - Evolution

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Trienens, M., Kurtz, J. and Wertheim, B. (2023). Rapid but narrow - Evolutionary adaptation and transcriptional response of Drosophila melanogaster to toxic mould. Mol Ecol. PubMed ID: 37066754
Insects have adapted to a multitude of environmental conditions, including the presence of xenobiotic noxious substances. Environmental microorganisms, particularly rich on ephemeral resources, employ these noxious chemicals in a chemical warfare against predators and competitors, driving co-evolutionary adaptations. In order to analyse how environmental microbes may be driving such evolutionary adaptations, this study experimentally evolved Drosophila melanogaster populations by exposing larvae to the toxin-producing mould Aspergillus nidulans that infests the flies' breeding substrate. To disentangle the effects of the mycotoxin Sterigmatocystin from other substrate modifications inflicted by the mould, the following four selection regimes were used: (i) control without fungus, (ii) A. nidulans wild type, (iii) a mutant of A. nidulans ΔlaeA with impaired toxin production, (iv) synthetic Sterigmatocystin. Experimental evolution was carried out in five independent D. melanogaster populations each, for a total of 11 generations. This evolution experiment was further combined with transcriptome analysis to identify evolutionary shifts in gene expression due to the selection regimes and mould confrontation. Populations that evolved in presence of the toxin-producing mould or the pure mycotoxin rapidly adapted to the respective conditions and showed higher viability in subsequent confrontations. Yet, mycotoxin-selected populations had no advantage in A. nidulans wild type confrontation. Moreover, distinctive changes in gene expression related to the selection-regime contrast were only associated with the toxin-producing-fungus regime and comprised a narrow set of genes. Thus, it needs the specific conditions of the selection agent to enable adaptation to the fungus.
Pelaez, J. N., Gloss, A. D., Goldman-Huertas, B., ..., Rodas, E., Liang, I. and Whiteman, N. K. (2023). Evolution of chemosensory and detoxification gene families across herbivorous Drosophilidae. bioRxiv. PubMed ID: 36993186
Herbivorous insects are exceptionally diverse, accounting for a quarter of all known eukaryotic species, but the genetic basis of adaptations that enabled this dietary transition remains poorly understood. Many studies have suggested that expansions and contractions of chemosensory and detoxification gene families - genes directly mediating interactions with plant chemical defenses - underlie successful plant colonization. However, this hypothesis has been challenging to test because the origins of herbivory in many lineages are ancient (>150 million years ago [mya]), obscuring genomic evolutionary patterns. This study characterized chemosensory and detoxification gene family evolution across Scaptomyza, a genus nested within Drosophila that includes a recently derived (<15 mya) herbivore lineage of mustard (Brassicales) specialists and carnation (Caryophyllaceae) specialists, and several non-herbivorous species. Comparative genomic analyses revealed that herbivorous Scaptomyza have among the smallest chemosensory and detoxification gene repertoires across 12 drosophilid species surveyed. Rates of gene turnover averaged across the herbivore clade were significantly higher than background rates in over half of the surveyed gene families. However, gene turnover was more limited along the ancestral herbivore branch, with only gustatory receptors and odorant binding proteins experiencing strong losses. The genes most significantly impacted by gene loss, duplication, or changes in selective constraint were those involved in detecting compounds associated with feeding on plants (bitter or electrophilic phytotoxins) or their ancestral diet (yeast and fruit volatiles). These results provide insight into the molecular and evolutionary mechanisms of plant-feeding adaptations and highlight strong gene candidates that have also been linked to other dietary transitions in Drosophila.
Verster, K. I., Cinege, G., Lipinszki, Z., Magyar, L. B., Kurucz, E., Tarnopol, R. L., Abraham, E., Darula, Z., Karageorgi, M., Tamsil, J. A., Akalu, S. M., Ando, I. and Whiteman, N. K. (2023). Evolution of insect innate immunity through domestication of bacterial toxins. Proc Natl Acad Sci U S A 120(16): e2218334120. PubMed ID: 37036995
Toxin cargo genes are often horizontally transferred by phages between bacterial species and are known to play an important role in the evolution of bacterial pathogenesis. This study shows how these same genes have been horizontally transferred from phage or bacteria to animals and have resulted in novel adaptations. It was discovered that two widespread bacterial genes encoding toxins of animal cells, cytolethal distending toxin subunit B (cdtB) and apoptosis-inducing protein of 56 kDa (aip56), were captured by insect genomes through horizontal gene transfer from bacteria or phages. To study the function of these genes in insects, focus was placed on Drosophila ananassae as a model. In the D. ananassae subgroup species, cdtB and aip56 are present as singular (cdtB) or fused copies (cdtB::aip56) on the second chromosome. cdtB and aip56 genes and encoded proteins were expressed by immune cells, some proteins were localized to the wasp embryo's serosa, and their expression increased following parasitoid wasp infection. Species of the ananassae subgroup are highly resistant to parasitoid wasps, and D. ananassae lines carrying null mutations in cdtB and aip56 toxin genes were observed to be more susceptible to parasitoids than the wild type. It is concluded that toxin cargo genes were captured by these insects millions of years ago and integrated as novel modules into their innate immune system. These modules now represent components of a heretofore undescribed defense response and are important for resistance to parasitoid wasps. Phage or bacterially derived eukaryotic toxin genes serve as macromutations that can spur the instantaneous evolution of novelty in animals.
Wang, Y., McNeil, P., Abdulazeez, R., Pascual, M., Johnston, S. E., Keightley, P. D. and Obbard, D. (2023). Variation in mutation, recombination, and transposition rates in Drosophila melanogaster and Drosophila simulans. Genome Res. PubMed ID: 37037625
The rates of mutation, recombination, and transposition are core parameters in models of evolution. They impact genetic diversity, responses to ongoing selection, and levels of genetic load. However, even for key evolutionary model species such as Drosophila melanogaster and D. simulans, few estimates of these parameters are available, and little idea is available of how rates vary between individuals, sexes, or populations. Knowledge of this variation is fundamental for parameterizing models of genome evolution. This study provides direct estimates of mutation, recombination, and transposition rates and their variation in a West African and a European population of D. melanogaster and a European population of D. simulans. Across 89 flies, 58 single nucleotide mutations, 286 crossovers, and 89 transposable elements (TE) insertions were observed. Compared to the European D. melanogaster, the West African population had a lower mutation rate (1.67 vs. 4.86 × 10-9 site-1 gen-1) and a lower transposition rate (8.99 vs. 23.36 × 10-5 copy-1 gen-1), but a higher recombination rate (3.44 vs. 2.06 cM/Mb). The European D. simulans population has a similar mutation rate to European D. melanogaster, but a significantly higher recombination rate and a lower, but not significantly different, transposition rate. Overall, paternal-derived mutations were found to be more frequent than maternal ones in both species. This study quantifies the variation in rates of mutation, recombination, and transposition among different populations and sexes, and the direct estimate of these parameters in D. melanogaster and D. simulans will benefit future studies in population and evolutionary genetics.
Kageyama, D., Harumoto, T., Nagamine, K., Fujiwara, A., Sugimoto, T. N., Jouraku, A., Tamura, M., Katoh, T. K. and Watada, M. (2023). A male-killing gene encoded by a symbiotic virus of Drosophila. Nat Commun 14(1): 1357. PubMed ID: 36914655
In most eukaryotes, biparentally inherited nuclear genomes and maternally inherited cytoplasmic genomes have different evolutionary interests. Strongly female-biased sex ratios that are repeatedly observed in various arthropods often result from the male-specific lethality (male-killing) induced by maternally inherited symbiotic bacteria such as Spiroplasma and Wolbachia. However, despite some plausible case reports wherein viruses are raised as male-killers, it is not well understood how viruses, having much smaller genomes than bacteria, are capable of inducing male-killing. This study showed that a maternally inherited double-stranded RNA (dsRNA) virus belonging to the family Partitiviridae (designated DbMKPV1) induces male-killing in Drosophila. DbMKPV1 localizes in the cytoplasm and possesses only four genes, i.e., one gene in each of the four genomic segments (dsRNA1-dsRNA4), in contrast to ca. 1000 or more genes possessed by Spiroplasma or Wolbachia. A protein (designated PVMKp1; 330 amino acids in size), encoded by a gene on the dsRNA4 segment, was shown to be necessary and sufficient for inducing male-killing. These results imply that male-killing genes can be easily acquired by symbiotic viruses through reassortment and that symbiotic viruses are hidden players in arthropod evolution. It is anticipated that host-manipulating genes possessed by symbiotic viruses can be utilized for controlling arthropods.
Narasimhan, A., Kapila, R., Meena, A. and Prasad, N. G. (2023). Consequences of adaptation to larval crowding on sexual and fecundity selection in Drosophila melanogaster. PubMed ID: J Evol Biol 36(4): 730-737. PubMed ID: 36946997
Sexual selection is a major force influencing the evolution of sexually reproducing species. Environmental factors such as larval density can manipulate adult condition and influence the direction and strength of sexual selection. While most studies on the influence of larval crowding on sexual selection are either correlational or single-generation manipulations, it is unclear how evolution under chronic larval crowding affects sexual selection. To answer this, the strength was measured of sexual selection on male and female Drosophila melanogaster that had evolved under chronic larval crowding for over 250 generations in the laboratory, along with their controls which had never experienced crowding, in a common garden high-density environment. Selection coefficients were measured on male mating success and sex-specific reproductive success, as separate estimates allowed dissection of sex-specific effects. Experimental evolution under chronic larval crowding was shown to decrease the strength of sexual and fecundity selection in males but not in females, relative to populations experiencing crowding for the first time. The effect of larval crowding in reducing reproductive success is almost twice in females than in males. This study highlights the importance of studying how evolution in a novel, stressful environment can shape adult fitness in organisms.

Thursday, June 29th - Cytoskeleton and Junctions

di Pietro, F., Osswald, M., De Las Heras, J. M., Cristo, I., Lopez-Gay, J., Wang, Z., Pelletier, S., Gaugue, I., Leroy, A., Martin, C., Morais-de-Sa, E. and Bellaiche, Y. (2023). Systematic analysis of RhoGEF/GAP localizations uncovers regulators of mechanosensing and junction formation during epithelial cell division. Curr Biol 33(5): 858-874.e857. PubMed ID: 36917931
Cell proliferation is central to epithelial tissue development, repair, and homeostasis. During cell division, small RhoGTPases control both actomyosin dynamics and cell-cell junction remodeling to faithfully segregate the genome while maintaining tissue polarity and integrity. To decipher the mechanisms of RhoGTPase spatiotemporal regulation during epithelial cell division, this study generated a transgenic fluorescently tagged library for the 48 Drosophila Rho guanine exchange factors (RhoGEFs) and GTPase-activating proteins (GAPs), and their endogenous distributions were systematically characterized by time-lapse microscopy. Therefore, candidate regulators of the interplay between actomyosin and junctional dynamics during epithelial cell division were unveiled. Building on these findings, it was established that the conserved RhoGEF Cysts and RhoGEF4 play sequential and distinct roles to couple cytokinesis with de novo junction formation. During ring contraction, Cysts via Rho1 participates in the neighbor mechanosensing response, promoting daughter-daughter cell membrane juxtaposition in preparation to de novo junction formation. Subsequently and upon midbody formation, RhoGEF4 via Rac acts in the dividing cell to ensure the withdrawal of the neighboring cell membranes, thus controlling de novo junction length and cell-cell arrangements upon cytokinesis. Altogether, these findings delineate how the RhoGTPases Rho and Rac are locally and temporally activated during epithelial cytokinesis, highlighting the RhoGEF/GAP library as a key resource to understand the broad range of biological processes regulated by RhoGTPases.
Gurley, N. J., Szymanski, R. A., Dowen, R. H., Butcher, T. A., Ishiyama, N. and Peifer, M. (2023). Exploring the evolution and function of Canoe's intrinsically disordered region in linking cell-cell junctions to the cytoskeleton during embryonic morphogenesis. bioRxiv. PubMed ID: 36945496
One central question for cell and developmental biologists is defining how epithelial cells can change shape and move during embryonic development without tearing tissues apart. This requires robust yet dynamic connections of cells to one another, via the cell-cell adherens junction, and of junctions to the actin and myosin cytoskeleton, which generates force. The last decade revealed that these connections involve a multivalent network of proteins, rather than a simple linear pathway. This studt focused on Drosophila Canoe, homolog of mammalian Afadin, as a model for defining the underlying mechanisms. Canoe and Afadin are complex, multidomain proteins that share multiple domains with defined and undefined binding partners. Both also share a long carboxy-terminal intrinsically disordered region (IDR), whose function is less well defined. IDRs are found in many proteins assembled into large multiprotein complexes. This study has combined bioinformatic analysis and the use of a series of canoe mutants with early stop codons to explore the evolution and function of the IDR. Bioinformatic analysis reveals that the IDRs of Canoe and Afadin differ dramatically in sequence and sequence properties. When shorter evolutionary time scales were examined, multiple conserved motifs were identified. Some of these are predicted by AlphaFold to be alpha-helical, and two correspond to known protein interaction sites for alpha-catenin and F-actin. Next the lesions were identified in a series of eighteen canoe mutants, which have early stop codons across the entire protein coding sequence. Analysis of their phenotypes are consistent with the idea that the IDR, including its C-terminal conserved motifs, are important for protein function. These data provide the foundation for further analysis of IDR function.
Khaitan, V., Shill, K., Chatterjee, P., Mukherjee, S. and Majumder, P. (2023). Singed and vinculin play redundant roles in cell migration by regulating F-actin. Dev Dyn. PubMed ID: 36912821
Drosophila Singed (mammalian Fascin) is an actin-binding protein that is known mainly for bundling parallel actin filaments. Among many functions of Singed, it is required for cell motility for both Drosophila and mammalian systems. Increased Fascin-1 levels positively correlate with greater metastasis and poor prognosis in human cancer. Border cell cluster, which forms and migrates during Drosophila egg chamber development, shows higher expression of Singed compared with other follicle cells. Interestingly, loss of singed in border cells does not lead to any effect other than delay. This work screened many actin-binding proteins in search of functional redundancy with Singed for border cell migration. Vinculin was found to work with Singed to regulate border cell migration, albeit mildly. Although Vinculin is known for anchoring F-actin to the membrane, knockdown of both singed and vinculin leads to a reduced level of F-actin and changes in protrusion characteristics in border cells. This study has also observed that they may act together to control microvilli length of brush border membrane vesicles and the shape of egg chambers in Drosophila. It is concluded that singed and vinculin work together to control F-actin and these interactions are consistent across multiple platforms.
Nakamura, M., Hui, J., Stjepic, V. and Parkhurst, S. M. (2023). Scar/WAVE has Rac GTPase-independent functions during cell wound repair. Sci Rep 13(1): 4763. PubMed ID: 36959278
Rho family GTPases regulate both linear and branched actin dynamics by activating downstream effectors to facilitate the assembly and function of complex cellular structures such as lamellipodia and contractile actomyosin rings. Wiskott-Aldrich Syndrome (WAS) family proteins are downstream effectors of Rho family GTPases that usually function in a one-to-one correspondence to regulate branched actin nucleation. In particular, the WAS protein Scar/WAVE has been shown to exhibit one-to-one correspondence with Rac GTPase. This study shows that Rac and SCAR are recruited to cell wounds in the Drosophila repair model and are required for the proper formation and maintenance of the dynamic actomyosin ring formed at the wound periphery. Interestingly, it was found that SCAR is recruited to wounds earlier than Rac and is still recruited to the wound periphery in the presence of a potent Rac inhibitor. It was also shown that while Rac is important for actin recruitment to the actomyosin ring, SCAR serves to organize the actomyosin ring and facilitate its anchoring to the overlying plasma membrane. These differing spatiotemporal recruitment patterns and wound repair phenotypes highlight the Rac-independent functions of SCAR and provide an exciting new context in which to investigate these newly uncovered SCAR functions.
Pradhan, R., Kumar, S. and Mathew, R. (2023). Lateral adherens junctions mediate a supracellular actomyosin cortex in Drosophila trachea. iScience 26(4): 106380. PubMed ID: 37009223
Drosophila trachea is a classical model for analyzing epithelial, especially tubular epithelial biology. Lateral E-cadherin mediated junctions were identified that encircle the cells just basal to the zonula adherens in the larval trachea. The lateral junction is associated with downstream adapters, including catenins, and has a distinct junctional actin cortex. The lateral cortex contributes to the development of a supracellular actomyosin mesh in the late larvae. Establishment of this cytoskeletal structure depends on lateral junction associated Rho1 and Cdc42 GTPases, and Arp and WASP pathways. The supracellular network takes the character of stress fibers along the AP axis in the early hours of pupation. It contributes to the shortening of the epithelial tube albeit in a manner redundant to ECM-mediated compression mechanism. In conclusion, this study shows the in vivo existence of functional lateral adherens junction and suggest a role for it in mediating dynamic cytoskeletal events during tissue scale morphogenesis.
Malin, J., Rosa Birriel, C. and Hatini, V. (2023). Pten, Pi3K and PtdIns(3,4,5)P (3) dynamics modulate pulsatile actin branching in Drosophila retina morphogenesis. bioRxiv. PubMed ID: 36993510
Epithelial remodeling of the Drosophila retina depends on the pulsatile contraction and expansion of apical contacts between the cells that form its hexagonal lattice. Phosphoinositide PI(3,4,5)P (3) (PIP (3)) accumulates around tricellular adherens junctions (tAJs) during contact expansion and dissipates during contraction, but with unknown function. This study found that manipulations of Pten or Pi3K that either decreased or increased PIP (3) resulted in shortened contacts and a disordered lattice, indicating a requirement for PIP (3) dynamics and turnover. These phenotypes are caused by a loss of protrusive branched actin, resulting from impaired activity of the Rac1 Rho GTPase and the WAVE regulatory complex (WRC). It was additionally found that during contact expansion, Pi3K moves into tAJs to promote the cyclical increase of PIP (3) in a spatially and temporally precise manner. Thus, dynamic regulation of PIP (3) by Pten and Pi3K controls the protrusive phase of junctional remodeling, which is essential for planar epithelial morphogenesis.

Wednesday, June 28th - Disease Model

Hagedorn, E., Bunnell, D., Henschel, B., Smith, D. L., Dickinson, S., Brown, A. W., De Luca, M., Turner, A. N. and Chtarbanova, S. (2023). RNA virus-mediated changes in organismal oxygen consumption rate in young and old Drosophila melanogaster males. Aging (Albany NY) 15(6): 1748-1767. PubMed ID: 36947702
Aging is accompanied by increased susceptibility to infections including with viral pathogens resulting in higher morbidity and mortality among the elderly. Significant changes in host metabolism can take place following virus infection. Efficient immune responses are energetically costly, and viruses divert host molecular resources to promote their own replication. Virus-induced metabolic reprogramming could impact infection outcomes, however, how this is affected by aging and impacts organismal survival remains poorly understood. RNA virus infection of Drosophila melanogaster with Flock House virus (FHV) is an effective model to study antiviral responses with age, where older flies die faster than younger flies due to impaired disease tolerance. Using this aged host-virus model, longitudinal, single-fly respirometry studies were conducted to determine if metabolism impacts infection outcomes. Analysis using linear mixed models on Oxygen Consumption Rate (OCR) following the first 72-hours post-infection showed that FHV modulates respiration, but age has no significant effect on OCR. However, the longitudinal assessment revealed that OCR in young flies progressively and significantly decreases, while OCR in aged flies remains constant throughout the three days of the experiment. Furthermore, it was found that the OCR signature at 24-hours varied in response to both experimental treatment and survival status. FHV-injected flies that died prior to 48- or 72-hours measurements had a lower OCR compared to survivors at 48-hours. These findings suggest the host's metabolic profile could influence the outcome of viral infections.
Mocci, I., Casu, M. A., Sogos, V., Liscia, A., Angius, R., Cadeddu, F., Fanti, M., Muroni, P., Talani, G., Diana, A., Collu, M. and Setzu, M. D. (2023). Effects of memantine on mania-like phenotypes exhibited by Drosophila Shaker mutants. CNS Neurosci Ther. PubMed ID: 36942502
Increased glutamate levels and electrolytic fluctuations have been observed in acutely manic patients. Despite some efficacy of the non-competitive NMDA receptor antagonist memantine (Mem), such as antidepressant-like and mood-stabilizer drugs in clinical studies, its specific mechanisms of action are still uncertain. The present study aims to better characterize the Drosophila melanogaster fly Shaker mutants (SH), as a translational model of manic episodes within bipolar disorder in humans, and to investigate the potential anti-manic properties of Mem. The findings showed typical behavioral abnormalities in SH, which mirrored with the overexpression of NMDAR-NR1 protein subunit, matched well to glutamate up-regulation. Such molecular features were associated to a significant reduction of SH brain volume in comparison to Wild Type strain flies (WT). This study reports on the ability of Mem treatment to ameliorate behavioral aberrations of SH (similar to that of Lithium), and its ability to reduce NMDAR-NR1 over-expression. These results show the involvement of the glutamatergic system in the SH, given the interaction between the Shaker channel and the NMDA receptor, suggesting this model as a promising tool for studying the neurobiology of bipolar disorders. Moreover, the results show Mem as a potential disease-modifying therapy, providing insight on new mechanisms of action.
Li, W., Pan, X., Li, M., Ling, L., Zhang, M., Liu, Z., Zhang, K., Guo, J. and Wang, H. (2023). Impact of age on the rotenone-induced sporadic Parkinson's disease model using Drosophila melanogaster. Neurosci Lett 805: 137187. PubMed ID: 36921666
Rotenone, a naturally occurring toxin, has been used to induce sporadic Parkinson's disease (PD) in Drosophila melanogaster for decades. However, the age of flies varies considerably between studies in this model. To investigate the impact of age on the rotenone-induced PD model, male flies were collected at the age of 1, 5, 7, and 10 days post-eclosion, respectively. Then, flies were immediately exposed to a feeding medium supplemented with 250 &mi;M rotenone for seven days. The motor ability of Drosophila was detected by negative geotaxis assay, and the number of dopamine (DA) neurons and tyrosine hydroxylase (TH) expression levels were evaluated. The results showed that both the motor deficits and mortality increased with age. The flies older than five days showed typical PD features, including the loss of DA neurons, decreased TH expression levels, and decreased locomotive ability. However, 1-day-old flies displayed an unstable motor deficit and little TH expression changes after seven days of rotenone exposure. Lastly, after 7 days of exposure to rotenone, the death rate of flies rapidly increased with increasing starting age. The death rates of 1-, 5-, 7-, and 10-days old flies were 10.0%, 22.8%, 41.5%, and 50.4%, respectively. The findings of this study suggest that age is a crucial factor impacting the Drosophila PD model. This information provides a reference for the age selection to use this model for future studies.
Liu, R., Xu, W., Zhu, H., Dong, Z., Dong, H. and Yin, S. (2023). Aging aggravates acetaminophen-induced acute liver injury and inflammation through inordinate C/EBPalpha-BMP9 crosstalk. Cell Biosci 13(1): 61. PubMed ID: 36945064
Previous studies have shown that bone morphogenetic protein 9 (BMP9) is almost exclusively produced in the liver and reaches tissues throughout the body as a secreted protein. However, the mechanism of BMP9 action and its role in aging-associated liver injury and inflammation are still unclear. Aging significantly aggravates acetaminophen (APAP)-induced acute liver injury (ALI). Increased expression of CCAAT/enhancer binding protein α (C/EBPα) and BMP9 was identified in aged livers and in hepatocytes and macrophages (MФs) isolated from aged mice. Further analysis revealed that excess BMP9 was directly related to APAP-induced hepatocyte injury and death, as evidenced by activated Drosophila Mothers against decapentaplegic protein 1/5/9 (SMAD1/5/9) signaling, an increased dead cell/total cell ratio, decreased levels of ATG3 and ATG7, blocked autophagy, increased senescence-associated beta-galactosidase (SA-β-Gal) activity, and a higher rate of senescence-associated secretory phenotype (SASP) acquisition. In contrast, Bmp9 knockout (Bmp9(-/-)) partially alleviated the aforementioned manifestations of BMP9 overexpression. Moreover, BMP9 expression was found to be regulated by C/EBPα in vitro and in vivo. Notably, BMP9 also downregulated autophagy through its effect on autophagy-related genes (ATG3 and ATG7) in MΦs, which was associated with aggravated liver injury and SASP acquisition. In summary, the present study highlights the crucial roles played by C/EBPα-BMP9 crosstalk and provides insights into the interrelationship between hepatocytes and MΦs during acute liver injury.
Kang, K. H., Han, J. E., Kim, H., Kim, S., Hong, Y. B., Yun, J., Nam, S. H., Choi, B. O. and Koh, H. (2023). PINK1 and Parkin Ameliorate the Loss of Motor Activity and Mitochondrial Dysfunction Induced by Peripheral Neuropathy-Associated HSPB8 Mutants in Drosophila Models. Biomedicines 11(3). PubMed ID: 36979812
Charcot-Marie-Tooth disease (CMT) is a group of inherited peripheral nerve disorders characterized by progressive muscle weakness and atrophy, sensory loss, foot deformities and steppage gait. Missense mutations in the gene encoding the small heat shock protein HSPB8 (HSP22) have been associated with hereditary neuropathies, including CMT. HSPB8 is a member of the small heat shock protein family sharing a highly conserved α-crystallin domain that is critical to its chaperone activity. This study modeled HSPB8 mutant-induced neuropathies in Drosophila. The overexpression of human HSPB8 mutants in Drosophila neurons produced no significant defect in fly development but led to a partial reduction in fly lifespan. Although these HSPB8 mutant genes failed to induce sensory abnormalities, they reduced the motor activity of flies and the mitochondrial functions in fly neuronal tissue. The motor defects and mitochondrial dysfunction were successfully restored by PINK1 and parkin, which are Parkinson's disease-associated genes that have critical roles in maintaining mitochondrial function and integrity. Consistently, kinetin riboside, a small molecule amplifying PINK1 activity, also rescued the loss of motor activity in the HSPB8 mutant model.
Lee, S., Jun, Y. W., Linares, G. R., Butler, B., Yuva-Adyemir, Y., Moore, J., Krishnan, G., Ruiz-Juarez, B., Santana, M., Pons, M., Silverman, N., Weng, Z., Ichida, J. K. and Gao, F. B. (2023). Downregulation of Hsp90 and the antimicrobial peptide Mtk suppresses poly(GR)-induced neurotoxicity in C9ORF72-ALS/FTD. Neuron. PubMed ID: 36931278
GGGGCC repeat expansion in the C9ORF72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Repeat RNAs can be translated into dipeptide repeat proteins, including poly(GR), whose mechanisms of action remain largely unknown. In an RNA-seq analysis of poly(GR) toxicity in Drosophila, it was found that several antimicrobial peptide genes, such as Metchnikowin (Mtk), and heat shock protein (Hsp) genes are activated. Mtk knockdown in the fly eye or in all neurons suppresses poly(GR) neurotoxicity. These findings suggest a cell-autonomous role of Mtk in neurodegeneration. Hsp90 knockdown partially rescues both poly(GR) toxicity in flies and neurodegeneration in C9ORF72 motor neurons derived from induced pluripotent stem cells (iPSCs). Topoisomerase II (TopoII) regulates poly(GR)-induced upregulation of Hsp90 and Mtk. TopoII knockdown also suppresses poly(GR) toxicity in Drosophila and improves survival of C9ORF72 iPSC-derived motor neurons. These results suggest potential novel therapeutic targets for C9ORF72-ALS/FTD.

Monday, June 26th - Adult Physiology and Metabolism

Camus, M. F., Rodriguez, E., Kotiadis, V., Carter, H. and Lane, N. (2023). Redox stress shortens lifespan through suppression of respiratory complex I in flies with mitonuclear incompatibilities. Exp Gerontol 175: 112158. PubMed ID: 36965604
Incompatibilities between mitochondrial and nuclear genes can perturb respiration, biosynthesis, signaling and gene expression. This study investigated whether mild mitonuclear incompatibilities alter the physiological response to redox stress induced by N-acetyl cysteine (NAC). Three Drosophila melanogaster lines were studied with mitochondrial genomes that were either coevolved (WT) or mildly mismatched (BAR, COX) to an isogenic nuclear background. Responses to NAC varied substantially with mitonuclear genotype, sex, tissue and dose. NAC caused infertility and high mortality in some groups, but not others. Using tissue-specific high-resolution fluorespirometry, it was shown that NAC did not alter H(2)O(2) flux but suppressed complex I-linked respiration in female flies, while maintaining a reduced glutathione pool. The high mortality in BAR females was associated with severe (>50 %) suppression of complex I-linked respiration, rising H(2)O(2) flux in the ovaries, and significant oxidation of the glutathione pool. The results suggest that redox stress is attenuated by the suppression of complex-I linked respiration, to the point of death in some mitonuclear lines. It is proposed that suppression of complex I-linked respiration is a general mechanism to maintain redox homeostasis in tissues, which could offset oxidative stress in ageing, producing a metabolic phenotype linked with epigenetic changes and age-related decline.
Jorgensen, L. B., Hansen, A. M., Willot, Q. and Overgaard, J. (2023). Balanced mitochondrial function at low temperature is linked to cold adaptation in Drosophila species. J Exp Biol 226(8). PubMed ID: 36939380
The ability of ectothermic animals to live in different thermal environments is closely associated with their capacity to maintain physiological homeostasis across diurnal and seasonal temperature fluctuations. For chill-susceptible insects, such as Drosophila, cold tolerance is tightly linked to ion and water homeostasis obtained through a regulated balance of active and passive transport. Active transport at low temperature requires a constant delivery of ATP and it is therefore hypothesized that cold-adapted Drosophila are characterized by superior mitochondrial capacity at low temperature relative to cold-sensitive species. To address this, how experimental temperatures from 1 to 19°C affected mitochondrial substrate oxidation was investigated in flight muscle of seven Drosophila species and compared it with a measure of species cold tolerance (CTmin, the temperature inducing cold coma). Mitochondrial oxygen consumption rates measured using a substrate-uncoupler-inhibitor titration (SUIT) protocol showed that cooling generally reduced oxygen consumption of the electron transport system across species, as was expected given thermodynamic effects. Complex I respiration is the primary consumer of oxygen at non-stressful temperatures, but low temperature decreases complex I respiration to a much greater extent in cold-sensitive species than in cold-adapted species. Accordingly, cold-induced reduction of complex I respiration correlates strongly with CTmin. The relative contribution of other substrates (proline, succinate and glycerol 3-phosphate) increased as temperature decreased, particularly in the cold-sensitive species. At present, it is unclear whether the oxidation of alternative substrates can be used to offset the effects of the temperature-sensitive complex I, and the potential functional consequences of such a substrate switch are discussed.
Bedont, J. L., Kolesnik, A., Pivarshev, P., Malik, D., Hsu, C. T., Weljie, A. and Sehgal, A. (2023). Chronic sleep loss sensitizes Drosophila melanogaster to nitrogen stress. Curr Biol. PubMed ID: 36965479
Chronic sleep loss profoundly impacts metabolic health and shortens lifespan, but studies of the mechanisms involved have focused largely on acute sleep deprivation. To identify metabolic consequences of chronically reduced sleep, unbiased metabolomics was conducted on heads of three adult Drosophila short-sleeping mutants with very different mechanisms of sleep loss: fumin (fmn), redeye (rye), and sleepless (sss). Common features included elevated ornithine and polyamines, with lipid, acyl-carnitine, and TCA cycle changes suggesting mitochondrial dysfunction. Studies of excretion demonstrate inefficient nitrogen elimination in adult sleep mutants, likely contributing to their polyamine accumulation. Increasing levels of polyamines, particularly putrescine, promote sleep in control flies but poison sleep mutants. This parallels the broadly enhanced toxicity of high dietary nitrogen load from protein in chronically sleep-restricted Drosophila, including both sleep mutants and flies with hyper-activated wake-promoting neurons. Together, these results implicate nitrogen stress as a novel mechanism linking chronic sleep loss to adverse health outcomes-and perhaps for linking food and sleep homeostasis at the cellular level in healthy organisms.
Dodge, R., Jones, E. W., Zhu, H., Obadia, B., Martinez, D. J., Wang, C., Aranda-Diaz, A., Aumiller, K., Liu, Z., Voltolini, M., Brodie, E. L., Huang, K. C., Carlson, J. M., Sivak, D. A., Spradling, A. C. and Ludington, W. B. (2023). A symbiotic physical niche in Drosophila melanogaster regulates stable association of a multi-species gut microbiota. Nat Commun 14(1): 1557. PubMed ID: 36944617
The gut is continuously invaded by diverse bacteria from the diet and the environment, yet microbiome composition is relatively stable over time for host species ranging from mammals to insects, suggesting host-specific factors may selectively maintain key species of bacteria. To investigate host specificity, gnotobiotic Drosophila, microbial pulse-chase protocols, and microscopy were used to investigate the stability of different strains of bacteria in the fly gut. A host-constructed physical niche in the foregut was shown to selectively binds bacteria with strain-level specificity, stabilizing their colonization. Primary colonizers saturate the niche and exclude secondary colonizers of the same strain, but initial colonization by Lactobacillus species physically remodels the niche through production of a glycan-rich secretion to favor secondary colonization by unrelated commensals in the Acetobacter genus. These results provide a mechanistic framework for understanding the establishment and stability of a multi-species intestinal microbiome.
Meichtry, L. B., da Silva, G. S., Londero, L., Mustafa Dahleh, M. M., Bortolotto, V. C., Araujo, S. M., Aparecida Musachio, E., da Silva, D. T., Emanuelli, T., Sigal Carriço, M. R., Roehrs, R., Guerra, G. P. and Prigol, M. (2023). Exposure to Trans Fat During the Developmental Period of Drosophila melanogaster Alters the Composition of Fatty Acids in the Head and Induces Depression-like Behavior. Neuroscience 519: 10-22. PubMed ID: 36933760
Given the importance of understanding the disorders caused by trans fatty acids (TFAs), this study sought to add different concentrations hydrogenated vegetable fat (HVF) to the diet of Drosophila melanogaster during the developmental period and evaluate the effects on neurobehavioral parameters. Longevity, hatching rate, and behavioral functions were assessed, such as negative geotaxis, forced swimming, light/dark, mating, and aggressiveness. The fatty acids (FAs) present in the heads of the flies were quantified as well as serotonin (5HT) and dopamine (DA) levels. The findings showed that flies that received HVF at all concentrations during development showed reduced longevity and hatching rates, in addition to increased depression-like, anxious-like, anhedonia-like, and aggressive behaviors. As for the biochemical parameters, there was a more significant presence of TFA in flies exposed to HVF at all concentrations evaluated and lower 5HT and DA levels. This study shows that HVF during the developmental phase can cause neurological changes and consequently induce behavioral disorders, thereby highlighting the importance of the type of FA offered in the early stages of life.
Scott, H., Novikov, B., Ugur, B., Allen, B., Mertsalov, I., Monagas-Valentin, P., Koff, M., Baas Robinson, S., Aoki, K., Veizaj, R., Lefeber, D. J., Tiemeyer, M., Bellen, H. and Panin, V. (2023). Glia-neuron coupling via a bipartite sialylation pathway promotes neural transmission and stress tolerance in Drosophila. Elife 12. PubMed ID: 36946697
Modification by sialylated glycans can affect protein functions, underlying mechanisms that control animal development and physiology. Sialylation relies on a dedicated pathway involving evolutionarily conserved enzymes, including CMP-sialic acid synthetase (CSAS) and sialyltransferase (SiaT) that mediate the activation of sialic acid and its transfer onto glycan termini, respectively. In Drosophila, CSAS and DSiaT genes function in the nervous system, affecting neural transmission and excitability. These genes were found to function in different cells: the function of CSAS is restricted to glia, while DSiaT functions in neurons. This partition of the sialylation pathway allows for regulation of neural functions via a glia-mediated control of neural sialylation. The sialylation genes were shown to be required for tolerance to heat and oxidative stress and for maintenance of the normal level of voltage-gated sodium channels. The results uncovered a unique bipartite sialylation pathway that mediates glia-neuron coupling and regulates neural excitability and stress tolerance.

riday, June 23rd - Larval and Adult Neural Development and Function

Kandimalla, P., Omoto, J. J., Hong, E. J. and Hartenstein, V. (2023). Lineages to circuits: the developmental and evolutionary architecture of information channels into the central complex. J Comp Physiol A Neuroethol Sens Neural Behav Physiol. PubMed ID: 36932234
The representation and integration of internal and external cues is crucial for any organism to execute appropriate behaviors. In insects, a highly conserved region of the brain, the central complex (CX), functions in the representation of spatial information and behavioral states, as well as the transformation of this information into desired navigational commands. How does this relatively invariant structure enable the incorporation of information from the diversity of anatomical, behavioral, and ecological niches occupied by insects? This study examined the input channels to the CX in the context of their development and evolution. Insect brains develop from ~100 neuroblasts per hemisphere that divide systematically to form "lineages" of sister neurons, that project to their target neuropils along anatomically characteristic tracts. Overlaying this developmental tract information onto the recently generated Drosophila "hemibrain" connectome and integrating this information with the anatomical and physiological recording of neurons in other species, this study observe neuropil and lineage-specific innervation, connectivity, and activity profiles in CX input channels. It is posited that the proliferative potential of neuroblasts and the lineage-based architecture of information channels enable the modification, over the evolutionary time scale, of neural networks across existing, novel, and deprecated modalities in a species-specific manner, thus forming the substrate for the evolution and diversification of insect navigational circuits.
Krantz, D. and Bonanno, S. (2023). Transcriptional changes in specific subsets of Drosophila neurons following inhibition of the serotonin transporter. Res Sq. PubMed ID: 36993644
The transcriptional effects of SSRIs and other serotonergic drugs remain unclear, in part due to the heterogeneity of postsynaptic cells, which may respond differently to changes in serotonergic signaling. Relatively simple model systems such as Drosophila afford more tractable microcircuits in which to investigate these changes in specific cell types. This study focused on the mushroom body, an insect brain structure heavily innervated by serotonin and comprised of multiple different but related subtypes of Kenyon cells. Fluorescence activated cell sorting of Kenyon cells, followed by either or bulk or single cell RNA sequencing, was used to explore the transcriptomic response of these cells to SERT inhibition. The effects of two different Drosophila Serotonin Transporter (dSERT) mutant alleles were compared as well as feeding the SSRI citalapram to adult flies. The genetic architecture associated with one of the mutants was found to contribute to significant artefactual changes in expression. Comparison of differential expression caused by loss of SERT during development versus aged, adult flies, suggests that changes in serotonergic signaling may have relatively stronger effects during development, consistent with behavioral studies in mice. Overall, these experiments revealed limited transcriptomic changes in Kenyon cells, but suggest that different subtypes may respond differently to SERT loss-of-function. Further work exploring the effects of SERT loss-of-function in other Drosophila circuits may be used help to elucidate how SSRIs differentially affect a variety of different neuronal subtypes both during development and in adults.
Pedigo, B. D., Powell, M., Bridgeford, E. W., Winding, M., Priebe, C. E. and Vogelstein, J. T. (2023). Generative network modeling reveals quantitative definitions of bilateral symmetry exhibited by a whole insect brain connectome. Elife 12. PubMed ID: 36976249
Comparing connectomes can help explain how neural connectivity is related to genetics, disease, development, learning, and behavior. However, making statistical inferences about the significance and nature of differences between two networks is an open problem, and such analysis has not been extensively applied to nanoscale connectomes. This study investigated this problem via a case study on the bilateral symmetry of a larval Drosophila brain connectome. Notions of 'bilateral symmetry' were translated to generative models of the network structure of the left and right hemispheres, allowing testing and refining of understanding of symmetry. Significant differences were found in connection probabilities both across the entire left and right networks and between specific cell types. By rescaling connection probabilities or removing certain edges based on weight, adjusted definitions are presented of bilateral symmetry exhibited by this connectome. This work shows how statistical inferences from networks can inform the study of connectomes, facilitating future comparisons of neural structures.
Nanda, S., Bhattacharjee, S., Cox, D. N. and Ascoli, G. A. (2023). Local microtubule and F-actin distributions fully determine the spatial geometry of Drosophila sensory dendritic arbors. bioRxiv. PubMed ID: 36909461
Dendritic morphology underlies the source and processing of neuronal signal inputs. Previous work demonstrated that microtubules and actin filaments are associated with arbor elongation and branching, fully constraining dendrogram topology. This study relate the local distribution of these two primary cytoskeletal components with dendritic spatial embedding. First, 167 sensory neurons from the Drosophila larva encompassing multiple cell classes and genotypes were reconstructed and analyzed. It was observed that branches with higher microtubule concentration are overall straighter and tend to deviate less from the direction of their parent branch. F-actin displays a similar effect on the angular deviation from the parent branch direction, but its influence on branch tortuosity varies by class and genotype. A computational model was created of dendritic morphology purely constrained by the cytoskeletal composition imaged from real neurons. The model quantitatively captures both spatial embedding and dendrogram topology across all tested neuron groups. These results suggest a common developmental mechanism regulating diverse morphologies, where the local cytoskeletal distribution can fully specify the overall emergent geometry of dendritic arbors.
Mangione, F., Titlow, J., Maclachlan, C., Gho, M., Davis, I., Collinson, L. and Tapon, N. (2023). Co-option of epidermal cells enables touch sensing. Nat Cell Biol 25(4): 540-549. PubMed ID: 36959505
The epidermis is equipped with specialized mechanosensory organs that enable the detection of tactile stimuli. By examining the differentiation of the tactile bristles, mechanosensory organs decorating the Drosophila adult epidermis, this study shows that neighbouring epidermal cells are essential for touch perception. Each mechanosensory bristle signals to the surrounding epidermis to co-opt a single epidermal cell, which this study named the F-Cell. Once specified, the F-Cell adopts a specialized morphology to ensheath each bristle. Functional assays reveal that adult mechanosensory bristles require association with the epidermal F-Cell for touch sensing. These findings underscore the importance of resident epidermal cells in the assembly of functional touch-sensitive organs.
Mi, T., Mack, J. O., Koolmees, W., Lyon, Q., Yochimowitz, L., Teng, Z. Q., Jiang, P., Montell, C. and Zhang, Y. V. (2023). Alkaline taste sensation through the alkaliphile chloride channel in Drosophila. Nat Metab 5(3): 466-480. PubMed ID: 36941450
The sense of taste is an important sentinel governing what should or should not be ingested by an animal, with high pH sensation playing a critical role in food selection. This study explored the molecular identities of taste receptors detecting the basic pH of food using Drosophila melanogaster as a model. A chloride channel has been identified, named alkaliphile (Alka), that is both necessary and sufficient for aversive taste responses to basic food. Alka forms a high-pH-gated chloride channel and is specifically expressed in a subset of gustatory receptor neurons (GRNs). Optogenetic activation of alka-expressing GRNs is sufficient to suppress attractive feeding responses to sucrose. Conversely, inactivation of these GRNs causes severe impairments in the aversion to high pH. Altogether, this discovery of Alka as an alkaline taste receptor lays the groundwork for future research on alkaline taste sensation in other animals.

Wednesday, June 21st - Embryonic Development

Lopez, C. H., Puliafito, A., Xu, Y., Lu, Z., Talia, S. D. and Vergassola, M. (2023). Two-fluid dynamics and micron-thin boundary layers shape cytoplasmic flows in early Drosophila embryos. bioRxiv. PubMed ID: 36993669
Cytoplasmic flows are widely emerging as key functional players in development. In early Drosophila embryos, flows drive the spreading of nuclei across the embryo. This study combined hydrodynamic modeling with quantitative imaging to develop a two-fluid model that features an active actomyosin gel and a passive viscous cytosol. Gel contractility is controlled by the cell cycle oscillator, the two fluids being coupled by friction. In addition to recapitulating experimental flow patterns, this model explains observations that remained elusive, and makes a series of new predictions. First, the model captures the vorticity of cytosolic flows, which highlights deviations from Stokes' flow that were observed experimentally but remained unexplained. Second, the model reveals strong differences in the gel and cytosol motion. In particular, a micron-sized boundary layer is predicted close to the cortex, where the gel slides tangentially whilst the cytosolic flow cannot slip. Third, the model unveils a mechanism that stabilizes the spreading of nuclei with respect to perturbations of their initial positions. This self-correcting mechanism is argued to be functionally important for proper nuclear spreading. Fourth, the model was used to analyze the effects of flows on the transport of the morphogen Bicoid, and the establishment of its gradients. Finally, the model predicts that the flow strength should be reduced if the shape of the domain is more round, which is experimentally confirmed in Drosophila mutants. Thus, the two-fluid model explains flows and nuclear positioning in early Drosophila, while making predictions that suggest novel future experiments.
Ho, E. K., Oatman, H. R., McFann, S. E., Yang, L., Johnson, H. E., Shvartsman, S. Y. and Toettcher, J. E. (2023). Dynamics of an incoherent feedforward loop drive ERK-dependent pattern formation in the early Drosophila embryo. bioRxiv. PubMed ID: 36945584
Positional information in developing tissues often takes the form of stripes of gene expression that mark the boundaries of a particular cell type or morphogenetic process. How stripes form is still in many cases poorly understood. This study used optogenetics and live-cell biosensors to investigate one such pattern: the posterior stripe of brachyenteron (byn) expression in the early Drosophila embryo. This byn stripe depends on interpretation of an upstream signal - a gradient of ERK kinase activity - and the expression of two target genes tailless (tll) and huckebein (hkb) that exert antagonistic control over byn. High or low doses of ERK signaling produce either transient or sustained byn expression, respectively. These ERK stimuli also regulate tll and hkb expression with distinct dynamics: tll transcription is rapidly induced under both low and high stimuli, whereas hkb transcription converts graded ERK inputs into an output switch with a variable time delay. Antagonistic regulatory paths acting on different timescales are hallmarks of an incoherent feedforward loop architecture, which is sufficient to explain transient or sustained byn dynamics and adds temporal complexity to the steady-state model of byn stripe formation. It was further shown that an all-or-none stimulus can be 'blurred' through intracellular diffusion to non-locally produce a stripe of byn gene expression. Overall, this study provides a blueprint for using optogenetic inputs to dissect developmental signal interpretation in space and time.
Fenelon, K. D., Gao, F., Borad, P., Abbasi, S., Pachter, L. and Koromila, T. (2023). Cell-specific occupancy dynamics between the pioneer-like factor Opa/ZIC and Ocelliless/OTX regulate early head development in embryos. Front Cell Dev Biol 11: 1126507. PubMed ID: 37051467
During development, embryonic patterning systems direct a set of initially uncommitted pluripotent cells to differentiate into a variety of cell types and tissues. A core network of transcription factors, such as Zelda/POU5F1, Odd-paired (Opa)/ZIC3 and Ocelliless (Oc)/OTX2, are conserved across animals. While Opa is essential for a second wave of zygotic activation after Zelda, it is unclear whether Opa drives head cell specification, in the Drosophila embryo. It is hypothesized that Opa and Oc are interacting with distinct cis-regulatory regions for shaping cell fates in the embryonic head. Super-resolution microscopy and meta-analysis of single-cell RNAseq datasets show that opa and oc overlapping expression domains are dynamic in the head region, with both factors being simultaneously transcribed at the blastula stage. Additionally, analysis of single-embryo RNAseq data reveals a subgroup of Opa-bound genes to be Opa-independent in the cellularized embryo. Interrogation of these genes against Oc ChIPseq combined with in situ data, suggests that Opa is competing with Oc for the regulation of a subgroup of genes later in gastrulation. Specifically, it was found that Oc binds to late, head-specific enhancers independently and activates them in a head-specific wave of zygotic transcription, suggesting distinct roles for Oc in the blastula and gastrula stages.
Colonnetta, M. M., Schedl, P. and Deshpande, G. (2023). Germline/soma distinction in Drosophila embryos requires regulators of zygotic genome activation. Elife 12. PubMed ID: 36598809
In Drosophila melanogaster embryos, somatic versus germline identity is the first cell fate decision. Zygotic genome activation (ZGA) orchestrates regionalized gene expression, imparting specific identity on somatic cells. ZGA begins with a minor wave that commences at nuclear cycle (NC)8 under the guidance of chromatin accessibility factors (Zelda, CLAMP, GAF), followed by the major wave during NC14. By contrast, primordial germ cell (PGC) specification requires maternally deposited and posteriorly anchored germline determinants. This is accomplished by a centrosome coordinated release and sequestration of germ plasm during the precocious cellularization of PGCs in NC10. This study reports a novel requirement for Zelda and CLAMP during the establishment of the germline/soma distinction. When their activity is compromised, PGC determinants are not properly sequestered, and specification is disrupted. Conversely, the spreading of PGC determinants from the posterior pole adversely influences transcription in the neighboring somatic nuclei. These reciprocal aberrations can be correlated with defects in centrosome duplication/separation that are known to induce inappropriate transmission of the germ plasm. Interestingly, consistent with the ability of bone morphogenetic protein (BMP) signaling to influence specification of embryonic PGCs, reduction in the transcript levels of a BMP family ligand, decapentaplegic (dpp), is exacerbated at the posterior pole.
Gehrels, E. W., Chakrabortty, B., Perrin, M. E., Merkel, M. and Lecuit, T. (2023). Curvature gradient drives polarized tissue flow in the Drosophila embryo. Proc Natl Acad Sci U S A 120(6): e2214205120. PubMed ID: 36724258
Tissue flow during morphogenesis is commonly driven by local constriction of cell cortices, which is caused by the activation of actomyosin contractility. This can lead to long-range flows due to tissue viscosity. However, in the absence of cell-intrinsic polarized forces or polarity in forces external to the tissue, these flows must be symmetric and centered around the region of contraction. Polarized tissue flows have been previously demonstrated to arise from the coupling of such contractile flows to points of increased friction or adhesion to external structures. However, this study shows with experiments and modeling that the onset of polarized tissue flow in early Drosophila morphogenesis occurs independent of adhesion and is instead driven by a geometric coupling of apical actomyosin contractility to tissue curvature. Particularly, the onset of polarized flow is driven by a mismatch between the position of apical myosin activation and the position of peak curvature at the posterior pole of the embryo. This work demonstrates how genetic and geometric information inherited from the mother interact to create polarized flow during embryo morphogenesis.
Ciabrelli, F., Rabbani, L., Cardamone, F., Zenk, F., Loser, E., Schächtle, M. A., Mazina, M., Loubiere, V. and Iovino, N. (2023). CBP and Gcn5 drive zygotic genome activation independently of their catalytic activity. Sci Adv 9(16): eadf2687. PubMed ID: 37083536
Zygotic genome activation (ZGA) is a crucial step of embryonic development. So far, little is known about the role of chromatin factors during this process. This study used an in vivo RNA interference reverse genetic screen to identify chromatin factors necessary for embryonic development in Drosophila melanogaster. The screen reveals that histone acetyltransferases (HATs) and histone deacetylases are crucial ZGA regulators. It was demonstrated that Nejire (CBP/EP300 ortholog) is essential for the acetylation of histone H3 lysine-18 and lysine-27, whereas Gcn5 (GCN5/PCAF ortholog) for lysine-9 of H3 at ZGA, with these marks being enriched at all actively transcribed genes. Nonetheless, these HATs activate distinct sets of genes. Unexpectedly, individual catalytic dead mutants of either Nejire or Gcn5 can activate zygotic transcription (ZGA) and transactivate a reporter gene in vitro. Together, these data identify Nejire and Gcn5 as key regulators of ZGA.

Tuesday, June 20th - Signaling

Gilhaus, K., Cepok, C., Kamm, D., Surmann, B., Nedvetsky, P. I., Emich, J., Sundukova, A., Saatkamp, K., Nusse, H., Klingauf, J., Wennmann, D. O., George, B., Krahn, M. P., Pavenstadt, H. J. and Vollenbroker, B. A. (2023). Activation of Hippo Pathway Damages Slit Diaphragm by Deprivation of Ajuba Proteins. J Am Soc Nephrol. PubMed ID: 36930055
The highly conserved Hippo pathway, which regulates organ growth and cell proliferation by inhibiting transcriptional cofactors YAP/TAZ, plays a special role in podocytes, where activation of the pathway leads to apoptosis. The Ajuba family proteins (Ajuba, LIM domain-containing protein 1 (LIMD1) and Wilms tumor protein 1-interacting protein [WTIP]) can bind and inactivate large tumor suppressor kinases 1 and 2, (LATS1/2) two of the Hippo pathway key kinases. WTIP, furthermore, connects the slit diaphragm (SD), the specialized cell-cell junction between podocytes, with the actin cytoskeleton. This study used garland cell nephrocytes of Drosophila to monitor the role of Ajuba proteins in Hippo pathway regulation and structural integrity of the SD. In nephrocytes, the Ajuba homolog Djub recruited Warts (LATS2 homolog) to the SD. Knockdown of Djub activated the Hippo pathway. Reciprocally, Hippo activation reduced the Djub level. Both Djub knockdown and Hippo activation led to morphological changes in the SD, rearrangement of the cortical actin cytoskeleton, and increased SD permeability. Knockdown of Warts or overexpression of constitutively active Yki prevented these effects. In podocytes, Hippo pathway activation or knockdown of YAP also decreased the level of Ajuba proteins. It is concluded that Ajuba proteins regulate the structure and function of the SD in nephrocytes, connecting the SD protein complex to the actin cytoskeleton and maintaining the Hippo pathway in an inactive state. Hippo pathway activation directly influencing Djub expression suggests a self-amplifying feedback mechanism.
Lei, Y., Huang, Y., Yang, K., Cao, X., Song, Y., Martín-Blanco, E. and Pastor-Pareja, J. C. (2023). FGF signaling promotes spreading of fat body precursors necessary for adult adipogenesis in Drosophila. PLoS Biol 21(3): e3002050. PubMed ID: 36947563
Knowledge of adipogenetic mechanisms is essential to understand and treat conditions affecting organismal metabolism and adipose tissue health. In Drosophila, mature adipose tissue (fat body) exists in larvae and adults. In contrast to the well-known development of the larval fat body from the embryonic mesoderm, adult adipogenesis has remained mysterious. Furthermore, conclusive proof of its physiological significance is lacking. This study shows that the adult fat body originates from a pool of undifferentiated mesodermal precursors that migrate from the thorax into the abdomen during metamorphosis. Through in vivo imaging, it was found that these precursors spread from the ventral midline and cover the inner surface of the abdomen in a process strikingly reminiscent of embryonic mesoderm migration, requiring fibroblast growth factor (FGF) signaling as well. FGF signaling guides migration dorsally and regulates adhesion to the substrate. After spreading is complete, precursor differentiation involves fat accumulation and cell fusion that produces mature binucleate and tetranucleate adipocytes. Finally, this study shows that flies where adult adipogenesis is impaired by knock down of FGF receptor Heartless or transcription factor Serpent display ectopic fat accumulation in oenocytes and decreased resistance to starvation. These results reveal that adult adipogenesis occurs de novo during metamorphosis and demonstrate its crucial physiological role.
Xia, B., Viswanatha, R., Hu, Y., Mohr, S. E. and Perrimon, N. (2023). Pooled genome-wide CRISPR activation screening for rapamycin resistance genes in Drosophila cells. Elife 12. PubMed ID: 37078570
Loss-of-function and gain-of-function genetic perturbations provide valuable insights into gene function. In Drosophila cells, while genome-wide loss-of-function screens have been extensively used to reveal mechanisms of a variety of biological processes, approaches for performing genome-wide gain-of-function screens are still lacking. This study describe a pooled CRISPR activation (CRISPRa) screening platform in Drosophila cells and apply this method to both focused and genome-wide screens to identify rapamycin resistance genes. The screens identified three genes as novel rapamycin resistance genes: a member of the SLC16 family of monocarboxylate transporters (CG8468), a member of the lipocalin protein family (CG5399), and a zinc finger C2H2 transcription factor (CG9932). Mechanistically, it was demonstrated that CG5399 overexpression activates the RTK-Akt-mTOR signaling pathway and that activation of insulin receptor (InR) by CG5399 requires cholesterol and clathrin-coated pits at the cell membrane. This study establishes a novel platform for functional genetic studies in Drosophila cells.
Zhao, N., Li, N. and Wang, T. (2023). PERK prevents rhodopsin degradation during retinitis pigmentosa by inhibiting IRE1-induced autophagy. J Cell Biol 222(5). PubMed ID: 37022709
Chronic endoplasmic reticulum (ER) stress is the underlying cause of many degenerative diseases, including autosomal dominant retinitis pigmentosa (adRP). In adRP, mutant rhodopsins accumulate and cause ER stress. This destabilizes wild-type rhodopsin and triggers photoreceptor cell degeneration. To reveal the mechanisms by which these mutant rhodopsins exert their dominant-negative effects, this study established an in vivo fluorescence reporter system to monitor mutant and wild-type rhodopsin in Drosophila. By performing a genome-wide genetic screen, PERK signaling was found to play a key role in maintaining rhodopsin homeostasis by attenuating IRE1 activities. Degradation of wild-type rhodopsin is mediated by selective autophagy of ER, which is induced by uncontrolled IRE1/XBP1 signaling and insufficient proteasome activities. Moreover, upregulation of PERK signaling prevents autophagy and suppresses retinal degeneration in the adRP model. These findings establish a pathological role for autophagy in this neurodegenerative condition and indicate that promoting PERK activity could be used to treat ER stress-related neuropathies, including adRP.
Carter, A. A., Ramsey, K. M., Hatem, C. L., Sherry, K. P., Majumdar, A. and Barrick, D. (2023). Structural features of the Notch ankyrin domain-Deltex WWE(2) domain heterodimer determined by NMR spectroscopy and functional implications. Structure. PubMed ID: 36977409
The Notch signaling pathway, an important cell fate determination pathway, is modulated by the ubiquitin ligase Deltex. This study investigated the structural basis for Deltex-Notch interaction. Nuclear magnetic resonance (NMR) spectroscopy was used to assign the backbone of the Drosophila Deltex WWE(2) domain and mapped the binding site of the Notch ankyrin (ANK) domain to the N-terminal WWE(A) motif. Using cultured Drosophila S2R+ cells, this study found that point substitutions within the ANK-binding surface of Deltex disrupt Deltex-mediated enhancement of Notch transcriptional activation and disrupt ANK binding in cells and in vitro. Likewise, ANK substitutions that disrupt Notch-Deltex heterodimer formation in vitro block disrupt Deltex-mediated stimulation of Notch transcription activation and diminish interaction with full-length Deltex in cells. Surprisingly, the Deltex-Notch intracellular domain (NICD) interaction is not disrupted by deletion of the Deltex WWE(2) domain, suggesting a secondary Notch-Deltex interaction. These results show the importance of the WWE(A):ANK interaction in enhancing Notch signaling.
Golubev, D. A., Zemskaya, N. V., Gorbunova, A. A., Kukuman, D. V., Moskalev, A. and Shaposhnikov, M. V. (2023). Studying the Geroprotective Properties of YAP/TAZ Signaling Inhibitors on Drosophila melanogaster Model. Int J Mol Sci 24(6). PubMed ID: 36983079
The transcriptional coactivators Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) are the main downstream effectors of the evolutionarily conserved Hippo signaling pathway. YAP/TAZ are implicated in the transcriptional regulation of target genes that are involved in a wide range of key biological processes affecting tissue homeostasis and play dual roles in the aging process, depending on the cellular and tissue context. The aim of the present study was to investigate whether pharmacological inhibitors of Yap/Taz increase the lifespan of Drosophila melanogaster. Real-time qRT-PCR was performed to measure the changes in the expression of Yki (Yorkie, the Drosophila homolog of YAP/TAZ) target genes. This study has revealed a lifespan-increasing effect of YAP/TAZ inhibitors that was mostly associated with decreased expression levels of the wg and E2f1 genes. However, further analysis is required to understand the link between the YAP/TAZ pathway and aging.

Friday, June 16th - Behavior

Rimniceanu, M., Currea, J. P. and Frye, M. A. (2023). Proprioception gates visual object fixation in flying flies. Curr Biol. PubMed ID: 37001520
Visual object tracking in animals as diverse as felines, frogs, and fish supports behaviors including predation, predator avoidance, and landscape navigation. Decades of experimental results show that a rigidly body-fixed tethered fly in a "virtual reality" visual flight simulator steers to follow the motion of a vertical bar, thereby "fixating" it on visual midline. This behavior likely reflects a desire to seek natural features such as plant stalks and has inspired algorithms for visual object tracking predicated on robust responses to bar velocity, particularly near visual midline. Using a modified flight simulator equipped with a magnetic pivot to allow frictionless turns about the yaw axis, it was discovered that bar fixation as well as smooth steering responses to bar velocity are attenuated or eliminated in yaw-free conditions. Body-fixed Drosophila melanogaster respond to bar oscillation on a stationary ground with frequency-matched wing kinematics and fixate the bar on midline. Yaw-free flies respond to the same stimulus by ignoring the bar and maintaining their original heading. These differences are driven by proprioceptive signals, rather than visual signals, as artificially "clamping" a bar in the periphery of a yaw-free fly has no effect. When presented with a bar and ground oscillating at different frequencies, a yaw-free fly follows the frequency of the ground only, whereas a body-fixed fly robustly steers at the frequencies of both the bar and ground. Threse findings support a model in which proprioceptive feedback promote active damping of high-gain optomotor responses to object motion.
Berger, M., Yapıcı, B. and Scholz, H. (2023). The function of ethanol in olfactory associative behaviors in Drosophila melanogaster larvae. PLoS One 18(3): e0276714. PubMed ID: 36913432
Drosophila melanogaster larvae develop on fermenting fruits with increasing ethanol concentrations. To address the relevance of ethanol in the behavioral response of the larvae, this study analyzed the function of ethanol in the context of olfactory associative behavior in Canton S and w1118 larvae. The motivation of larvae to move toward or out of an ethanol-containing substrate depends on the ethanol concentration and the genotype. Ethanol in the substrate reduces the attraction to odorant cues in the environment. Relatively short repetitive exposures to ethanol, which are comparable in their duration to reinforcer representation in olfactory associative learning and memory paradigms, result in positive or negative association with the paired odorant or indifference to it. The outcome depends on the order in which the reinforcer is presented during training, the genotype and the presence of the reinforcer during the test. Independent of the order of odorant presentation during training, Canton S and w1118 larvae do not form a positive or negative association with the odorant when ethanol is not present in the test context. When ethanol is present in the test, w1118 larvae show aversion to an odorant paired with a naturally occurring ethanol concentration of 5%. These results provide insights into the parameters influencing olfactory associative behaviors using ethanol as a reinforcer in Drosophila larvae and indicate that short exposures to ethanol might not uncover the positive rewarding properties of ethanol for developing larvae.
Sun, X., Nose, A. and Kohsaka, H. (2023). A vacuum-actuated soft robot inspired by Drosophila larvae to study kinetics of crawling behaviour. PLoS One 18(4): e0283316. PubMed ID: 37018174
Peristalsis, a motion generated by the propagation of muscular contraction along the body axis, is one of the most common locomotion patterns in limbless animals. While the kinematics of peristalsis has been examined intensively, its kinetics remains unclear, partially due to the lack of suitable physical models to simulate the locomotion patterns and inner drive in soft-bodied animals. Inspired by a soft-bodied animal, Drosophila larvae, this study propose a vacuum-actuated soft robot mimicking its crawling behaviour. The soft structure, made of hyperelastic silicone rubber, was designed to imitate the larval segmental hydrostatic structure. Referring to a numerical simulation by the finite element method, the dynamical change in the vacuum pressure in each segment was controlled accordingly, and the soft robots could exhibit peristaltic locomotion. The soft robots successfully reproduced two previous experimental phenomena on fly larvae: 1. Crawling speed in backward crawling is slower than in forward crawling. 2. Elongation of either the segmental contraction duration or intersegmental phase delay makes peristaltic crawling slow. Furthermore, this experimental results provided a novel prediction for the role of the contraction force in controlling the speed of peristaltic locomotion. These observations indicate that soft robots could serve to examine the kinetics of crawling behaviour in soft-bodied animals.
Ko, T., Murakami, H., Kobayashi, S., Kamikouchi, A. and Ishimoto, H. (2023). Behavioral screening of sleep-promoting effects of human intestinal and food-associated bacteria on Drosophila melanogaster. Genes Cells. PubMed ID: 36914986
Commensal microbes influence various aspects of vertebrate and invertebrate brain function. It has been previously reported that Lactiplantibacillus plantarum SBT2227 promotes sleep in the fruit fly, Drosophila melanogaster. However, how widely the sleep-promoting effects are conserved in gut bacterial species remains unknown. This study orally administered human intestinal and food-associated bacterial species (39 in total) to flies and investigated their effects on sleep. Six species of bacteria were found to have significant sleep-promoting effects. Of these, Bifidobacterium adolescentis, which had the greatest sleep-promoting effect, was further investigated and it was found that the strength of the sleep effect varied among strains of the same bacterial species. The B. adolescentis strains BA2786 and BA003 showed strong and weak effects on sleep, respectively. Transcriptome characteristics compared between the heads of flies treated with BA2786 or BA003 revealed that the gene expression of the insulin-like receptor (InR) was increased in BA2786-fed flies. Furthermore, a heterozygous mutation in InR suppressed the sleep-promoting effect of BA2786. These results suggest that orally administered sleep-promoting bacteria (at least BA2786), may act on insulin signaling to modulate brain function for sleep.
Zhao, J., Xi, S., Li, Y., Guo, A. and Wu, Z. (2023). A fly inspired solution to looming detection for collision avoidance. iScience 26(4): 106337. PubMed ID: 37035005
Dodging rapidly approaching objects is a fundamental skill for both animals and intelligent robots. Flies are adept at high-speed collision avoidance. However, it remains unclear whether the fly algorithm can be extracted and is applicable to real-time machine vision. This study developed a computational model inspired by the looming detection circuit recently identified in Drosophila. These results suggest that in the face of considerably noisy local motion signals, the key for the fly circuit to achieve accurate detection is attributed to two computation strategies: population encoding and nonlinear integration. The model is further shown to be an effective algorithm for collision avoidance by virtual robot tests. The algorithm is characterized by practical flexibility, whose looming detection parameters can be modulated depending on factors such as the body size of the robots. The model sheds light on the potential of the concise fly algorithm in real-time applications.
Jiang, N. J., Chang, H., Weisflog, J., Eberl, F., Veit, D., Weniger, K., Hansson, B. S. and Knaden, M. (2023). Ozone exposure disrupts insect sexual communication. Nat Commun 14(1): 1186. PubMed ID: 36918554
Insect sexual communication often relies upon sex pheromones. Most insect pheromones, however, contain carbon-carbon double bonds and potentially degrade by oxidation. This study shows that frequently reported increased levels of Anthropocenic ozone can oxidize all described male-specific pheromones of Drosophila melanogaster, resulting in reduced amounts of pheromones such as cis-Vaccenyl Acetate and (Z)-7-Tricosene. At the same time female acceptance of ozone-exposed males is significantly delayed. Interestingly, groups of ozone-exposed males also exhibit significantly increased levels of male-male courtship behaviour. When repeating similar experiments with nine other drosophilid species, pheromone degradation and/or disrupted sex recognition were observed in eight of them. The data suggest that Anthropocenic levels of ozone can extensively oxidize double bonds in a variety of insect pheromones, thereby leading to deviations in sexual recognition.

Thursday, June 15th - Adult Neural Development and Function

Galindo, S. E., Shin, G. J., Millard, S. S. and Grueber, W. B. (2023). Regulated alternative splicing of Dscam2 is required for somatosensory circuit wiring. bioRxiv. PubMed ID: 36909552
Axon and dendrite placement and connectivity is guided by a wide range of secreted and surface molecules in the developing nervous system. Nevertheless, the extraordinary complexity of connections in the brain requires that this repertoire be further diversified to precisely and uniquely regulate cell-cell interactions. One important mechanism for molecular diversification is alternative splicing. Drosophila Down syndrome cell adhesion molecule (Dscam2) undergoes cell type-specific alternative splicing to produce two isoform-specific homophilic binding proteins. Regulated alternative splicing of Dscam2 is important for dendrite and axon patterning, but how this translates to circuit wiring and animal behavior is not well understood. This study examined the role of cell-type specific expression of Dscam2 isoforms in regulating synaptic partner selection in the larval somatosensory system. Synaptic partners in the nociceptive circuit express different Dscam2 isoforms. Forcing synaptic partners to express a common isoform resulted in nociceptive axon patterning defects and attenuated nocifensive behaviors, indicating that a role for Dscam2 alternative splicing is to ensure that synaptic partners do not express matching isoforms. These results point to a model in which regulated alternative splicing of Dscam2 across populations of neurons restricts connectivity to specific partners and prevents inappropriate synaptic connections.
Deanhardt, B., Duan, Q., Du, C., Soeder, C., Morlote, A., Garg, D., Saha, A., Jones, C. D. and Volkan, P. C. (2023). Social experience and pheromone receptor activity reprogram gene expression in sensory neurons. G3 (Bethesda). PubMed ID: 36972331
Social experience and pheromone signaling in olfactory neurons affect neuronal responses and male courtship behaviors in Drosophila. Previous work has shown that social experience and pheromone signaling modulate chromatin around behavioral switch gene fruitless, which encodes a transcription factor necessary and sufficient for male sexual behaviors. To identify the molecular mechanisms driving social experience-dependent changes in neuronal responses, RNA-seq was performed from antennal samples of mutants in pheromone receptors and fruitless, as well as grouped or isolated wild-type males. Genes affecting neuronal physiology and function, such as neurotransmitter receptors, ion channels, ion and membrane transporters, and odorant binding proteins are differentially regulated by social context and pheromone signaling. While this study found that loss of pheromone detection only has small effects on differential promoter and exon usage within fruitless gene, many of the differentially regulated genes have Fruitless binding sites or are bound by Fruitless in the nervous system. Recent studies showed that social experience and juvenile hormone signaling co-regulate fruitless chromatin to modify pheromone responses in olfactory neurons. Interestingly, genes involved in juvenile hormone metabolism are also misregulated in different social contexts and mutant backgrounds. These results suggest that modulation of neuronal activity and behaviors in response to social experience and pheromone signaling likely arise due to large-scale changes in transcriptional programs for neuronal function downstream of behavioral switch gene function.
Chen, C. L., Aymanns, F., Minegishi, R., Matsuda, V. D. V., Talabot, N., GUnel, S., Dickson, B. J. and Ramdya, P. (2023). Ascending neurons convey behavioral state to integrative sensory and action selection brain regions. Nat Neurosci 26(4): 682-695. PubMed ID: 36959417
Knowing one's own behavioral state has long been theorized as critical for contextualizing dynamic sensory cues and identifying appropriate future behaviors. Ascending neurons (ANs) in the motor system that project to the brain are well positioned to provide such behavioral state signals. However, what ANs encode and where they convey these signals remains largely unknown. Through large-scale functional imaging in behaving animals and morphological quantification, this study reports the behavioral encoding and brain targeting of hundreds of genetically identifiable ANs in the adult fly, Drosophila melanogaster. It is revealed that ANs encode behavioral states, specifically conveying self-motion to the anterior ventrolateral protocerebrum, an integrative sensory hub, as well as discrete actions to the gnathal ganglia, a locus for action selection. Additionally, AN projection patterns within the motor system are predictive of their encoding. Thus, ascending populations are well poised to inform distinct brain hubs of self-motion and ongoing behaviors and may provide an important substrate for computations that are required for adaptive behavior.
Chen, N., Zhang, Y., Rivera-Rodriguez, E. J., Yu, A. D., Hobin, M., Rosbash, M. and Griffith, L. C. (2023). Widespread post-transcriptional regulation of co-transmission. bioRxiv. PubMed ID: 36909471
While neurotransmitter identity was once considered singular and immutable for mature neurons, it is now appreciated that one neuron can release multiple neuroactive substances (co-transmission) whose identities can even change over time. To explore the mechanisms that tune the suite of transmitters a neuron releases, this study developed transcriptional and translational reporters for cholinergic, glutamatergic, and GABAergic signaling in Drosophila. Many glutamatergic and GABAergic cells also transcribe cholinergic genes, but fail to accumulate cholinergic effector proteins. Suppression of cholinergic signaling involves posttranscriptional regulation of cholinergic transcripts by the microRNA miR-190; chronic loss of miR-190 function allows expression of cholinergic machinery, reducing and fragmenting sleep. Using a "translation-trap" strategy this study shows that neurons in these populations have episodes of transient translation of cholinergic proteins, demonstrating that suppression of co-transmission is actively modulated. Posttranscriptional restriction of fast transmitter co-transmission provides a mechanism allowing reversible tuning of neuronal output. It is concluded that colinergic co-transmission in large populations of glutamatergic and GABAergic neurons in the Drosophila adult brain is controlled by miR-190.
Yin, J. C. P., Cui, E., Hardin, P. E. and Zhou, H. (2023). Circadian disruption of memory consolidation in Drosophila. Front Syst Neurosci 17: 1129152. PubMed ID: 37034015
The role of the circadian system in memory formation is an important question in neurobiology. Despite this hypothesis being intuitively appealing, the existing data is confusing. Recent work in Drosophila has helped to clarify certain aspects of the problem, but the emerging sense is that the likely mechanisms are more complex than originally conceptualized. This report, identifies a post-training window of time (during consolidation) when the circadian clock and its components are involved in memory formation. In the broader context, the data suggest that circadian biology might have multiple roles during memory formation. Testing for its roles at multiple timepoints, and in different cells, will be necessary to resolve some of the conflicting data.
Hafez, O. A., Escribano, B., Ziegler, R. L., Hirtz, J. J., Niebur, E. and Pielage, J. (2023). The cellular architecture of memory modules in Drosophila supports stochastic input integration. Elife 12. PubMed ID: 36916672
The ability to associate neutral stimuli with valence information and to store these associations as memories forms the basis for decision making. To determine the underlying computational principles, this study built a realistic computational model of a central decision module within the Drosophila mushroom body (MB), the fly's center for learning and memory. The model combines the electron microscopy-based architecture of one MB output neuron (MBON-α3), the synaptic connectivity of its 948 presynaptic Kenyon cells (KCs), and its membrane properties obtained from patch-clamp recordings. This neuron is electrotonically compact and that synaptic input corresponding to simulated odor input robustly drives its spiking behavior. Therefore, sparse innervation by KCs can efficiently control and modulate MBON activity in response to learning with minimal requirements on the specificity of synaptic localization. This architecture allows efficient storage of large numbers of memories using the flexible stochastic connectivity of the circuit.

Wednesday, June 14th - Disease Models

Alassaf, M. and Rajan, A. (2023). Diet-Induced Glial Insulin Resistance Impairs The Clearance Of Neuronal Debris. bioRxiv. PubMed ID: 36945507
Obesity significantly increases the risk of developing neurodegenerative disorders, yet the precise mechanisms underlying this connection remain unclear. Defects in glial phagocytic function are a key feature of neurodegenerative disorders, as delayed clearance of neuronal debris can result in inflammation, neuronal death, and poor nervous system recovery. Mounting evidence indicates that glial function can affect feeding behavior, weight, and systemic metabolism, suggesting that diet may play a role in regulating glial function. While it is appreciated that glial cells are insulin sensitive, whether obesogenic diets can induce glial insulin resistance and thereby impair glial phagocytic function remains unknown. In this study, using a Drosophila model, it is shown that a chronic obesogenic diet induces glial insulin resistance and impairs the clearance of neuronal debris. Specifically, obesogenic diet exposure downregulates the basal and injury-induced expression of the glia-associated phagocytic receptor, Draper. Constitutive activation of systemic insulin release from Drosophila Insulin-producing cells (IPCs) mimics the effect of diet-induced obesity on glial draper expression. In contrast, genetically attenuating systemic insulin release from the IPCs rescues diet-induced glial insulin resistance and draper expression. Significantly, this study shows that genetically stimulating Phosphoinositide 3-kinase (PI3K), a downstream effector of Insulin receptor signaling, rescues HSD-induced glial defects. This study established that obesogenic diets impair glial phagocytic function and delays the clearance of neuronal debris.
Chaves, N. S. G., Janner, D. E., Poetini, M. R., Fernandes, E. J., de Almeida, F. P., Musachio, E. A. S., Reginaldo, J. C., Dahleh, M. M. M., de Carvalho, A. S., Leimann, F. V., Gonçalves, O. H., Ramborger, B. P., Roehrs, R., Prigol, M. and Guerra, G. P. (2023). β-carotene-loaded nanoparticles protect against neuromotor damage, oxidative stress, and dopamine deficits in a model of Parkinson's disease in Drosophila melanogaster. Comp Biochem Physiol C Toxicol Pharmacol 268: 109615. PubMed ID: 36940893
β-carotene-loaded nanoparticles improves absorption by increasing bioavailability. The Drosophila melanogaster model of Parkinson's disease must be helpful in investigating potential neuroprotective effects. Four groups of four-day-old flies were exposed to: (1) control; (2) diet containing rotenone (500 &mu,M); (3) &bets;-carotene-loaded nanoparticles (20 μM); (4) β-carotene-loaded nanoparticles and rotenone for 7 days. Then, the percentage of survival, geotaxis tests, open field, aversive phototaxis and food consumption were evaluated. At the end of the behaviors, the analyses of the levels of reactive species (ROS), thiobarbituric acid reactive substances (TBARS), catalase (CAT) and superoxide dismutase (SOD) activity was carried out, as well as an evaluation of the levels of dopamine and acetylcholinesterase (AChE) activity, in the head of flies. Nanoparticles loaded with β-carotene were able to improve motor function, memory, survival and also restored the oxidative stress indicators (CAT, SOD, ROS and TBARS), dopamine levels, AChE activity after exposure to rotenone. Overall, nanoparticles loaded with β-carotene showed significant neuroprotective effect against damage induced by the Parkinson-like disease model, emerging as a possible treatment. Overall, β-carotene-loaded nanoparticles presented significant neuroprotective effect against damage induced by model of Parkinson-like disease, emerging as a possible treatment.
Gignac, S. J., MacCharles, K. R., Fu, K., Bonaparte, K., Akarsu, G., Barrett, T. W., Verheyen, E. M. and Richman, J. M. (2023). Mechanistic studies in Drosophila and chicken give new insights into functions of DVL1 in dominant Robinow syndrome. Dis Model Mech 16(4). PubMed ID: 36916233
The study of rare genetic diseases provides valuable insights into human gene function. The autosomal dominant or autosomal recessive forms of Robinow syndrome are genetically heterogeneous, and the common theme is that all the mutations lie in genes in Wnt signaling pathways. Cases diagnosed with Robinow syndrome do survive to adulthood with distinct skeletal phenotypes, including limb shortening and craniofacial abnormalities. This study focused on mutations in dishevelled 1 (DVL1), an intracellular adaptor protein that is required for both canonical (β-catenin-dependent) or non-canonical (requiring small GTPases and JNK) Wnt signaling. Human wild-type DVL1 or DVL1 variants were expressed alongside the endogenous genome of chicken and Drosophila. This design is strategically suited to test for functional differences between mutant and wild-type human proteins in relevant developmental contexts. The expression of variant forms of DVL1 produced a major disorganization of cartilage and Drosophila wing morphology compared to expression of wild-type DVL1. Moreover, the variants caused a loss of canonical and gain of non-canonical Wnt signaling in several assays. These data point to future therapies that might correct the levels of Wnt signaling, thus improving skeletal growth.
Borg, R., Purkiss, A., Cacciottolo, R., Herrera, P. and Cauchi, R. J. (2023). Loss of amyotrophic lateral sclerosis risk factor SCFD1 causes motor dysfunction in Drosophila. Neurobiol Aging 126: 67-76. PubMed ID: 36944290
Amyotrophic lateral sclerosis (ALS) is a progressive neuromuscular disease mostly resulting from a complex interplay between genetic, environmental and lifestyle factors. Common genetic variants in the Sec1 Family Domain Containing 1 (SCFD1) gene have been associated with increased ALS risk in the most extensive genome-wide association study (GWAS). SCFD1 was also identified as a top-most significant expression Quantitative Trait Locus (eQTL) for ALS. Whether loss of SCFD1 function directly contributes to motor system dysfunction remains unresolved. This study shows that moderate gene silencing of Slh, the Drosophila orthologue of SCFD1, is sufficient to cause climbing and flight defects in adult flies. A more severe knockdown induced a significant reduction in larval mobility and profound neuromuscular junction (NMJ) deficits prior to death before metamorphosis. RNA-seq revealed downregulation of genes encoding chaperones that mediate protein folding downstream of Slh ablation. These findings support the notion that loss of SCFD1 function is a meaningful contributor to ALS and disease predisposition may result from erosion of the mechanisms protecting against misfolding and protein aggregation.
Guo, S., Zhang, S., Zhuang, Y., Xie, F., Wang, R., Kong, X., Zhang, Q., Feng, Y., Gao, H., Kong, X. and Liu, T. (2023). Muscle PARP1 inhibition extends lifespan through AMPKalpha PARylation and activation in Drosophila.. J Clin Invest. PubMed ID: 36976648 Proc Natl Acad Sci U S A 120(13): e2213857120. PubMed ID: 36947517
Poly(ADP-ribose) polymerase-1 (PARP1) has been reported to play an important role in longevity. This study showed that the knockdown of the PARP1 extended the lifespan of Drosophila, with particular emphasis on the skeletal muscle. The muscle-specific mutant Drosophila exhibited resistance to starvation and oxidative stress, as well as an increased ability to climb, with enhanced mitochondrial biogenesis and activity at an older age. Mechanistically, the inhibition of PARP1 increases the activity of AMP-activated protein kinase alpha (AMPKalpha) and mitochondrial turnover. PARP1 could interact with AMPKalpha and then regulate it via poly(ADP ribosyl)ation (PARylation) at residues E155 and E195. Double knockdown of PARP1 and AMPKα, specifically in muscle, could counteract the effects of PARP1 inhibition in Drosophila. Finally, it was shown that increasing lifespan via maintaining mitochondrial network homeostasis required intact PTEN induced kinase 1 (PINK1). Taken together, these data indicate that the interplay between PARP1 and AMPKalpha can manipulate mitochondrial turnover, and be targeted to promote longevity.
Gehin, C., Lone, M. A., Lee, W., Capolupo, L., Ho, S., Adeyemi, A. M., Gerkes, E. H., Stegmann, A. P., ..., Hornemann, T., D'Angelo, G. and Gennarino, V. A. (2023). CERT1 mutations perturb human development by disrupting sphingolipid homeostasis. J Clin Invest. PubMed ID: 36976648
Neural differentiation, synaptic transmission, and action potential propagation depend on membrane sphingolipids, whose metabolism is tightly regulated. Mutations in the ceramide transporter CERT (CERT1), which is involved in sphingolipid biosynthesis, are associated with intellectual disability, but the pathogenic mechanism remains obscure. This study characterize 31 individuals with de novo missense variants in CERT1. Several variants fall into a previously uncharacterized dimeric helical domain that enables CERT homeostatic inactivation, without which sphingolipid production goes unchecked. The clinical severity reflects the degree to which CERT autoregulation is disrupted, and inhibiting CERT pharmacologically corrects morphological and motor abnormalities in a Drosophila model of the disease, which was called CerTra syndrome. These findings uncover a central role for CERT autoregulation in the control of the sphingolipid biosynthetic flux, provide unexpected insight into the structural organisation of CERT, and suggest a possible therapeutic approach for CerTra syndrome patients.

Tuesday June 13th - RNAs and Transposons

Merenciano, M. and Gonzalez, J. (2023). The Interplay Between Developmental Stage and Environment Underlies the Adaptive Effect of a Natural Transposable Element Insertion. Mol Biol Evol 40(3). PubMed ID: 36811953
Establishing causal links between adaptive mutations and ecologically relevant phenotypes is key to understanding the process of adaptation, which is a central goal in evolutionary biology with applications for conservation, medicine, and agriculture. Yet despite recent progress, the number of identified causal adaptive mutations remains limited. Linking genetic variation to fitness-related effects is complicated by gene-by-gene and gene-by-environment interactions, among other processes. Transposable elements, which are often ignored in the quest for the genetic basis of adaptive evolution, are a genome-wide source of regulatory elements across organisms that can potentially result in adaptive phenotypes. This work combined gene expression, in vivo reporter assays, CRISPR/Cas9 genome editing, and survival experiments to characterize in detail the molecular and phenotypic consequences of a natural Drosophila melanogaster transposable element insertion: the roo solo-LTR FBti0019985. This transposable element provides an alternative promoter to the transcription factor Lime, involved in cold- and immune-stress responses. The effect of FBti0019985 on Lime expression were found to depend on the interplay between the developmental stage and environmental condition. A causal link between the presence of FBti0019985 and increased survival to cold- and immune-stress was established. The results exemplify how several developmental stages and environmental conditions need to be considered to characterize the molecular and functional effects of a genetic variant, and add to the growing body of evidence that transposable elements can induce complex mutations with ecologically relevant effects.
Yang, D., Xiao, F., Yuan, Y., Li, J., Wang, S., Fan, X., Ni, Q., Li, Y., Zhang, M., Gu, X., Yan, T., Yang, M. and He, Z. (2023). The Expression Pattern of tRNA-Derived Small RNAs in Adult Drosophila and the Function of tRF-Trp-CCA-014-H3C4 Network Analysis. Int J Mol Sci 24(7). PubMed ID: 37047149
tRNA-derived small RNAs (tsRNAs) are derived from tRNA and include tRNA halves (tiRNAs) and tRNA fragments (tRFs). tsRNAs have been implicated in a variety of important biological functions, such as cell growth, transcriptional regulation, and apoptosis. Emerging evidence has shown that Ago1-guided and Ago2-guided tsRNAs are expressed at 3 and 30 days in Drosophila and that tRF biogenesis in fruit flies affects tRNA processing and tRNA methylation. In the present study, tsRNAs of young (7 days) and old (42 days) Drosophila were sequenced and their expression characteristics were analysed. Then, a specific tRF (named tRF-Trp-CCA-014) was determined and was found to be conserved in fruit flies, mice, and humans. The expression patterns of tRF-Trp-CCA-014 in different tissues and stages of fruit flies and mice, and mouse NIH/3T3 cells were detected. Furthermore, mouse embryonic fibroblast NIH/3T3 cells were used as a model to analyse the function and targets of tRF-Trp-CCA-014. The results showed that the number of tsRNAs at 42 days (417) was more than at 7 days (288); thus, it was enriched with age. tRFs-1 were the most enriched, followed by 5'-tRFs and 3'-tRFs. Twenty-one differentially expressed tsRNAs were identified between 7 days and 42 days. RNA-seq data showed that most differentially expressed genes were involved in the immune system, cancer: overview, and signal translation. Furthermore, tRF-Trp-CCA-014 was found to bind to the 3'UTR of H3C4. The results suggest that the H3C4 gene is the target of tRF-Trp-CCA-014. This study will advance the current understanding of tRF roles and their implication in Drosophila and mouse studies.
Thompson, M. K., Ceccarelli, A., Ish-Horowicz, D. and Davis, I. (2023). Dynamically regulated transcription factors are encoded by highly unstable mRNAs in the Drosophila larval brain. RNA. PubMed ID: 37041032
The level of each RNA species depends on the balance between its rates of production and decay. Although previous studies have measured RNA decay across the genome in tissue culture and single-celled organisms, few experiments have been performed in intact complex tissues and organs. It is therefore unclear whether the determinants of RNA decay found in cultured cells are preserved in an intact tissue, and whether they differ between neighboring cell types and are regulated during development. To address these questions, this study measured RNA synthesis and decay rates genome wide via metabolic labeling of whole cultured Drosophila larval brains using 4-thiouridine. This analysis revealed that decay rates span a range of more than 100-fold, and that RNA stability is linked to gene function, with mRNAs encoding transcription factors being much less stable than mRNAs involved in core metabolic functions. Surprisingly, among transcription factor mRNAs there was a clear demarcation between more widely used transcription factors and those that are expressed only transiently during development. mRNAs encoding transient transcription factors are among the least stable in the brain. These mRNAs are characterized by epigenetic silencing in most cell types, as shown by their enrichment with the histone modification H3K27me3. This data suggests the presence of an mRNA destabilizing mechanism targeted to these transiently expressed transcription factors to allow their levels to be regulated rapidly with high precision. This study also demonstrates a general method for measuring mRNA transcription and decay rates in intact organs or tissues, offering insights into the role of mRNA stability in the regulation of complex developmental programs.
Eugenio, A. T., Marialva, M. S. P. and Beldade, P. (2023). Effects of Wolbachia on Transposable Element Expression Vary Between Drosophila melanogaster Host Genotypes. Genome Biol Evol 15(3). PubMed ID: 36929176
Transposable elements (TEs) are repetitive DNA sequences capable of changing position in host genomes, thereby causing mutations. TE insertions typically have deleterious effects but they can also be beneficial. Increasing evidence of the contribution of TEs to adaptive evolution further raises interest in understanding what factors impact TE activity. Based on previous studies associating the bacterial endosymbiont Wolbachia with changes in the abundance of piRNAs, a mechanism for TE repression, and to transposition of specific TEs, it was hypothesized that Wolbachia infection would interfere with TE activity. This hypothesis was tested by studying the expression of 14 TEs in a panel of 25 Drosophila melanogaster host genotypes, naturally infected with Wolbachia and annotated for TE insertions. The host genotypes differed significantly in Wolbachia titers inside individual flies, with broad-sense heritability around 20%, and in the number of TE insertions, which depended greatly on TE identity. By removing Wolbachia from the target host genotypes, a panel was generated of 25 pairs of Wolbachia-positive and Wolbachia-negative lines in which transcription levels were quantified for the target TEs. Variation was found in TE expression that was dependent on Wolbachia status, TE identity, and host genotype. Comparing between pairs of Wolbachia-positive and Wolbachia-negative flies, it was found that Wolbachia removal affected TE expression in 21.1% of the TE-genotype combinations tested, with up to 2.3 times differences in the median level of transcript. These data show that Wolbachia can impact TE activity in host genomes, underscoring the importance this endosymbiont can have in the generation of genetic novelty in hosts.
Verma, D., Hegde, V., Kirkpatrick, J. and Carlomagno, T. (2023). The EJC disassembly factor PYM is an intrinsically disordered protein and forms a fuzzy complex with RNA. Front Mol Biosci 10: 1148653. PubMed ID: 37065448
The discovery of several functional interactions where one or even both partners remain disordered has demonstrated that specific interactions do not necessarily require well-defined intermolecular interfaces. This study describes a fuzzy protein-RNA complex formed by the intrinsically unfolded protein PYM and RNA. PYM is a cytosolic protein, which has been reported to bind the exon junction complex (EJC). In the process of Oskar mRNA localization in Drosophila melanogaster, removal of the first intron and deposition of the EJC are essential, while PYM is required to recycle the EJC components after localization has been accomplished. This study demonstrates that the first 160 amino acids of PYM (PYM(1-160)) are intrinsically disordered. PYM(1-160) binds RNA independently of its nucleotide sequence, forming a fuzzy protein-RNA complex that is incompatible with PYM's function as an EJC recycling factor. It is proposed that the role of RNA binding consists in down-regulating PYM activity by blocking the EJC interaction surface of PYM until localization has been accomplished. It is suggested that the largely unstructured character of PYM may act to enable binding to a variety of diverse interaction partners, such as multiple RNA sequences and the EJC proteins Y14 and Mago.
Yamazaki, H., Namba, Y., Kuriyama, S., Nishida, K. M., Kajiya, A. and Siomi, M. C. (2023). Bombyx Vasa sequesters transposon mRNAs in nuage via phase separation requiring RNA binding and self-association. Nat Commun 14(1): 1942. PubMed ID: 37029111
Bombyx Vasa (BmVasa) assembles non-membranous organelle, nuage or Vasa bodies, in germ cells, known as the center for Siwi-dependent transposon silencing and concomitant Ago3-piRISC biogenesis. However, details of the body assembly remain unclear. This study shows that the N-terminal intrinsically disordered region (N-IDR) and RNA helicase domain of BmVasa are responsible for self-association and RNA binding, respectively, but N-IDR is also required for full RNA-binding activity. Both domains are essential for Vasa body assembly in vivo and droplet formation in vitro via phase separation. FAST-iCLIP reveals that BmVasa preferentially binds transposon mRNAs. Loss of Siwi function derepresses transposons but has marginal effects on BmVasa-RNA binding. This study shows that BmVasa assembles nuage by phase separation via its ability to self-associate and bind newly exported transposon mRNAs. This unique property of BmVasa allows transposon mRNAs to be sequestered and enriched in nuage, resulting in effective Siwi-dependent transposon repression and Ago3-piRISC biogenesis.

Monday June 12th - Chromatin

McPherson, J. E., Grossmann, L. C., Armstrong, R. L., Kwon, E., Salzler, H. R., Matera, A. G., McKay, D. J. and Duronio, R. J. (2023). Reduced histone gene copy number disrupts Drosophila Polycomb function. bioRxiv. PubMed ID: 37034607
The chromatin of animal cells contains two types of histones: canonical histones that are expressed during S phase of the cell cycle to package the newly replicated genome, and variant histones with specialized functions that are expressed throughout the cell cycle and in non-proliferating cells. Determining whether and how canonical and variant histones cooperate to regulate genome function is integral to understanding how chromatin-based processes affect normal and pathological development. This study demonstrates that variant histone H3.3 is essential for Drosophila development only when canonical histone gene copy number is reduced, suggesting that coordination between canonical H3.2 and variant H3.3 expression is necessary to provide sufficient H3 protein for normal genome function. To identify genes that depend upon, or are involved in, this coordinate regulation was screened for heterozygous chromosome 3 deficiencies that impair development of flies bearing reduced H3.2 and H3.3 gene copy number. Two regions of chromosome 3 were identified that conferred this phenotype, one of which contains the Polycomb gene, which is necessary for establishing domains of facultative chromatin that repress master regulator genes during development. It was further found that reduction in Polycomb dosage decreases viability of animals with no H3.3 gene copies. Moreover, heterozygous Polycomb mutations result in de-repression of the Polycomb target gene Ubx and cause ectopic sex combs when either canonical or variant H3 gene copy number is also reduced. It is concluded that Polycomb-mediated facultative heterochromatin function is compromised when canonical and variant H3 gene copy number falls below a critical threshold.
Park, J. I., Bell, G. W. and Yamashita, Y. M. (2023). Derepression of Y-linked multicopy protamine-like genes interferes with sperm nuclear compaction in D. melanogaster. Proc Natl Acad Sci U S A 120(16): e2220576120. PubMed ID: 37036962
Across species, sperm maturation involves the dramatic reconfiguration of chromatin into highly compact nuclei that enhance hydrodynamic ability and ensure paternal genomic integrity. This process is mediated by the replacement of histones by sperm nuclear basic proteins, also referred to as protamines. In humans, a carefully balanced dosage between two known protamine genes is required for optimal fertility. However, it remains unknown how their proper balance is regulated and how defects in balance may lead to compromised fertility. This study shows that a nucleolar protein, modulo, a homolog of nucleolin, mediates the histone-to-protamine transition during Drosophila spermatogenesis. modulo mutants display nuclear compaction defects during late spermatogenesis due to decreased expression of autosomal protamine genes (including Mst77F) and derepression of Y-linked multicopy Mst77F homologs (Mst77Y), leading to the mutant's known sterility. Overexpression of Mst77Y in a wild-type background is sufficient to cause nuclear compaction defects, similar to modulo mutant, indicating that Mst77Y is a dominant-negative variant interfering with the process of histone-to-protamine transition. Interestingly, ectopic overexpression of Mst77Y caused decompaction of X-bearing spermatids nuclei more frequently than Y-bearing spermatid nuclei, although this did not greatly affect the sex ratio of offspring. It was further shown that modulo regulates these protamine genes at the step of transcript polyadenylation. It is concluded that the regulation of protamines mediated by modulo, ensuring the expression of functional ones while repressing dominant-negative ones, is critical for male fertility.
MacPherson, R. A., Shankar, V., Anholt, R. R. H. and Mackay, T. F. C. (2023). Genetic and Genomic Analyses of Drosophila melanogaster Models of Chromatin Modification Disorders. Genetics. PubMed ID: 37036413
Switch/Sucrose Non-Fermentable (SWI/SNF)-related intellectual disability disorders (SSRIDDs) and Cornelia de Lange syndrome are rare syndromic neurodevelopmental disorders with overlapping clinical phenotypes. SSRIDDs are associated with the BAF (Brahma-Related Gene-1 Associated Factor) complex, whereas CdLS is a disorder of chromatin modification associated with the cohesin complex. This study used RNA interference in Drosophila melanogaster to reduce expression of six genes (brm, osa, Snr1, SMC1, SMC3, vtd) orthologous to human genes associated with SSRIDDs and CdLS. These fly models exhibit changes in sleep, activity, startle behavior (a proxy for sensorimotor integration) and brain morphology. Whole genome RNA sequencing identified 9,657 differentially expressed genes (FDR < 0.05), 156 of which are differentially expressed in both sexes in SSRIDD- and CdLS-specific analyses, including Bap60, which is orthologous to SMARCD1, a SSRIDD-associated BAF component. k-means clustering reveals genes co-regulated within and across SSRIDD and CdLS fly models. RNAi-mediated reduction of expression of six genes co-regulated with focal genes brm, osa, and/or Snr1 recapitulated changes in behavior of the focal genes. Based on the assumption that fundamental biological processes are evolutionarily conserved, Drosophila models can be used to understand underlying molecular effects of variants in chromatin-modification pathways and may aid in discovery of drugs that ameliorate deleterious phenotypic effects.
Puerto, M., Shukla, M., Bujosa, P., Perez-Roldan, J., Tamirisa, S., Sole, C., de Nadal, E., Posas, F., Azorin, F. and Rowley, M. J. (2023). Somatic chromosome pairing has a determinant impact on 3D chromatin organization. bioRxiv. PubMed ID: 37034722
In the nucleus, chromatin is intricately structured into multiple layers of 3D organization important for genome activity. How distinct layers influence each other is not well understood. In particular, the contribution of chromosome pairing to 3D chromatin organization has been largely neglected. This study addresses this question in Drosophila, an organism that shows robust chromosome pairing in interphasic somatic cells. The extent of chromosome pairing depends on the balance between pairing and anti-pairing factors, with the anti-pairing activity of the CAP-H2 condensin II subunit being the best documented. This study identifieds the zinc-finger protein Z4 as a strong anti-pairer that interacts with and mediates the chromatin binding of CAP-H2. It is also reported that hyperosmotic cellular stress induces fast and reversible chromosome unpairing that depends on Z4/CAP-H2. And, most important, by combining Z4 depletion and osmostress, this study shows that chromosome pairing reinforces intrachromosomal 3D interactions. On the one hand, pairing facilitates RNAPII occupancy that correlates with enhanced intragenic gene-loop interactions. In addition, acting at a distance, pairing reinforces chromatin-loop interactions mediated by Polycomb (Pc). In contrast, chromosome pairing does not affect which genomic intervals segregate to active (A) and inactive (B) compartments, with only minimal effects on the strength of A-A compartmental interactions. Altogether, these results unveil the intimate interplay between inter-chromosomal and intra-chromosomal 3D interactions, unraveling the interwoven relationship between different layers of chromatin organization and the essential contribution of chromosome pairing.
Zhao, T., Wang, M., Li, Z., Li, H., Yuan, D., Zhang, X., Guo, M., Qian, W. and Cheng, D. (2023). Wds-Mediated H3K4me3 Modification Regulates Lipid Synthesis and Transport in Drosophila. Int J Mol Sci 24(7). PubMed ID: 37047100
Lipid homeostasis is essential for insect growth and development. The complex of proteins associated with Set 1 (COMPASS)-catalyzed Histone 3 lysine 4 trimethylation (H3K4me3) epigenetically activates gene transcription and is involved in various biological processes, but the role and molecular mechanism of H3K4me3 modification in lipid homeostasis remains largely unknown. The present study showed in Drosophila that fat body-specific knockdown of will die slowly (Wds) as one of the COMPASS complex components caused a decrease in lipid droplet (LD) size and triglyceride (TG) levels. Mechanistically, Wds-mediated H3K4me3 modification in the fat body targeted several lipogenic genes involved in lipid synthesis and the Lpp gene associated with lipid transport to promote their expressions; the transcription factor heat shock factor (Hsf) could interact with Wds to modulate H3K4me3 modification within the promoters of these targets; and fat body-specific knockdown of Hsf phenocopied the effects of Wds knockdown on lipid homeostasis in the fat body. Moreover, fat body-specific knockdown of Wds or Hsf reduced high-fat diet (HFD)-induced oversized LDs and high TG levels. Altogether, this study reveals that Wds-mediated H3K4me3 modification is required for lipid homeostasis during Drosophila development and provides novel insights into the epigenetic regulation of insect lipid metabolism.
Amiad Pavlov, D., Unnikannan, C. P., Lorber, D., Bajpai, G., Olender, T., Stoops, E., Reuveny, A., Safran, S. and Volk, T. (2023). The LINC Complex Inhibits Excessive Chromatin Repression. Cells 12(6). PubMed ID: 36980273
The Linker of Nucleoskeleton and Cytoskeleton (LINC) complex transduces nuclear mechanical inputs suggested to control chromatin organization and gene expression; however, the underlying mechanism is currently unclear. This study has shown that the LINC complex is needed to minimize chromatin repression in muscle tissue, where the nuclei are exposed to significant mechanical inputs during muscle contraction. To this end, the genomic binding profiles of Polycomb, Heterochromatin Protein1 (HP1a) repressors, and of RNA-Pol II were studied in Drosophila larval muscles lacking functional LINC complex. A significant increase in the binding of Polycomb and parallel reduction of RNA-Pol-II binding to a set of muscle genes was observed. Consistently, enhanced tri-methylated H3K9 and H3K27 repressive modifications and reduced chromatin activation by H3K9 acetylation were found. Furthermore, larger tri-methylated H3K27me3 repressive clusters, and chromatin redistribution from the nuclear periphery towards nuclear center, were detected in live LINC mutant larval muscles. Computer simulation indicated that the observed dissociation of the chromatin from the nuclear envelope promotes growth of tri-methylated H3K27 repressive clusters. Thus, it is suggested that by promoting chromatin-nuclear envelope binding, the LINC complex restricts the size of repressive H3K27 tri-methylated clusters, thereby limiting the binding of Polycomb transcription repressor, directing robust transcription in muscle fibers.

Friday June 9th - Larval and Adult PhySiology and Metabolism

Onuma, T., Yamauchi, T., Kosakamoto, H., Kadoguchi, H., Kuraishi, T., Murakami, T., Mori, H., Miura, M. and Obata, F. (2023). Recognition of commensal bacterial peptidoglycans defines Drosophila gut homeostasis and lifespan. PLoS Genet 19(4): e1010709. PubMed ID: 37023169
Commensal microbes in animals have a profound impact on tissue homeostasis, stress resistance, and ageing. Previous work has shown in Drosophila melanogaster that Acetobacter persici is a member of the gut microbiota that promotes ageing and shortens fly lifespan. However, the molecular mechanism by which this specific bacterial species changes lifespan and physiology remains unclear. The difficulty in studying longevity using gnotobiotic flies is the high risk of contamination during ageing. To overcome this technical challenge, a bacteria-conditioned diet was used, enriched with bacterial products and cell wall components. This study demonstrated that an A. persici-conditioned diet shortens lifespan and increases intestinal stem cell (ISC) proliferation. Feeding adult flies a diet conditioned with A. persici, but not with Lactiplantibacillus plantarum, can decrease lifespan but increase resistance to paraquat or oral infection of Pseudomonas entomophila, indicating that the bacterium alters the trade-off between lifespan and host defence. A transcriptomic analysis using fly intestine revealed that A. persici preferably induces antimicrobial peptides (AMPs), while L. plantarum upregulates amidase peptidoglycan recognition proteins (PGRPs). The specific induction of these Imd target genes by peptidoglycans from two bacterial species is due to the stimulation of the receptor PGRP-LC in the anterior midgut for AMPs or PGRP-LE from the posterior midgut for amidase PGRPs. Heat-killed A. persici also shortens lifespan and increases ISC proliferation via PGRP-LC, but it is not sufficient to alter the stress resistance. this study emphasizes the significance of peptidoglycan specificity in determining the gut bacterial impact on healthspan. It also unveils the postbiotic effect of specific gut bacterial species, which turns flies into a "live fast, die young" lifestyle.
Wen, D. T., Gao, Y. H., Wang, J., Wang, S., Zhong, Q. and Hou, W. Q. (2023). Role of muscle FOXO gene in exercise against the skeletal muscle and cardiac age-related defects and mortality caused by high-salt intake in Drosophila. Genes Nutr 18(1): 6. PubMed ID: 36997839
FOXO has long been associated with aging, exercise, and tissue homeostasis, but it remains unclear what the role is of the muscle FOXO gene in exercise (E) against high-salt intake(HSI)-induced age-related defects of the skeletal muscle, heart, and mortality. In this research, overexpression and RNAi of the FOXO gene in the skeletal and heart muscle of Drosophila were constructed by building Mhc-GAL4/FOXO-UAS-overexpression and Mhc-GAL4/FOXO-UAS-RNAi system. The skeletal muscle and heart function, the balance of oxidation and antioxidant, and mitochondrial homeostasis were measured. The results showed that exercise reversed the age-related decline in climbing ability and downregulation of muscle FOXO expression induced by HSI. Muscle-specific FOXO-RNAi (FOXO-RNAi) and -overexpression (FOXO-OE) promoted or slowed the age-related decline in climbing ability, heart function, and skeletal muscle and heart structure damage, which was accompanied by the inhibition or activation of FOXO/PGC-1α/SDH and FOXO/SOD pathway activity, and oxidative stress (ROS) increased or decreased in both skeletal muscle and heart. The protective effect of exercise on the skeletal muscle and heart was blocked by FOXO-RNAi in aged HSI flies. FOXO-OE prolonged its lifespan, but it did not resist the HSI-induced lifespan shortening. Exercise did not improve HSI-induced lifespan shortening in FOXO-RNAi flies. Therefore, current results confirmed that the muscle FOXO gene played a vital role in exercise against age-related defects of the skeletal muscle and heart induced by HSI because it determined the activity of muscle FOXO/SOD and FOXO/PGC-1α/SDH pathways. The muscle FOXO gene also played an important role in exercise against HSI-induced mortality in aging flies.
Nikolopoulos, N., Matos, R., Ravaud, S., Courtin, P., Akherraz, H., Palussiere, S., Gueguen-Chaignon, V., Salomon-Mallet, M., Guillot, A., Guerardel, Y., Chapot-Chartier, M. P., Grangeasse, C. and Leulier, F. (2023). Structure-function analysis of Lactiplantibacillus plantarum DltE reveals D-alanylated lipoteichoic acids as direct cues supporting Drosophila juvenile growth. Elife 12. PubMed ID: 37042660
Metazoans establish mutually beneficial interactions with their resident microorganisms. However, understanding of the microbial cues contributing to host physiology remains elusive. Previously, a bacterial machinery was identified that is encoded by the dlt operon involved in Drosophila melanogaster's juvenile growth promotion by Lactiplantibacillus plantarum. Using crystallography combined with biochemical and cellular approaches, the physiological role was investigated of an uncharacterized protein (DltE) encoded by this operon. Lipoteichoic acids (LTAs) but not wall teichoic acids are D-alanylated in Lactiplantibacillus plantarum(NC8) cell envelope, and it was demonstrated that DltE is a D-Ala carboxyesterase removing D-Ala from LTA. Using the mutualistic association of L. plantarum(NC8) and Drosophila melanogaster as a symbiosis model, it was establish that D-alanylated LTAs (D-Ala-LTAs) are direct cues supporting intestinal peptidase expression and juvenile growth in Drosophila. These results pave the way to probing the contribution of D-Ala-LTAs to host physiology in other symbiotic models.
Aguilar, P., Dag, B., Carazo, P. and Sultanova, Z. (2023). Sex-specific paternal age effects on offspring quality in Drosophila melanogaster. J Evol Biol 36(4): 720-729. PubMed ID: 36946550
Advanced paternal age has been repeatedly shown to modulate offspring quality via male- and/or female-driven processes, and there are theoretical reasons to expect that some of these effects can be sex-specific. For example, sex allocation theory predicts that, when mated with low-condition males, mothers should invest more in their daughters compared to their sons. This is because male fitness is generally more condition-dependent and more variable than female fitness, which makes it less risky to invest in female offspring. This study explored whether paternal age can affect the quality and quantity of offspring in a sex-specific way using Drosophila melanogaster as a model organism. In order to understand the contribution of male-driven processes on paternal age effects, the seminal vesicle size of young and older males was measured, and its relationship with reproductive success and offspring quality was explored. Older males had lower competitive reproductive success, as expected, but there was no difference between the offspring sex ratio of young and older males. However, it was found that paternal age caused an increase in offspring quality (i.e., offspring weight), and that this increase was more marked in daughters than sons. Different male- and female-driven processes that may explain such sex-specific paternal age effects are discussed.
Pandey, A., Galeone, A., Han, S. Y., Story, B. A., Consonni, G., Mueller, W. F., Steinmetz, L. M., Vaccari, T. and Jafar-Nejad, H. (2023). Gut barrier defects, increased intestinal innate immune response, and enhanced lipid catabolism drive lethality in N -glycanase 1 deficient Drosophila. bioRxiv. PubMed ID: 37066398
Intestinal barrier dysfunction leads to inflammation and associated metabolic changes. However, the relative impact of infectious versus non-infectious mechanisms on animal health in the context of barrier dysfunction is not well understood. This study established that loss of Drosophila N -glycanase 1 (Pngl) leads to gut barrier defects, which cause starvation and increased JNK activity. These defects result in Foxo overactivation, which induces a hyperactive innate immune response and lipid catabolism, thereby contributing to lethality associated with loss of Pngl. Notably, germ-free rearing of Pngl mutants did not rescue lethality. In contrast, raising Pngl mutants on isocaloric, fat-rich diets improved animal survival in a dosage-dependent manner. These data indicate that Pngl functions in Drosophila larvae to establish the gut barrier, and that the immune and metabolic consequences of loss of Pngl are primarily mediated through non-infectious mechanisms.
Zhang, Q., Zheng, H., Yang, S., Feng, T., Jie, M., Chen, H. and Jiang, H. (2023). Bub1 and Bub3 regulate metabolic adaptation via macrolipophagy in Drosophila. Cell Rep 42(4): 112343. PubMed ID: 37027296
Lipophagy, the process of selective catabolism of lipid droplets (LDs) by autophagy, maintains lipid homeostasis and provides cellular energy under metabolic adaptation, yet its underlying mechanism remains largely ambiguous. This study shows that the Bub1-Bub3 complex, the crucial regulator involved in the whole process of chromosome alignment and separation during mitosis, controls the fasting-induced lipid catabolism in the fat body (FB) of Drosophila. Bidirectional deviations of the Bub1 or Bub3 level affect the consumption of triacylglycerol (TAG) of fat bodies and the survival rate of adult flies under starving. Moreover, Bub1 and Bub3 work together to attenuate lipid degradation via macrolipophagy upon fasting. Thus, this study uncovered physiological roles of the Bub1-Bub3 complex on metabolic adaptation and lipid metabolism beyond their canonical mitotic functions, providing insights into the in vivo functions and molecular mechanisms of macrolipophagy during nutrient deprivation.

Thursday June 8th - Disease Models

Zhu, Y., Lobato, A. G., Rebelo, A. P., Canic, T., Ortiz Vega, N., Tao, X., Syed, S., Yanick, C., Saporta, M., Shy, M., Perfetti, R., Shendelman, S., Zuchner, S. L. and Zhai, R. G. (2023). Sorbitol reduction via AT-007 (govorestat) ameliorates synaptic dysfunction and neurodegeneration in models of sorbitol dehydrogenase deficiency. JCI Insight. PubMed ID: 37014713
Sorbitol dehydrogenase (SORD) deficiency has been identified as the most frequent autosomal recessive form of hereditary neuropathy. Loss of SORD causes high sorbitol levels in tissues due to the inability to convert sorbitol to fructose in the two-step polyol pathway, leading to degenerative neuropathy. The underlying mechanisms of sorbitol-induced degeneration have not been fully elucidated, and no current FDA-approved therapeutic options are available to reduce sorbitol levels in the nervous system. In a Drosophila model of SORD deficiency, synaptic degeneration in the brain, neurotransmission defect, locomotor impairment, and structural abnormalities in the neuromuscular junctions were shown. In addition, it was found reduced ATP production in the brain and reactive oxygen species accumulation in the central nervous system (CNS) and muscle, indicating mitochondrial dysfunction. Applied Therapeutics, Inc has developed a CNS-penetrant next-generation aldose reductase inhibitor (ARI), AT-007 (govorestat), which inhibits the conversion of glucose to sorbitol. AT-007 significantly reduced sorbitol levels in patient-derived fibroblasts, iPSC-derived motor neurons, and Drosophila brains. AT-007 feeding in Sord-deficient Drosophila mitigated synaptic degeneration and significantly improved synaptic transduction, locomotor activity, and mitochondrial function. Moreover, AT-007 treatment significantly reduced ROS accumulation in Drosophila CNS, muscle, and patient-derived fibroblasts. These findings uncover the molecular and cellular pathophysiology of SORD neuropathy and provide a potential treatment strategy for patients with SORD deficiency.
Tepe, B., Macke, E. L., Niceta, M., Weisz Hubshman, M., Kanca, O., Schultz-Rogers, L., Zarate, Y. A., Schaefer, G. B., Granadillo De Luque, J. L., Wegner, D. J., Cogne, B., Gilbert-Dussardier, B., Le Guillou, X., Wagner, E. J., Pais, L. S., Neil, J. E., Mochida, G. H., Walsh, C. A., Magal, N., Drasinover, V., Shohat, M., Schwab, T., Schmitz, C., Clark, K., Fine, A., Lanpher, B., Gavrilova, R., Blanc, P., Burglen, L., Afenjar, A., Steel, D., Kurian, M. A., Prabhakar, P., Goswein, S., Di Donato, N., Bertini, E. S., Wangler, M. F., Yamamoto, S., Tartaglia, M., Klee, E. W. and Bellen, H. J. (2023). Bi-allelic variants in INTS11 are associated with a complex neurological disorder. Am J Hum Genet. PubMed ID: 37054711
The Integrator complex is a multi-subunit protein complex that regulates the processing of nascent RNAs transcribed by RNA polymerase II (RNAPII), including small nuclear RNAs, enhancer RNAs, telomeric RNAs, viral RNAs, and protein-coding mRNAs. Integrator subunit 11 (INTS11) is the catalytic subunit that cleaves nascent RNAs, but, to date, mutations in this subunit have not been linked to human disease. This study describes 15 individuals from 10 unrelated families with bi-allelic variants in INTS11 who present with global developmental and language delay, intellectual disability, impaired motor development, and brain atrophy. Consistent with human observations, this study has found that the fly ortholog of INTS11, dIntS11, is essential and expressed in the central nervous systems in a subset of neurons and most glia in larval and adult stages. Using Drosophila as a model, the effect of seven variants was investigated. Two (p.Arg17Leu and p.His414Tyr) fail to rescue the lethality of null mutants, indicating that they are strong loss-of-function variants. Furthermore, it was found that five variants (p.Gly55Ser, p.Leu138Phe, p.Lys396Glu, p.Val517Met, and p.Ile553Glu) rescue lethality but cause a shortened lifespan and bang sensitivity and affect locomotor activity, indicating that they are partial loss-of-function variants. Altogether, these results provide compelling evidence that integrity of the Integrator RNA endonuclease is critical for brain development.
Wang, X., Li, J., Zhang, W., Wang, F., Wu, Y., Guo, Y., Wang, D., Yu, X., Li, A., Li, F. and Xie, Y. (2023). IGFBP-3 promotes cachexia-associated lipid loss by suppressing insulin-like growth factor/insulin signaling. Chin Med J (Engl). PubMed ID: 37014770
Progressive lipid loss of adipose tissue is a major feature of cancer-associated cachexia. In addition to systemic immune/inflammatory effects in response to tumor progression, tumor-secreted cachectic ligands also play essential roles in tumor-induced lipid loss. However, the mechanisms of tumor-adipose tissue interaction in lipid homeostasis are not fully understood. The yki-gut tumors were induced in fruit flies. Lipid metabolic assays were performed to investigate the lipolysis level of different types of insulin-like growth factor binding protein-3 (IGFBP-3) treated cells. Immunoblotting was used to display phenotypes of tumor cells and adipocytes. Quantitative polymerase chain reaction (qPCR) analysis was carried out to examine the gene expression levels such as Acc1, Acly, and Fasn. This study revealed that tumor-derived IGFBP-3 was an important ligand directly causing lipid loss in matured adipocytes. IGFBP-3, which is highly expressed in cachectic tumor cells, antagonized insulin/IGF-like signaling (IIS) and impaired the balance between lipolysis and lipogenesis in 3T3-L1 adipocytes. Conditioned medium from cachectic tumor cells, such as Capan-1 and C26 cells, contained excessive IGFBP-3 that potently induced lipolysis in adipocyted. Notably, neutralization of IGFBP-3 by neutralizing antibody in the conditioned medium of cachectic tumor cells significantly alleviated the lipolytic effect and restored lipid storage in adipocytes. Furthermore, cachectic tumor cells were resistant to IGFBP-3 inhibition of IIS, ensuring their escape from IGFBP-3-associated growth suppression. Finally, cachectic tumor-derived ImpL2, the IGFBP-3 homolog, also impaired lipid homeostasis of host cells in an established cancer-cachexia model in Drosophila. Most importantly, IGFBP-3 was highly expressed in cancer tissues in pancreatic and colorectal cancer patients, especially higher in the sera of cachectic cancer patients than non-cachexia cancer patients. This study demonstrates that tumor-derived IGFBP-3 plays a critical role in cachexia-associated lipid loss and could be a biomarker for diagnosis of cachexia in cancer patients.
Zhao, W., Zhang, Y., Lin, S., Li, Y., Zhu, A. J., Shi, H. and Liu, M. (2023). Identification of Ubr1 as an amino acid sensor of steatosis in liver and muscle. J Cachexia Sarcopenia Muscle. PubMed ID: 37057345
Malnutrition is implicated in human metabolic disorders, including hepatic steatosis and myosteatosis. The corresponding nutrient signals and sensors as well as signalling pathways have not yet been well studied. This study aimed to unravel the nutrient-sensing mechanisms in the pathogenesis of steatosis. Perilipin 2 (Plin2), a lipid droplet (LD) protein-inhibiting lipolysis, is associated with steatosis in liver and muscle. Taking advantage of the Gal4-UAS system, this study used the Drosophila melanogaster wing imaginal disc as an in vivo model to study the regulation of Plin2 proteostasis and LD homeostasis. Drosophila Schneider 2 (S2) cells were used for western blotting, immunoprecipitation assays, amino acid-binding assays and ubiquitination assays to further investigate the regulatory mechanisms of Plin2 in response to nutrient signals. Mouse AML12 hepatocytes, human JHH-7 and SNU-475 hepatoma cells were used for immunofluorescence, western blotting and immunoprecipitation to demonstrate that the mode of Plin2 regulation is evolutionarily conserved. In addition, proteins were purified from HEK293 cells, and in vitro cell-free systems in amino acid-binding assays, pulldown assays and ubiquitination assays were reconstituted to directly demonstrate the molecular mechanism by which Ubr1 senses amino acids to regulate Plin2 proteostasis. As a lipolysis inhibitor, Plin2 was significantly elevated in liver and muscle in patients with steatosis. Consistently, it was found that the ubiquitin moiety can be conjugated to any Lys residue in Plin2, ensuring robust clearance of Plin2 by protein degradation. It was further demonstrated that the E3 ubiquitin ligase Ubr1 targets Plin2 for degradation in an amino acid-dependent manner. Ubr1 uses two canonical substrate-binding pockets, independent of each other, to bind basic and bulky hydrophobic amino acids, respectively. Mechanistically, amino acid binding allosterically activates Ubr1 by alleviating Ubr1's auto-inhibition. In the absence of amino acids, or when the amino acid-binding capacity of Ubr1 is diminished, Ubr1-mediated Plin2 degradation is inactivated, leading to steatosis. This study has identified Ubr1 as an amino acid sensor regulating Plin2 proteostasis, bridging the knowledge gap between steatosis and nutrient sensing. This work may provide new strategies for the prevention and treatment of steatosis.
Zhao, Y., Xuan, H., Shen, C., Liu, P., Han, J. J. and Yu, W. (2022). Immunosuppression Induced by Brain-Specific HDAC6 Knockdown Improves Aging Performance in Drosophila melanogaster. Phenomics 2(3): 194-200. PubMed ID: 36939772
HDAC6 is involved in several biological processes related to aging-associated diseases. However, it was unknown whether HDAC6 could directly regulate lifespan and healthspan. This study found that HDAC6 knockdown induced transcriptome changes to attenuate the aging changes in the Drosophila head, particularly on the inflammation and innate immunity-related genes. Whole-body knockdown of HDAC6 extended lifespan in the fly, furthermore brain-specific knockdown of HDAC6 extended both lifespan and healthspan in the fly. These results established HDAC6 as a lifespan regulator and provided a potential anti-aging target.
Xiong, Y., Cheng, Q., Li, Y., Han, Y., Sun, X. and Liu, L. (2023). Vimar/RAP1GDS1 promotes acceleration of brain aging after flies and mice reach middle age. Commun Biol 6(1): 420. PubMed ID: 37061660
Brain aging may accelerate after rodents reach middle age. However, the endogenous mediator that promotes this acceleration is unknown. It is predicted that the mediator may be expressed after an organism reaches middle age and dysregulates mitochondrial function. In the neurons of wild-type Drosophila (flies), it was observed that mitochondria were fragmented in aged flies, and this fragmentation was associated with mitochondrial calcium overload. In a previous study, it was found that mitochondrial fragmentation induced by calcium overload was reversed by the loss of Vimar, which forms a complex with Miro. Interestingly, Vimar expression was increased after the flies reached middle age. Overexpression of Vimar in neurons resulted in premature aging and mitochondrial calcium overload. In contrast, downregulation of Vimar in flies older than middle age promoted healthy aging. As the mouse homolog of Vimar, RAP1GDS1 expression was found to be increased after mice reached middle age; RAP1GDS1-transgenic and RAP1GDS1-knockdown mice displayed similar responses to flies with overexpressed and reduced Vimar expression, respectively. This research provides genetic evidence of a conserved endogenous mediator that promotes accelerated brain aging.

Wednesday June 7th - Adult Neural Development and Function

Wang, F., Ruppell, K. T., Zhou, S., Qu, Y., Gong, J., Shang, Y., Wu, J., Liu, X., Diao, W., Li, Y. and Xiang, Y. (2023). Gliotransmission and adenosine signaling promote axon regeneration. Dev Cell. PubMed ID: 37028426
How glia control axon regeneration remains incompletely understood. This study investigated glial regulation of regenerative ability differences of closely related Drosophila larval sensory neuron subtypes. Axotomy elicits Ca(2+) signals in ensheathing glia, which activates regenerative neurons through the gliotransmitter adenosine and mounts axon regenerative programs. However, non-regenerative neurons do not respond to glial stimulation or adenosine. Such neuronal subtype-specific responses result from specific expressions of adenosine receptors in regenerative neurons. Disrupting gliotransmission impedes axon regeneration of regenerative neurons, and ectopic adenosine receptor expression in non-regenerative neurons suffices to activate regenerative programs and induce axon regeneration. Furthermore, stimulating gliotransmission or activating the mammalian ortholog of Drosophila adenosine receptors in retinal ganglion cells (RGCs) promotes axon regrowth after optic nerve crush in adult mice. Altogether, these findings demonstrate that gliotransmission orchestrates neuronal subtype-specific axon regeneration in Drosophila and suggest that targeting gliotransmission or adenosine signaling is a strategy for mammalian central nervous system repair.
Lazar, A. A., Liu, T. and Yeh, C. H. (2023). The functional logic of odor information processing in the Drosophila antennal lobe. PLoS Comput Biol 19(4): e1011043. PubMed ID: 37083547
Recent advances in molecular transduction of odorants in the Olfactory Sensory Neurons (OSNs) of the Drosophila Antenna have shown that the odorant object identity is multiplicatively coupled with the odorant concentration waveform. The resulting combinatorial neural code is a confounding representation of odorant semantic information (identity) and syntactic information (concentration). By analyzing single-channel physiology recordings at the output of the Antennal Lobe (AL) (AL), this study found that the Projection Neuron responses can be decomposed into a concentration-invariant component, and two transient components boosting the positive/negative concentration contrast that indicate onset/offset timing information of the odorant object. It is hypothesized that the concentration-invariant component, in the multi-channel context, is the recovered odorant identity vector presented between onset/offset timing events. A model was developed of LN pathways in the Antennal Lobe termed the differential Divisive Normalization Processors (DNPs) that robustly extracts the semantics (the identity of the odorant object) and the ON/OFF semantic timing events indicating the presence/absence of an odorant object. For real-time processing with spiking PN models, this study showed that the phase-space of the biological spike generator of the PN offers an intuit perspective for the representation of recovered odorant semantics, and the dynamics induced by the odorant semantic timing events were examined. Finally, theoretical and computational evidence is provided for the functional logic of the AL as a robust ON-OFF odorant object identity recovery processor across odorant identities, concentration amplitudes and waveform profiles.
Vaikakkara Chithran, A., Allan, D. W. and O'Connor, T. P. (2023). Adult expression of Semaphorins and Plexins is essential for motor neuron survival. Sci Rep 13(1): 5894. PubMed ID: 37041188
Axon guidance cues direct the growth and steering of neuronal growth cones, thus guiding the axons to their targets during development. Nonetheless, after axons have reached their targets and established functional circuits, many mature neurons continue to express these developmental cues. The role of axon guidance cues in the adult nervous system has not been fully elucidated. Using the expression pattern data available on FlyBase, this study found that more than 96% of the guidance genes that are expressed in the Drosophila melanogaster embryo continue to be expressed in adults. The GeneSwitch and TARGET systems were used to spatiotemporally knockdown the expression of these guidance genes selectively in the adult neurons, once the development was completed. An RNA interference (RNAi) screen was performed against 44 guidance genes in the adult Drosophila nervous system, and 14 genes were identified that are required for adult survival and normal motility. Additionally, it was shown that adult expression of Semaphorins and Plexins in motor neurons is necessary for neuronal survival, indicating that guidance genes have critical functions in the mature nervous system.
Serna-Morales, E., Sanchez-Sanchez, B. J., Marcotti, S., Nichols, A., Bhargava, A., Dragu, A., Hirvonen, L. M., Diaz-de-la-Loza, M. D., Mink, M., Cox, S., Rayfield, E., Lee, R. M., Hobson, C. M., Chew, T. L. and Stramer, B. M. (2023). Extracellular matrix assembly stress initiates Drosophila central nervous system morphogenesis. Dev Cell. PubMed ID: 37086718
Forces controlling tissue morphogenesis are attributed to cellular-driven activities, and any role for extracellular matrix (ECM) is assumed to be passive. However, all polymer networks, including ECM, can develop autonomous stresses during their assembly. This study examine the morphogenetic function of an ECM before reaching homeostatic equilibrium by analyzing de novo ECM assembly during Drosophila ventral nerve cord (VNC) condensation. Asymmetric VNC shortening and a rapid decrease in surface area correlate with the exponential assembly of collagen IV (Col4) surrounding the tissue. Concomitantly, a transient developmentally induced Col4 gradient leads to coherent long-range flow of ECM, which equilibrates the Col4 network. Finite element analysis and perturbation of Col4 network formation through the generation of dominant Col4 mutations that affect assembly reveal that VNC morphodynamics is partially driven by a sudden increase in ECM-driven surface tension. These data suggest that ECM assembly stress and associated network instabilities can actively participate in tissue morphogenesis.
Sakamura, S., Hsu, F. Y., Tsujita, A., Abubaker, M. B., Chiang, A. S. and Matsuno, K. (2023). Ecdysone signaling determines lateral polarity and remodels neurites to form Drosophila's left-right brain asymmetry. Cell Rep: 112337. PubMed ID: 37044096
Left-right (LR) asymmetry of the brain is fundamental to its higher-order functions. The Drosophila brain's asymmetrical body (AB) consists of a structural pair arborized from AB neurons and is larger on the right side than the left. This study found that the AB initially forms LR symmetrically and then develops LR asymmetrically by neurite remodeling that is specific to the left AB and is dynamin dependent. Additionally, neuronal ecdysone signaling inhibition randomizes AB laterality, suggesting that ecdysone signaling determines AB's LR polarity. Given that AB's LR asymmetry relates to memory formation, this research establishes AB as a valuable model for studying LR asymmetry and higher-order brain function relationships.
Veen, K., Nguyen, P. K., Froldi, F., Dong, Q., Alvarez-Ochoa, E., Harvey, K. F., McMullen, J. P., Marshall, O., Jusuf, P. R. and Cheng, L. Y. (2023). Dedifferentiation-derived neural stem cells exhibit perturbed temporal progression. EMBO Rep: e55837. PubMed ID: 37039033
Dedifferentiation is the reversion of mature cells to a stem cell-like fate, whereby gene expression programs are altered and genes associated with multipotency are (re)expressed. Misexpression of multipotency factors and pathways causes the formation of ectopic neural stem cells (NSCs). Whether dedifferentiated NSCs faithfully produce the correct number and types of progeny, or undergo timely terminal differentiation, has not been assessed. This study shows that ectopic NSCs induced via bHLH transcription factor Deadpan (Dpn) expression fail to undergo appropriate temporal progression by constantly expressing mid-temporal transcription factor(tTF), Sloppy-paired 1/2 (Slp). Consequently, this resulted in impaired terminal differenation and generated an excess of Twin of eyeless (Toy)-positive neurons at the expense of Reversed polarity (Repo)-positive glial cells. Preference for a mid-temporal fate in these ectopic NSCs is concordant with an enriched binding of Dpn at mid-tTF loci and a depletion of Dpn binding at early- and late-tTF loci. Retriggering the temporal series via manipulation of the temporal series or cell cycle is sufficient to reinstate neuronal diversity and timely termination.

Tuesday, June 6th - Disease models

Sousa, E. S. R., Sousa, A. D., Vieira, J. and Vieira, C. P. (2023). The Josephin domain (JD) containing proteins are predicted to bind to the same interactors: Implications for spinocerebellar ataxia type 3 (SCA3) studies using Drosophila melanogaster mutants. Front Mol Neurosci 16: 1140719. PubMed ID: 37008788
Spinocerebellar ataxia type 3, also known as Machado-Joseph disease (SCA3/ MJD), is the most frequent polyglutamine (polyQ) neurodegenerative disorder. It is caused by a pathogenic expansion of the polyQ tract, located at the C-terminal region of the protein encoded by the ATXN3 gene. This gene codes for a deubiquitinating enzyme (DUB) that belongs to a gene family, that in humans is composed by three more genes (ATXN3L, JOSD1, and JOSD2), that define two gene lineages (the ATXN3 and the Josephins). These proteins have in common the N-terminal catalytic domain (Josephin domain, JD), that in Josephins is the only domain present. In ATXN3 knock-out mouse and nematode models, the SCA3 neurodegeneration phenotype is not, however, reproduced, suggesting that in the genome of these species there are other genes that are able to compensate for the lack of ATXN3. Moreover, in mutant Drosophila melanogaster, where the only JD protein is coded by a Josephin-like gene, expression of the expanded human ATXN3 gene reproduces multiple aspects of the SCA3 phenotype, in contrast with the results of the expression of the wild type human form. In order to explain these findings, phylogenetic, as well as, protein-protein docking inferences are here performed. This study showed multiple losses of JD containing genes across the animal kingdom, suggesting partial functional redundancy of these genes. Accordingly, it is predicted that the JD is essential for binding with ataxin-3 and proteins of the Josephin lineages, and that D. melanogaster mutants are a good model of SCA3 despite the absence of a gene from the ATXN3 lineage. The molecular recognition regions of the ataxin-3 binding and those predicted for the Josephins are, however, different. Different binding regions between the two ataxin-3 forms (wild-type (wt) and expanded (exp)) are reported. The interactors that show an increase in the interaction strength with exp ataxin-3, are enriched in extrinsic components of mitochondrial outer membrane and endoplasmatic reticulum membrane. On the other hand, the group of interactors that show a decrease in the interaction strength with exp ataxin-3 is significantly enriched in extrinsic component of cytoplasm.
Stubbs, K., Batchelor, B., Sivanantharajah, L., Sealey, M., Ramirez-Moreno, M., Ruiz, E., Richardson, B., Perry, V. H., Newman, T. A. and Mudher, A. (2023). Tau-mediated axonal degeneration is prevented by activation of the Wld(S) pathway. Brain Commun 5(2): fcad052. PubMed ID: 37013175
Tauopathy is characterized by neuronal dysfunction and degeneration occurring as a result of changes to the microtubule-associated protein tau (see Drosophila Tau). The neuronal changes evident in tauopathy bear striking morphological resemblance to those reported in models of Wallerian degeneration (see Drosophila Walenda). The mechanisms underpinning Wallerian degeneration are not fully understood although it can be delayed by the expression of the slow Wallerian degeneration (Wld(S)) protein, which has also been demonstrated to delay axonal degeneration in some models of neurodegenerative disease. Given the morphological similarities between tauopathy and Wallerian degeneration, this study investigated whether tau-mediated phenotypes can be modulated by co-expression of Wld(S). In a Drosophila model of tauopathy in which expression of human 0N3R tau protein leads to progressive age-dependent phenotypes, Wld(S) was expressed with and without activation of the downstream pathway. The olfactory receptor neuron circuit OR47b was used for these studies in adults, and the larval motor neuron system was employed in larvae. Tau phenotypes studied included neurodegeneration, axonal transport, synaptic deficits and locomotor behaviour. Impact on total tau was ascertained by assessing total, phosphorylated and misfolded tau levels by immunohistochemistry. Activation of the pathway downstream of Wld(S) completely suppressed tau-mediated degeneration. This protective effect was evident even if the pathway downstream of Wld(S) was activated several weeks after tau-mediated degeneration had become established. Though total tau levels were not altered, the protected neurons displayed significantly reduced MC1 immunoreactivity suggestive of clearance of misfolded tau, as well as a trend for a decline in tau species phosphorylated at the AT8 and PHF1 epitopes. In contrast, Wld(S) expression without activation of the downstream protective pathway did not rescue tau-mediated degeneration in adults or improve tau-mediated neuronal dysfunction including deficits in axonal transport, synaptic alterations and locomotor behaviour in tau-expressing larvae. This collectively implies that the pathway mediating the protective effect of Wld(S) intersects with the mechanism(s) of degeneration initiated by tau and can effectively halt tau-mediated degeneration at both early and late stages. Understanding the mechanisms underpinning this protection could identify much-needed disease-modifying targets for tauopathies.
Nait-Saidi, R., Chartier, A., Abgueguen, E., Guedat, P. and Simonelig, M. (2023). The small compound Icerguastat reduces muscle defects in oculopharyngeal muscular dystrophy through the PERK pathway of the unfolded protein response. Open Biol 13(4): 230008. PubMed ID: 37042114
Oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant disease characterized by the progressive degeneration of specific muscles. OPMD is due to a mutation in the gene encoding poly(A) binding protein nuclear 1 (PABPN1) leading to a stretch of 11 to 18 alanines at N-terminus of the protein, instead of 10 alanines in the normal protein. This alanine tract extension induces the misfolding and aggregation of PABPN1 in muscle nuclei. In this study, using Drosophila OPMD models, it was shown that the unfolded protein response (UPR) is activated in OPMD upon endoplasmic reticulum stress. Mutations in components of the PERK branch of the UPR reduce muscle degeneration and PABPN1 aggregation characteristic of the disease. This study shows that oral treatment of OPMD flies with Icerguastat (previously IFB-088), a Guanabenz acetate derivative that shows lower side effects, also decreases muscle degeneration and PABPN1 aggregation. Furthermore, the positive effect of Icerguastat depends on GADD34, a key component of the phosphatase complex in the PERK branch of the UPR. This study reveals a major contribution of the ER stress in OPMD pathogenesis and provides a proof-of-concept for Icerguastat interest in future pharmacological treatments of OPMD.
Stewart, T. R. M., Foley, J. R., Holbert, C. E., Khomutov, M. A., Rastkari, N., Tao, X., Khomutov, A. R., Zhai, R. G. and Casero, R. A. (2023). Difluoromethylornithine rebalances aberrant polyamine ratios in Snyder-Robinson syndrome: mechanism of action and therapeutic potential. bioRxiv. PubMed ID: 37034775
Snyder-Robinson Syndrome (SRS) is caused by mutations in the spermine synthase (SMS) gene, the enzyme product of which converts the polyamine spermidine into spermine. Affecting primarily males, common manifestations of SRS include intellectual disability, osteoporosis, hypotonic musculature, and seizures, along with other more variable symptoms. Currently, medical management focuses on treating these symptoms without addressing the underlying molecular cause of the disease. Reduced SMS catalytic activity in cells of SRS patients causes the accumulation of spermidine, while spermine levels are reduced. The resulting exaggeration in spermidine-to-spermine ratio is a biochemical hallmark of SRS that tends to correlate with symptom severity in the patient. These studies aim to pharmacologically manipulate polyamine metabolism to correct this polyamine imbalance and investigate the potential of this approach as a therapeutic strategy for affected individuals. This study reports the use of difluoromethylornithine (DFMO; eflornithine), an FDA-approved inhibitor of polyamine biosynthesis, in re-establishing normal spermidine-to-spermine ratios in SRS patient cells. Through mechanistic studies, it was demonstrated that, while reducing spermidine biosynthesis, DFMO also stimulates the conversion of existing spermidine into spermine in cell lines with hypomorphic variants of SMS. Further, DFMO treatment induces a compensatory uptake of exogenous polyamines, including spermine and spermine mimetics, cooperatively reducing spermidine and increasing spermine levels. In a Drosophila SRS model characterized by reduced lifespan, adding DFMO to the feed extended lifespan. As nearly all known SRS patient mutations are hypomorphic, these studies form a foundation for future translational studies with significant therapeutic potential.
Tafesh-Edwards, G., Kalukin, A., Bunnell, D., Chtarbanova, S. and Eleftherianos, I. (2023). Temperature and sex shape Zika virus pathogenicity in the adult Brat (cheesehead) brain: A Drosophila model for virus-associated neurological diseases. iScience 26(4): 106424. PubMed ID: 37009222
Severe neurological complications affecting brain growth and function have been well documented in newborn and adult patients infected by Zika virus (ZIKV), but the underlying mechanisms remain unknown. This study used a Drosophila melanogaster mutant, cheesehead (chs), with a mutation in the brain tumor (brat) locus that exhibits both aberrant continued proliferation and progressive neurodegeneration in the adult brain. This study reports that temperature variability is a key driver of ZIKV pathogenesis, thereby altering host mortality and causing motor dysfunction in a sex-dependent manner. Furthermore, it was shown that ZIKV is largely localized to the brat (chs) brain and activates the RNAi and apoptotic immune responses. These findings establish an in vivo model to study host innate immune responses and highlight the need of evaluating neurodegenerative deficits as a potential comorbidity in ZIKV-infected adults.
Rosado-Ramos, R., Pocas, G. M., Marques, D., Foito, A., D, M. S., Lopes-da-Silva, M., Gonçalves, L. G., Menezes, R., Ottens, M., Stewart, D., Ibanez de Opakua, A., Zweckstetter, M., Seabra, M. C., Mendes, C. S., Outeiro, T. F., Domingos, P. M. and Santos, C. N. (2023). Genipin prevents alpha-synuclein aggregation and toxicity by affecting endocytosis, metabolism and lipid storage. Nat Commun 14(1): 1918. PubMed ID: 37024503
Parkinson's Disease (PD) is a common neurodegenerative disorder affecting millions of people worldwide for which there are only symptomatic therapies. Small molecules able to target key pathological processes in PD have emerged as interesting options for modifying disease progression. It has been previously shown that a (poly)phenol-enriched fraction (PEF) of Corema album L. leaf extract modulates central events in PD pathogenesis, namely α-synuclein (αSyn) toxicity, aggregation and clearance. PEF was now subjected to a bio-guided fractionation with the aim of identifying the critical bioactive compound. Genipin, an iridoid, which relieves αSyn toxicity and aggregation was identified. Furthermore, genipin promotes metabolic alterations and modulates lipid storage and endocytosis. Importantly, genipin was able to prevent the motor deficits caused by the overexpression of αSyn in a Drosophila melanogaster model of PD. These findings widens the possibility for the exploitation of genipin for PD therapeutics.

Monday June 5th - Transposons and RNA

Popovic, R., Yu, Y., Leal, N. S., Fedele, G., Loh, S. H. Y. and Martins, L. M. (2023). Upregulation of Tribbles decreases body weight and increases sleep duration. Dis Model Mech 16(4). PubMed ID: 37083954
Eukaryotic Tribbles proteins are pseudoenzymes that regulate multiple aspects of intracellular signalling. Both Drosophila melanogaster and mammalian members of this family of pseudokinases act as negative regulators of insulin signalling. Mammalian tribbles pseudokinase (TRIB) genes have also been linked to insulin resistance and type 2 diabetes mellitus. Type 2 diabetes mellitus is associated with increased body weight, sleep problems and increased long-term mortality. This study investigated how manipulating the expression of Tribbles impacts body weight, sleep and mortality. Overexpression of Drosophila tribbles (trbl) in the fly fat body reduces both body weight and lifespan in adult flies without affecting food intake. Furthermore, it decreases the levels of Drosophila insulin-like peptide 2 (DILP2; ILP2) and increases night-time sleep. The three genes encoding TRIBs of mammals, TRIB1, TRIB2 and TRIB3, show both common and unique features. As the three human TRIB genes share features with Drosophila trbl, this study further explored the links between TRIB genetic variants and both body weight and sleep in the human population. Associations were identified between the polymorphisms and expression levels of the pseudokinases and markers of body weight and sleep duration. It is concluded that Tribbles pseudokinases are involved in the control of body weight, lifespan and sleep.
Sood, C., Nahid, M. A., Branham, K. R., Pahl, M. C., Doyle, S. E. and Siegrist, S. E. (2023). Delta-dependent Notch activation closes the early neuroblast temporal program to promote lineage progression and neurogenesis termination in Drosophila. bioRxiv. PubMed ID: 37034719
Neuroblasts in Drosophila divide asymmetrically, sequentially expressing a series of intrinsic factors to generate a diversity of neuron types. These intrinsic factors known as temporal factors dictate timing of neuroblast transitions in response to steroid hormone signaling and specify early versus late temporal fates in neuroblast neuron progeny. After completing their temporal programs, neuroblasts differentiate or die, finalizing both neuron number and type within each neuroblast lineage. From a screen aimed at identifying genes required to terminate neuroblast divisions, this study identified Notch and Notch pathway components. When Notch is knocked down, neuroblasts maintain early temporal factor expression longer, delay late temporal factor expression, and continue dividing into adulthood. This study found that Delta, expressed in cortex glia, neuroblasts, and after division, their GMC progeny, regulates neuroblast Notch activity. Delta in neuroblasts is expressed high early, low late, and is controlled by the intrinsic temporal program: early factor Imp promotes Delta, late factors Syp/E93 reduce Delta. Thus, in addition to systemic steroid hormone cues, forward lineage progression is controlled by local cell-cell signaling between neuroblasts and their cortex glia/GMC neighbors: Delta transactivates Notch in neuroblasts bringing the early temporal program and early temporal factor expression to a close.
Song, T., Qin, W., Lai, Z., Li, H., Li, D., Wang, B., Deng, W., Wang, T., Wang, L. and Huang, R. (2023). Dietary cysteine drives body fat loss via FMRFamide signaling in Drosophila and mouse. Cell Res. PubMed ID: 37055592
Obesity imposes a global health threat and calls for safe and effective therapeutic options. This study found that protein-rich diet significantly reduced body fat storage in fruit flies, which was largely attributed to dietary cysteine intake. Mechanistically, dietary cysteine increased the production of a neuropeptide FMRFamide (FMRFa). Enhanced FMRFa activity simultaneously promoted energy expenditure and suppressed food intake through its cognate receptor (FMRFaR), both contributing to the fat loss effect. In the fat body, FMRFa signaling promoted lipolysis by increasing PKA and lipase activity. In sweet-sensing gustatory neurons, FMRFa signaling suppressed appetitive perception and hence food intake. This study also demonstrated that dietary cysteine worked in a similar way in mice via neuropeptide FF (NPFF) signaling, a mammalian RFamide peptide. In addition, dietary cysteine or FMRFa/NPFF administration provided protective effect against metabolic stress in flies and mice without xal abnormalities. Therefore, this study reveals a novel target for the development of safe and effective therapies against obesity and related metabolic diseases.
Fulford, A. D., Enderle, L., Rusch, J., Hodzic, D., Holder, M. V., Earl, A., Oh, R. H., Tapon, N. and McNeill, H. (2023). Expanded directly binds conserved regions of Fat to restrain growth via the Hippo pathway. J Cell Biol 222(5). PubMed ID: 37071483
The Hippo pathway is a conserved and critical regulator of tissue growth. The FERM protein Expanded is a key signaling hub that promotes activation of the Hippo pathway, thereby inhibiting the transcriptional co-activator Yorkie. Previous work identified the polarity determinant Crumbs as a primary regulator of Expanded. This study showed that the giant cadherin Fat also regulates Expanded directly and independently of Crumbs. Direct binding between Expanded and a highly conserved region of the Fat cytoplasmic domain recruits Expanded to the apicolateral junctional zone and stabilizes Expanded. In vivo deletion of Expanded binding regions in Fat causes loss of apical Expanded and promotes tissue overgrowth. Unexpectedly, this study found Fat can bind its ligand Dachsous via interactions of their cytoplasmic domains, in addition to the known extracellular interactions. Importantly, Expanded is stabilized by Fat independently of Dachsous binding. These data provide new mechanistic insights into how Fat regulates Expanded, and how Hippo signaling is regulated during organ growth.
Mo, D., Liu, C., Chen, Y., Cheng, X., Shen, J., Zhao, L. and Zhang, J. (2023). The mitochondrial ribosomal protein mRpL4 regulates Notch signaling. EMBO Rep: e55764. PubMed ID: 37009823
Mitochondrial ribosomal proteins (MRPs) assemble as specialized ribosome to synthesize mtDNA-encoded proteins, which are essential for mitochondrial bioenergetic and metabolic processes. MRPs are required for fundamental cellular activities during animal development, but their roles beyond mitochondrial protein translation are poorly understood. This study reports a conserved role of the mitochondrial ribosomal protein L4 (mRpL4) in Notch signaling. Genetic analyses demonstrate that mRpL4 is required in the Notch signal-receiving cells to permit target gene transcription during Drosophila wing development. mRpL4 physically and genetically interacts with the WD40 repeat protein Wings apart (Wap) and activates the transcription of Notch signaling targets. This study shows that human mRpL4 is capable of replacing fly mRpL4 during wing development. Furthermore, knockout of mRpL4 in zebrafish leads to downregulated expression of Notch signaling components. Thus, this study has discovered a previously unknown function of mRpL4 during animal development.
Rhodes-Mordov, E., Brandwine-Shemmer, T., Zaguri, R., Gutorov, R., Peters, M. and Minke, B. (2023). Diacylglycerol Activates the Drosophila Light Sensitive Channel TRPL Expressed in HEK Cells. Int J Mol Sci 24(7). PubMed ID: 37047261
Physiological activation by light of the Drosophila TRP and TRP-like (TRPL) channels requires the activation of phospholipase Cβ (PLC). The hydrolysis of phosphatidylinositol 4,5, bisphosphate (PIP(2)) by PLC is a crucial step in the still-unclear light activation, while the generation of Diacylglycerol (DAG) by PLC seems to be involved. This study re-examined the ability of a DAG analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG) to activate the TRPL channels expressed in HEK cells. Unlike previous studies, this study added OAG into the cytosol via a patch-clamp pipette and observed robust activation of the expressed TRPL channels. However, TRPL channel activation was much slower than the physiologically activated TRPL by light. Therefore, a picosecond-fast optically activated DAG analogue, OptoDArG, was used. Inactive OptoDArG was added into the intracellular solution with the patch-clamp pipette, and it slowly accumulated on the surface membrane of the recorded HEK cell in the dark. A fast application of intense UV light to the recorded cell resulted in a robust and relatively fast TRPL-dependent current that was greatly accelerated by the constitutively active TRPL(F557I) pore-region mutation. However, this current of the mutant channel was still considerably slower than the native light-induced TRPL current, suggesting that DAG alone is not sufficient for TRPL channel activation under physiological conditions.

Friday June 2nd - Transposons and RNA

Narita, H., Shima, T., Iizuka, R. and Uemura, S. (2023). N-terminal region of Drosophila melanogaster Argonaute2 forms amyloid-like aggregates. BMC Biol 21(1): 78. PubMed ID: 37072852
Argonaute proteins play a central role in RNA silencing by forming protein-small RNA complexes responsible for the silencing process. While most Argonaute proteins have a short N-terminal region, Argonaute2 in Drosophila melanogaster (DmAgo2) harbors a long and unique N-terminal region. Previous in vitro biochemical studies have shown that the loss of this region does not impair the RNA silencing activity of the complex. However, an N-terminal mutant of Drosophila melanogaster has demonstrated abnormal RNA silencing activity. To explore the causes of this discrepancy between in vitro and in vivo studies, this study investigated the biophysical properties of the region. The N-terminal region is highly rich in glutamine and glycine residues, which is a well-known property for prion-like domains, a subclass of amyloid-forming peptides. Therefore, the possibility of the N-terminal region functioning as an amyloid was tested. In silico and biochemical assays demonstrated that the N-terminal region exhibits amyloid-specific properties. The region indeed formed aggregates that were not dissociated even in the presence of sodium dodecyl sulfate. Also, the aggregates enhanced the fluorescence intensity of thioflavin-T, an amyloid detection reagent. The kinetics of the aggregation followed that of typical amyloid formation exhibiting self-propagating activity. Furthermore, this study directly visualized the aggregation process of the N-terminal region under fluorescence microscopy and found that the aggregations took fractal or fibril shapes. Together, the results indicate that the N-terminal region can form amyloid-like aggregates. Many other amyloid-forming peptides have been reported to modulate the function of proteins through their aggregation. Therefore, these findings raise the possibility that aggregation of the N-terminal region regulates the RNA silencing activity of DmAgo2.
Jedlicka, P., Tokan, V., Kejnovska, I., Hobza, R. and Kejnovsky, E. (2023). Telomeric retrotransposons show propensity to form G-quadruplexes in various eukaryotic species. Mob DNA 14(1): 3. PubMed ID: 37038191
Canonical telomeres (telomerase-synthetised) are readily forming G-quadruplexes (G4) on the G-rich strand. However, there are examples of non-canonical telomeres among eukaryotes where telomeric tandem repeats are invaded by specific retrotransposons. Drosophila melanogaster represents an extreme example with telomeres composed solely by three retrotransposons-Het-A, TAHRE and TART (HTT). Even though non-canonical telomeres often show strand biased G-distribution, the evidence for the G4-forming potential is limited. Using circular dichroism spectroscopy and UV absorption melting assay this study has verified in vitro G4-formation in the HTT elements of D. melanogaster. Namely 3 in Het-A, 8 in TART and 2 in TAHRE. All the G4s are asymmetrically distributed as in canonical telomeres. Bioinformatic analysis showed that asymmetric distribution of potential quadruplex sequences (PQS) is common in telomeric retrotransposons in other Drosophila species. Most of the PQS are located in the gag gene where PQS density correlates with higher DNA sequence conservation and codon selection favoring G4-forming potential. The importance of G4s in non-canonical telomeres is further supported by analysis of telomere-associated retrotransposons from various eukaryotic species including green algae, Diplomonadida, fungi, insects and vertebrates. Virtually all analyzed telomere-associated retrotransposons contained PQS, frequently with asymmetric strand distribution. Comparison with non-telomeric elements showed independent selection of PQS-rich elements from four distinct LINE clades. These findings of strand-biased G4-forming motifs in telomere-associated retrotransposons from various eukaryotic species support the G4-formation as one of the prerequisites for the recruitment of specific retrotransposons to chromosome ends and call for further experimental studies.
Fan, Y. J., Ding, Z., Zhang, Y., Su, R., Yue, J. L., Liang, A. M., Huang, Q. W., Meng, Y. R., Li, M., Xue, Y. and Xu, Y. Z. (2023). Sex-lethal regulates back-splicing and generation of the sex-differentially expressed circular RNAs. Nucleic Acids Res. PubMed ID: 37070178
Conversely to canonical splicing, back-splicing connects the upstream 3' splice site (SS) with a downstream 5'SS and generates exonic circular RNAs (circRNAs) that are widely identified and have regulatory functions in eukaryotic gene expression. However, sex-specific back-splicing in Drosophila has not been investigated and its regulation remains unclear. This study performed multiple RNA analyses of a variety sex-specific Drosophila samples and identified over ten thousand circular RNAs, in which hundreds are sex-differentially and -specifically back-spliced. Intriguingly, w expression of Sxl, an RNA-binding protein encoded by Sex-lethal (Sxl), the master Drosophila sex-determination gene that is only spliced into functional proteins in females, promoted back-splicing of many female-differential circRNAs in the male S2 cells, whereas expression of a Sxl mutant (SXLRRM) did not promote those events. Using a monoclonal antibody, this study further obtained the transcriptome-wide RNA-binding sites of Sxl through PAR-CLIP. After splicing assay of mini-genes with mutations in the Sxl-binding sites, it esd revealed that Sxl-binding on flanking exons and introns of pre-mRNAs facilitates back-splicing, whereas Sxl-binding on the circRNA exons inhibits back-splicing. This study provides strong evidence that Sxl has a regulatory role in back-splicing to generate sex-specific and -differential circRNAs, as well as in the initiation of sex-determination cascade through canonical forward-splicing.
Schneider, B. K., Sun, S., Lee, M., Li, W., Skvir, N., Neretti, N., Vijg, J. and Secombe, J. (2023). Expression of retrotransposons contributes to aging in Drosophila. Genetics. PubMed ID: 37084379
Retrotransposons are a class of transposable elements capable of self-replication and insertion into new genomic locations. Across species, the mobilization of retrotransposons in somatic cells has been suggested to contribute to the cell and tissue functional decline that occurs during aging. Retrotransposons are broadly expressed across cell types, and de novo insertions have been observed to correlate with tumorigenesis. However, the extent to which new retrotransposon insertions occur during normal aging and their effect on cellular and animal function remains understudied. This study used a single nucleus whole genome sequencing approach in Drosophila to directly test whether transposon insertions increase with age in somatic cells. Analyses of nuclei from thoraces and indirect flight muscles using a newly developed pipeline, Retrofind, revealed no significant increase in the number of transposon insertions with age. Despite this, reducing the expression of two different retrotransposons, 412 and Roo, extended lifespan, but did not alter indicators of health such as stress resistance. This suggests a key role for transposon expression and not insertion in regulating longevity. Transcriptomic analyses revealed similar changes to gene expression in 412 and Roo knockdown flies and highlighted changes to genes involved in proteolysis and immune function as potential contributors to the observed changes in longevity. Combined, these data show a clear link between retrotransposon expression and aging.
Cao, J., Yu, T., Xu, B., Hu, Z., Zhang, X. O., Theurkauf, W. E. and Weng, Z. (2023). Epigenetic and chromosomal features drive transposon insertion in Drosophila melanogaster. Nucleic Acids Res 51(5): 2066-2086. PubMed ID: 36762470
Transposons are mobile genetic elements prevalent in the genomes of most species. The distribution of transposons within a genome reflects the actions of two opposing processes: initial insertion site selection, and selective pressure from the host. By analyzing whole-genome sequencing data from transposon-activated Drosophila melanogaster, 43,316 de novo and 237 germline insertions were identified from four long-terminal-repeat (LTR) transposons, one LINE transposon (I-element), and one DNA transposon (P-element). All transposon types favored insertion into promoters de novo, but otherwise displayed distinct insertion patterns. De novo and germline P-element insertions preferred replication origins, often landing in a narrow region around transcription start sites and in regions of high chromatin accessibility. De novo LTR transposon insertions preferred regions with high H3K36me3, promoters and exons of active genes; within genes, LTR insertion frequency correlated with gene expression. De novo I-element insertion density increased with distance from the centromere. Germline I-element and LTR transposon insertions were depleted in promoters and exons, suggesting strong selective pressure to remove transposons from functional elements. Transposon movement is associated with genome evolution and disease; therefore, these results can improve understanding of genome and disease biology.
Sheng, P., Li, L., Li, T., Wang, Y., Hiers, N. M., Mejia, J. S., Sanchez, J. S., Zhou, L. and Xie, M. (2023). Screening of Drosophila microRNA-degradation sequences reveals Argonaute1 mRNA's role in regulating miR-999. Nat Commun 14(1): 2108. PubMed ID: 37055443
MicroRNAs (miRNA) load onto AGO proteins to target mRNAs for translational repression or degradation. However, miRNA degradation can be triggered when extensively base-paired with target RNAs, which induces confirmational change of AGO and recruitment of ZSWIM8 ubiquitin ligase to mark AGO for proteasomal degradation. This target RNA-directed miRNA degradation (TDMD) mechanism appears to be evolutionarily conserved, but recent studies have focused on mammalian systems. This study performed AGO1-CLASH in Drosophila S2 cells, with Dora (ortholog of vertebrate ZSWIM8) knockout mediated by CRISPR-Cas9 to identify five TDMD triggers (sequences that can induce miRNA degradation). Interestingly, one trigger in the 3' UTR of AGO1 mRNA induces miR-999 degradation. CRISPR-Cas9 knockout of the AGO1 trigger in S2 cells and in Drosophila specifically elevates miR-999, with concurrent repression of the miR-999 targets. AGO1 trigger knockout flies respond poorly to hydrogen peroxide-induced stress, demonstrating the physiological importance of this TDMD event.

Thursday June 1st - Adult Development

Buffry, A. D., Kittelmann, S. and McGregor, A. P. (2023). Characterisation of the role and regulation of Ultrabithorax in sculpting fine-scale leg morphology. Front Cell Dev Biol 11: 1119221. PubMed ID: 36861038
Hox genes are expressed during embryogenesis and determine the regional identity of animal bodies along the antero-posterior axis. However, they also function post-embryonically to sculpt fine-scale morphology. To better understand how Hox genes are integrated into post-embryonic gene regulatory networks, this study further analysed the role and regulation of Ultrabithorax (Ubx) during leg development in Drosophila melanogaster. Ubx regulates several aspects of bristle and trichome patterning on the femurs of the second (T2) and third (T3) leg pairs. Repression of trichomes in the proximal posterior region of the T2 femur by Ubx is likely mediated by activation of the expression of microRNA-92a and microRNA-92b by this Hox protein. Furthermore, this study identified a novel enhancer of Ubx that recapitulates the temporal and regional activity of this gene in T2 and T3 legs. Transcription factor (TF) binding motif analysis was used in regions of accessible chromatin in T2 leg cells to predict and functionally test TFs that may regulate the Ubx leg enhancer.The role of the Ubx co-factors Homothorax (Hth) and Extradenticle (Exd) in T2 and T3 femurs was also tested. Several TFs were found that may act upstream or in concert with Ubx to modulate trichome patterning along the proximo-distal axis of developing femurs and that the repression of trichomes also requires Hth and Exd. Taken together these results provide insights into how Ubx is integrated into a post-embryonic gene regulatory network to determine fine-scale leg morphology.
Chen, M., Gao, E., Lin, G., Shen, J. and Wang, D. (2023). The transcription factor optomotor-blind restricts apterous expression through TrxG and PcG genes. Dev Biol 497: 59-67. PubMed ID: 36907311
The establishment of body pattern is a fundamental process in developmental biology. In Drosophila, the wing disc is subdivided into dorsal (D) and ventral (V) compartments by the D/V boundary. The dorsal fate is adopted by expressing the selector gene apterous (ap). ap expression is regulated by three combinational cis-regulatory modules which are activated by EGFR pathway, Ap-Vg auto-regulatory and epigenetic mechanisms. This study found that the Tbx family transcription factor Optomotor-blind (Omb) restricted ap expression in the ventral compartment. Loss of omb induced autonomous initiation of ap expression in the middle third instar larvae in the ventral compartment. Oppositely, over-activation of omb inhibited ap in the medial pouch. All three enhancers apE, apDV and apP were upregulated in omb null mutants, indicating a combinational regulation of ap modulators. However, Omb affected ap expression neither by directly regulating EGFR signaling, nor via Vg regulation. Therefore, a genetic screen of epigenetic regulators, including the Trithorax group (TrxG) and Polycomb group (PcG) genes was performed. Knocking down the TrxG gene kohtalo (kto), domino (dom) or expressing the PcG gene grainy head (grh), the ectopic ap in omb mutants was repressed. The inhibition of apDV by kto knockdown and grh activation could contribute to ap repression. Moreover, Omb and the EGFR pathway are genetically parallel in ap regulation in the ventral compartment. Collectively, Omb is a repressive signal for ap expression in the ventral compartment, which requires TrxG and PcG genes.
Chakraborty, A., Peterson, N. G., King, J. S., Gross, R. T., Pla, M. M., Thennavan, A., Zhou, K. C., DeLuca, S., Bursac, N., Bowles, D. E., Wolf, M. J. and Fox, D. T. (2023). Conserved Chamber-Specific Polyploidy Maintains Heart Function in Drosophila. bioRxiv. PubMed ID: 36798187
Developmentally programmed polyploidy (whole-genome-duplication) of cardiomyocytes is common across evolution. Functions of such polyploidy are essentially unknown. This study reveals roles for precise polyploidy levels in cardiac tissue. A conserved asymmetry is found in polyploidy level between cardiac chambers in Drosophila larvae and humans. In Drosophila , differential Insulin Receptor (InR) sensitivity leads the heart chamber to reach a higher ploidy/cell size relative to the aorta chamber. Cardiac ploidy-reduced animals exhibit reduced heart chamber size, stroke volume, cardiac output, and acceleration of circulating hemocytes. These Drosophila phenotypes mimic systemic human heart failure. Using human donor hearts, this study revealed asymmetry in nuclear volume (ploidy) and insulin signaling between the left ventricle and atrium. Tese results identify productive and likely conserved roles for polyploidy in cardiac chambers and suggest precise ploidy levels sculpt many developing tissues. These findings of productive cardiomyocyte polyploidy impact efforts to block developmental polyploidy to improve heart injury recovery.
Rathore, S., Meece, M., Charlton-Perkins, M., Cook, T. A. and Buschbeck, E. K. (2023). Probing the conserved roles of cut in the development and function of optically different insect compound eyes. Front Cell Dev Biol 11: 1104620. PubMed ID: 37065850
Astonishing functional diversity exists among arthropod eyes, yet eye development relies on deeply conserved genes. This phenomenon is best understood for early events, whereas fewer investigations have focused on the influence of later transcriptional regulators on diverse eye organizations and the contribution of critical support cells, such as Semper cells (SCs). As SCs in Drosophila melanogaster secrete the lens and function as glia, they are critical components of ommatidia. In this study RNAi-based knockdowns were performed of the transcription factor cut (CUX in vertebrates), a marker of SCs, the function of which has remained untested in these cell types. To probe for the conserved roles of cut, two optically different compound eyes were tested: the apposition optics of D. melanogaster and the superposition optics of the diving beetle Thermonectus marmoratus. In both cases, multiple aspects of ocular formation were disrupted, including lens facet organization and optics as well as photoreceptor morphogenesis. Together, these findings support the possibility of a generalized role for SCs in arthropod ommatidial form and function and introduces Cut as a central player in mediating this role.
Schultheis, D. and Frasch, M. (2023).. Longitudinal visceral muscles in Drosophila fully dedifferentiate and fragment prior to their reestablishment during metamorphosis. MicroPubl Biol 2023. PubMed ID: 37008728
Although the Drosophila longitudinal visceral muscles have been shown to undergo major morphological changes during the transition from larval to adult gut musculature, there have been conflicting views as to whether these muscles persist as such during metamorphosis or whether they are built anew. This study presents an independent analysis using HLH54Fb-eGFP as a cell type specific marker, which strengthens the proposition that the syncytial larval longitudinal gut muscles completely dedifferentiate and fragment into mononucleated myoblasts during pupariation before they fuse again and redifferentiate to form the adult longitudinal gut muscles.
Ostale, C. M., Vega-Cuesta, P., Gonzalez, T., Lopez-Varea, A. and de Celis, J. F. (2023). RNAi screen in the Drosophila wing of genes encoding proteins related to cytoskeleton organization and cell division. Dev Biol 498: 61-76. PubMed ID: 37015290
Cell division and cytoskeleton organization are fundamental processes participating in the development of Drosophila imaginal discs. This manuscript describes the phenotypes in the adult fly wing generated by knockdowns of 85% of Drosophila genes encoding proteins likely related to the regulation of cell division and cytoskeleton organization. A molecular classification of these proteins was compiled into classes that describe their expected or known main biochemical characteristics, as well as mRNA expression in the wing disc and likely protein subcellular localization for a subset of these genes. Finally, this study analyzed in more detail one protein family of cytoskeleton genes (Arp2/3 complex), and define the consequences of interfering with cell division for wing growth and patterning.
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