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Tuesday, August 31st, 2021 - Evolution

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Lafuente, E., Alves, F., King, J. G., Peralta, C. M. and Beldade, P. (2021). Many ways to make darker flies: Intra- and interspecific variation in Drosophila body pigmentation components. Ecol Evol 11(12): 8136-8155. PubMed ID: 34188876
Summary:
Body pigmentation is an evolutionarily diversified and ecologically relevant trait with substantial variation within and between species, and important roles in animal survival and reproduction. Insect pigmentation, in particular, provides some of the most compelling examples of adaptive evolution, including its ecological significance and genetic bases. Pigmentation includes multiple aspects of color and color pattern that may vary more or less independently, and can be under different selective pressures. This study decomposed Drosophila thorax and abdominal pigmentation, a valuable eco-evo-devo model, into distinct measurable traits related to color and color pattern. Intra- and interspecific variation for those traits was investigated, and its different sources were assessed. For each body part, overall darkness was measured, as well as four other pigmentation properties distinguishing between background color and color of the darker pattern elements that decorate each body part. By focusing on two standard D. melanogaster laboratory populations, this study showed that pigmentation components vary and covary in distinct manners depending on sex, genetic background, and temperature during development. Studying three natural populations of D. melanogaster along a latitudinal cline and five other Drosophila species, it was shown that evolution of lighter or darker bodies can be achieved by changing distinct component traits. The results paint a much more complex picture of body pigmentation variation than previous studies could uncover, including patterns of sexual dimorphism, thermal plasticity, and interspecific diversity. These findings underscore the value of detailed quantitative phenotyping and analysis of different sources of variation for a better understanding of phenotypic variation and diversification, and the ecological pressures and genetic mechanisms underlying them.
Poikela, N., Tyukmaeva, V., Hoikkala, A. and Kankare, M. (2021). Multiple paths to cold tolerance: the role of environmental cues, morphological traits and the circadian clock gene vrille. BMC Ecol Evol 21(1): 117. PubMed ID: 34112109
Summary:
Tracing the association between insect cold tolerance and latitudinally and locally varying environmental conditions, as well as key morphological traits and molecular mechanisms, is essential for understanding the processes involved in adaptation. These issues were explored in two closely-related species, Drosophila montana and Drosophila flavomontana, originating from diverse climatic locations across several latitudes on the coastal and mountainous regions of North America were investigated. The association between sequence variation in one of the key circadian clock genes, vrille, and cold tolerance in both species was investigated. Finally, the impact of vrille on fly cold tolerance and cold acclimation ability was investigated by silencing it with RNA interference in D. montana. A principal component analysis (PCA) was performed on variables representing bioclimatic conditions on the study sites and latitude was used as a proxy of photoperiod. PC1 separated the mountainous continental sites from the coastal ones based on temperature variability and precipitation, while PC2 arranged the sites based on summer and annual mean temperatures. Cold tolerance tests showed D. montana to be more cold-tolerant than D. flavomontana and chill coma resistance (CT(min)) of this species showed an association with PC2. Both the sequence analysis and RNAi study on vrille suggested this gene to play an essential role in D. montana cold resistance and acclimation, but not in recovery time. This study demonstrates the complexity of insect cold tolerance and emphasizes the need to trace its association with multiple environmental variables and morphological traits to identify potential agents of natural selection. It also shows that a circadian clock gene vrille is essential both for short- and long-term cold acclimation, potentially elucidating the connection between circadian clock system and cold tolerance.
Nishiguchi, S. and Oda, H. (2021). Structural variability and dynamics in the ectodomain of an ancestral-type classical cadherin revealed by AFM imaging. J Cell Sci 134(14). PubMed ID: 34152409
Summary:
Type III cadherin represents the ancestral form of classical cadherin in bilaterian metazoans. Drosophila possesses type III and type IVa cadherins, known as DN-cadherin and DE-cadherin, respectively. Mature DN- and DE-cadherins have 15 and 7 extracellular cadherin domain (EC) repeats, respectively, with DN-cadherin EC6-EC11 homologous to DE-cadherin EC1-EC6. These EC repeats contain predicted complete or partial Ca2+-free inter-EC linkers that potentially contribute to adhesion. Comparative structure-function studies of DN- and DE-cadherins may help in understanding the ancestral and derived states of classical cadherin-mediated adhesion mechanisms. Using bead aggregation assays, this study found that DN-cadherin EC1-EC11 and DE-cadherin EC1-EC6 exhibit Ca2+-dependent adhesive properties. Using high-speed atomic force microscopy (HS-AFM) imaging in solution, both DN- and DE-cadherin ectodomains were shown to share a common morphological framework consisting of a strand-like and a globule-like portion. Furthermore, the DN-cadherin EC repeats are highly variable, flexible in morphology and have at least three bendable sites, one of which is located in EC6-EC11 and can act as a flexible hinge. These findings provide insights into diversification of classical cadherin-mediated adhesion mechanisms.
Patlar, B., Jayaswal, V., Ranz, J. M. and Civetta, A. (2021). Nonadaptive molecular evolution of seminal fluid proteins in Drosophila. Evolution. PubMed ID: 34184267
Summary:
Seminal fluid proteins (SFPs) are a group of reproductive proteins that are among the most evolutionarily divergent known. As SFPs can impact male and female fitness, these proteins have been proposed to evolve under postcopulatory sexual selection (PCSS). However, the fast change of the SFPs can also result from nonadaptive evolution, and the extent to which selective constraints prevent SFPs rapid evolution remains unknown. Using intra- and interspecific sequence information, along with genomics and functional data, this study examine the molecular evolution of approximately 300 SFPs in Drosophila. It was found that 50-57% of the SFP genes, depending on the population examined, are evolving under relaxed selection. Only 7-12% showed evidence of positive selection, with no evidence supporting other forms of PCSS, and 35-37% of the SFP genes were selectively constrained. Further, despite associations of positive selection with gene location on the X chromosome and protease activity, the analysis of additional genomic and functional features revealed their lack of influence on SFPs evolving under positive selection. These results highlight a lack of sufficient evidence to claim that most SFPs are driven to evolve rapidly by PCSS while identifying genomic and functional attributes that influence different modes of SFPs evolution.
Peluffo, A. E., Hamdani, M., Vargas-Valderrama, A., David, J. R., Mallard, F., Graner, F. and Courtier-Orgogozo, V. (2021). A morphological trait involved in reproductive isolation between Drosophila sister species is sensitive to temperature. Ecol Evol 11(12): 7492-7506. PubMed ID: 34188829
Summary:
Male genitalia are usually extremely divergent between closely related species, but relatively constant within one species. This study examined the effect of temperature on the shape of the ventral branches, a male genital structure involved in reproductive isolation, in the sister species Drosophila santomea and Drosophila yakuba. A semi-automatic measurement machine learning pipeline was designed that can reliably identify curvatures and landmarks based on manually digitized contours of the ventral branches. With this method, it was observed that temperature does not affect ventral branches in D. yakuba but that in D. santomea ventral branches tend to morph into a D. yakuba-like shape at lower temperature. Male genitalia structures involved in reproductive isolation can be relatively variable within one species and can resemble the shape of closely related species' genitalia through plasticity to temperature. These results suggest that reproductive isolation mechanisms can be dependent on the environmental context.
Dai, A., Wang, Y., Greenberg, A., Liufu, Z. and Tang, T. (2021). Rapid Evolution of Autosomal Binding Sites of the Dosage Compensation Complex in Drosophila melanogaster and Its Association With Transcription Divergence. Front Genet 12: 675027. PubMed ID: 34194473
Summary:
How pleiotropy influences evolution of protein sequence remains unclear. The male-specific lethal (MSL) complex in Drosophila mediates dosage compensation by 2-fold upregulation of the X chromosome in males. Nevertheless, several MSL proteins also bind autosomes and likely perform functions not related to dosage compensation. The evolution of MOF, MSL1, and MSL2 binding sites was studied in Drosophila melanogaster and its close relative Drosophila simulans. Pervasive expansion of the MSL binding sites were found in D. melanogaster, particularly on autosomes. The majority of these newly-bound regions are unlikely to function in dosage compensation and associated with an increase in expression divergence between D. melanogaster and D. simulans. While dosage-compensation related sites show clear signatures of adaptive evolution, these signatures are even more marked among autosomal regions. This study points to an intriguing avenue of investigation of pleiotropy as a mechanism promoting rapid protein sequence evolution.

Monday, August 30th - Adult neural development and function

Wang, Q., Xu, P., Andreazza, F., Liu, Y., Nomura, Y., Duran, P., Jiang, L., Chen, M., Takamatsu, G., Ihara, M., Matsuda, K., Isaacs, R., Oliveira, E. E., Du, Y. and Dong, K. (2021). Identification of multiple odorant receptors essential for pyrethrum repellency in Drosophila melanogaster. PLoS Genet 17(7): e1009677. PubMed ID: 34237075
Summary:
Pyrethrum extract from dry flowers of Tanacetum cinerariifolium (formally Chrysanthemum cinerariifolium) has been used globally as a popular insect repellent against arthropod pests for thousands of years. However, the mechanistic basis of pyrethrum repellency remains unknown. This study found that pyrethrum spatially repels and activates olfactory responses in Drosophila melanogaster, a genetically tractable model insect, and the closely-related D. suzukii which is a serious invasive fruit crop pest. The discovery of spatial pyrethrum repellency and olfactory response to pyrethrum in D. melanogaster facilitated identification of four odorant receptors, Or7a, Or42b, Or59b and Or98a that are responsive to pyrethrum. Further analysis showed that the first three Ors are activated by pyrethrins, the major insecticidal components in pyrethrum, whereas Or98a is activated by (E)-β-farnesene (EBF), a sesquiterpene and a minor component in pyrethrum. Importantly, knockout of Or7a, Or59b or Or98a individually abolished fly avoidance to pyrethrum, while knockout of Or42b had no effect, demonstrating that simultaneous activation of Or7a, Or59b and Or98a is required for pyrethrum repellency in D. melanogaster. This study provides insights into the molecular basis of repellency of one of the most ancient and globally used insect repellents. Identification of pyrethrum-responsive Ors opens the door to develop new synthetic insect repellent mixtures that are highly effective and broad-spectrum.
Basak, R., Sutradhar, S. and Howard, J. (2021). Focal laser stimulation of fly nociceptors activates distinct axonal and dendritic Ca(2+) signals. Biophys J. PubMed ID: 34175294
Summary:
Drosophila class IV neurons are polymodal nociceptors that detect noxious mechanical, thermal, optical, and chemical stimuli. Escape behaviors in response to attacks by parasitoid wasps are dependent on class IV cells, whose highly branched dendritic arbors form a fine meshwork that is thought to enable detection of the wasp's needle-like ovipositor barb. To understand how mechanical stimuli trigger cellular responses, a focused 405-nm laser was used to create highly localized lesions to probe the precise position needed to evoke responses. By imaging calcium signals in dendrites, axons, and soma in response to stimuli of varying positions, intensities, and spatial profiles, it was discovered that there are two distinct nociceptive pathways. Direct stimulation to dendrites (the contact pathway) produces calcium responses in axons, dendrites, and the cell body, whereas stimulation adjacent to the dendrite (the noncontact pathway) produces calcium responses in the axons only. The noncontact pathway is interpreted as damage to adjacent cells releasing diffusible molecules that act on the dendrites. Axonal responses have higher sensitivities and shorter latencies. In contrast, dendritic responses have lower sensitivities and longer latencies. Stimulation of finer, distal dendrites leads to smaller responses than stimulation of coarser, proximal dendrites, as expected if the contact response depends on the geometric overlap of the laser profile and the dendrite diameter. Because the axon signals to the central nervous system to trigger escape behaviors, it is proposed that the density of the dendritic meshwork is high not only to enable direct contact with the ovipositor but also to enable neuronal activation via diffusing signals from damaged surrounding cells. Dendritic contact evokes responses throughout the dendritic arbor, even to regions distant and distal from the stimulus. These dendrite-wide calcium signals may facilitate hyperalgesia or cellular morphological changes after dendritic damage.
Li, F., Lo, T. Y., Miles, L., Wang, Q., Noristani, H. N., Li, D., Niu, J., Trombley, S., Goldshteyn, J. I., Wang, C., Wang, S., Qiu, J., Pogoda, K., Mandal, K., Brewster, M., Rompolas, P., He, Y., Janmey, P. A., Thomas, G. M., Li, S. and Song, Y. (2021). The Atr-Chek1 pathway inhibits axon regeneration in response to Piezo-dependent mechanosensation. Nat Commun 12(1): 3845. PubMed ID: 34158506
Summary:
Atr is a serine/threonine kinase, known to sense single-stranded DNA breaks and activate the DNA damage checkpoint by phosphorylating Chek1 (Grapes in Drosophila), which inhibits Cdc25, causing cell cycle arrest. This pathway has not been implicated in neuroregeneration. This study shows that in Drosophila sensory neurons removing Atr or Chek1, or overexpressing Cdc25 promotes regeneration, whereas Atr or Chek1 overexpression, or Cdc25 knockdown impedes regeneration. Inhibiting the Atr-associated checkpoint complex in neurons promotes regeneration and improves synapse/behavioral recovery after CNS injury. Independent of DNA damage, Atr responds to the mechanical stimulus elicited during regeneration, via the mechanosensitive ion channel Piezo and its downstream NO signaling. Sensory neuron-specific knockout of Atr in adult mice, or pharmacological inhibition of Atr-Chek1 in mammalian neurons in vitro and in flies in vivo enhances regeneration. These findings reveal the Piezo-Atr-Chek1-Cdc25 axis as an evolutionarily conserved inhibitory mechanism for regeneration, and identify potential therapeutic targets for treating nervous system trauma.
Kinold, J. C., Brenner, M. and Aberle, H. (2021). Misregulation of Drosophila Sidestep leads to uncontrolled wiring of the adult neuromuscular system and severe locomotion defects. Front Neural Circuits 15: 658791. PubMed ID: 34149366
Summary:
Holometabolic organisms undergo extensive remodelling of their neuromuscular system during metamorphosis. Relatively, little is known whether or not the embryonic guidance of molecules and axonal growth mechanisms are re-activated for the innervation of a very different set of adult muscles. This study shows that the axonal attractant Sidestep (Side) is re-expressed during Drosophila metamorphosis and is indispensable for neuromuscular wiring. Mutations in side cause severe innervation defects in all legs. Neuromuscular junctions (NMJs) show a reduced density or are completely absent at multi-fibre muscles. Misinnervation strongly impedes, but does not completely abolish motor behaviours, including walking, flying, or grooming. Overexpression of Side in developing muscles induces similar innervation defects; for example, at indirect flight muscles, it causes flightlessness. Since muscle-specific overexpression of Side is unlikely to affect the central circuits, the resulting phenotypes seem to correlate with faulty muscle wiring. It was further shown that mutations in beaten path Ia (beat), a receptor for Side, results in similar weaker adult innervation and locomotion phenotypes, indicating that embryonic guidance pathways seem to be reactivated during metamorphosis.
Jacob, P. F., Vargas-Gutierrez, P., Okray, Z., Vietti-Michelina, S., Felsenberg, J. and Waddell, S. (2021). Prior experience conditionally inhibits the expression of new learning in Drosophila. Curr Biol. PubMed ID: 34146482
Summary:
Prior experience of a stimulus can inhibit subsequent acquisition or expression of a learned association of that stimulus. However, the neuronal manifestations of this learning effect, named latent inhibition (LI), are poorly understood. This study shows that prior odor exposure can produce context-dependent LI of later appetitive olfactory memory performance in Drosophila. Odor pre-exposure forms a short-lived aversive memory whose lone expression lacks context-dependence. Acquisition of odor pre-exposure memory requires aversively reinforcing dopaminergic neurons that innervate two mushroom body compartments-one group of which exhibits increasing activity with successive odor experience. Odor-specific responses of the corresponding mushroom body output neurons are suppressed, and their output is necessary for expression of both pre-exposure memory and LI of appetitive memory. Therefore, odor pre-exposure attaches negative valence to the odor itself, and LI of appetitive memory results from a temporary and context-dependent retrieval deficit imposed by competition with the parallel short-lived aversive memory.
Zhao, B., Sun, J., Li, Q. and Zhong, Y. (2021). Differential conditioning produces merged long-term memory in Drosophila. Elife 10. PubMed ID: 34279222
Summary:
Multiple spaced trials of aversive differential conditioning can produce two independent long-term memories (LTMs) of opposite valence. One is an aversive memory for avoiding the conditioned stimulus (CS+), and the other is a safety memory for approaching the non-conditioned stimulus (CS-). This study shows that a single trial of aversive differential conditioning yields one merged LTM (mLTM) for avoiding both CS+ and CS-. Such mLTM can be detected after sequential exposures to the shock-paired CS+ and unpaired CS-, and be retrieved by either CS+ or CS-. The formation of mLTM relies on triggering aversive-reinforcing dopaminergic neurons and subsequent new protein synthesis. Expressing mLTM involves αβ Kenyon cells and corresponding approach-directing mushroom body output neurons (MBONs), in which similar-amplitude long-term depression of responses to CS+ and CS- seems to signal the mLTM. These results suggest that animals can develop distinct strategies for occasional and repeated threatening experiences.

Friday, August 27th - Chromatin

Mitra, R., Richhariya, S., Jayakumar, S., Notani, D. and Hasan, G. (2021). IP3-mediated Ca2+ signals regulate larval to pupal transition under nutrient stress through the H3K36 methyltransferase Set2. Development 148(11). PubMed ID: 34117888
Summary:
dPersistent loss of dietary protein usually signals a shutdown of key metabolic pathways. In Drosophila larvae that have reached a 'critical weight' and can pupariate to form viable adults, such a metabolic shutdown would needlessly lead to death. Inositol 1,4,5-trisphosphate-mediated calcium (IP3/Ca2+) release in some interneurons (vGlutVGN6341) allows Drosophila larvae to pupariate on a protein-deficient diet by partially circumventing this shutdown through upregulation of neuropeptide signaling and the expression of ecdysone synthesis genes. This study shows that IP3/Ca2+ signals in vGlutVGN6341 neurons drive expression of Set2, a gene encoding Drosophila Histone 3 Lysine 36 methyltransferase. Furthermore, Set2 expression is required for larvae to pupariate in the absence of dietary protein. IP3/Ca2+ signal-driven Set2 expression upregulates key Ca2+-signaling genes through a novel positive-feedback loop. Transcriptomic studies, coupled with analysis of existing ChIP-seq datasets, identified genes from larval and pupal stages that normally exhibit robust H3K36 trimethyl marks on their gene bodies and concomitantly undergo stronger downregulation by knockdown of either the intracellular Ca2+ release channel IP3R or Set2. IP3/Ca2+ signals thus regulate gene expression through Set2-mediated H3K36 marks on select neuronal genes for the larval to pupal transition.
Wei, K. H., Chan, C. and Bachtrog, D. (2021). Establishment of H3K9me3-dependent heterochromatin during embryogenesis in Drosophila miranda. Elife 10. PubMed ID: 34128466
Summary:
Heterochromatin is a key architectural feature of eukaryotic genomes crucial for silencing of repetitive elements. During Drosophila embryonic cellularization, heterochromatin rapidly appears over repetitive sequences, but the molecular details of how heterochromatin is established are poorly understood. This study mapped the genome-wide distribution of H3K9me3-dependent heterochromatin in individual embryos of Drosophila miranda at precisely staged developmental time points. Canonical H3K9me3 enrichment was found to be established prior to cellularization and matures into stable and broad heterochromatin domains through development. Intriguingly, initial nucleation sites of H3K9me3 enrichment appear as early as embryonic stage 3 over transposable elements (TEs) and progressively broaden, consistent with spreading to neighboring nucleosomes. The earliest nucleation sites are limited to specific regions of a small number of recently active retrotransposon families and often appear over promoter and 5' regions of LTR retrotransposons, while late nucleation sites develop broadly across the entirety of most TEs. Interestingly, early nucleating TEs are strongly associated with abundant maternal piRNAs and show early zygotic transcription. These results support a model of piRNA-associated co-transcriptional silencing while also suggesting additional mechanisms for site-restricted H3K9me3 nucleation at TEs in pre-cellular Drosophila embryos.
Stephenson, R. A., Thomalla, J. M., Chen, L., Kolkhof, P., White, R. P., Beller, M. and Welte, M. A. (2021). Sequestration to lipid droplets promotes histone availability by preventing turnover of excess histones. Development. PubMed ID: 34286844
Summary:
Because both dearth and overabundance of histones result in cellular defects, histone synthesis and demand are typically tightly coupled. In Drosophila embryos, histones H2B/H2A/H2Av accumulate on lipid droplets (LDs), cytoplasmic fat storage organelles. Without LD-binding, maternally provided H2B/H2A/H2Av are absent, but how LDs ensure histone storage is unclear. Using quantitative imaging, this study uncover when during oogenesis these histones accumulate, and which step of accumulation is LD-dependent. LDs originate in nurse cells (NCs) and are transported to the oocyte. Although H2Av accumulates on LDs in NCs, the majority of the final H2Av pool is synthesized in oocytes. LDs promote intercellular transport of the histone-anchor Jabba and thus its presence in the ooplasm. Ooplasmic Jabba then prevents H2Av degradation, safeguarding the H2Av stockpile. These findings provide insight into the mechanism for establishing histone stores during Drosophila oogenesis and shed light on the function of LDs as protein-sequestration sites.
Jordan, W. and Larschan, E. (2021). The zinc finger protein CLAMP promotes long-range chromatin interactions that mediate dosage compensation of the Drosophila male X-chromosome. Epigenetics Chromatin 14(1): 29. PubMed ID: 34187599
Summary:
Drosophila dosage compensation is an important model system for defining how active chromatin domains are formed. The male-specific lethal dosage compensation complex (MSLc) increases transcript levels of genes along the length of the single male X-chromosome to equalize with that expressed from the two female X-chromosomes. The strongest binding sites for MSLc cluster together in three-dimensional space largely independent of MSLc because clustering occurs in both sexes. CLAMP, a non-sex specific, ubiquitous zinc finger protein, binds synergistically with MSLc to enrich the occupancy of both factors on the male X-chromosome. This study demonstrates that CLAMP promotes the observed three-dimensional clustering of MSLc binding sites. Moreover, the X-enriched CLAMP protein more strongly promotes longer-range three-dimensional interactions on the X-chromosome than autosomes. Genome-wide, CLAMP promotes three-dimensional interactions between active chromatin regions together with other insulator proteins. This study has defined how long-range interactions which are modulated by a locally enriched ubiquitous transcription factor promote hyper-activation of the X-chromosome to mediate dosage compensation.
Melnikova, L. S., Molodina, V. V., Kostyuchenko, M. V., Georgiev, P. G. and Golovnin, A. K. (2021). The BEAF-32 Protein Directly Interacts with Z4/putzig and Chriz/Chromator Proteins in Drosophila melanogaster. Dokl Biochem Biophys 498(1): 184-189. PubMed ID: 34189647
Summary:
In Drosophila, the BEAF-32, Z4/putzig, and Chriz/Chromator proteins colocalize in the interbands of polytene chromosomes. It was assumed that these proteins can form a complex that affects the structure of chromatin. However, the mechanism of the formation of such a complex has not been studied. This study proved for the first time that the BEAF-32, Z4/putzig, and Chriz/Chromator proteins interact directly with each other and localized the protein domains that provide multiple protein-protein interactions. Based on the data obtained, a model was developed of the mechanism of the formation the BEAF/Z4/Chriz complex and its recruitment to chromatin.
Khalisova, K. Y., Osadchiy, I. S., Georgiev, P. G. and Maksimenko, O. G. (2021). TTK Isoforms Interact with Two Regions of the Mep-1 Protein of Drosophila melanogaster. Dokl Biochem Biophys 498(1): 177-179. PubMed ID: 34189645
Summary:
The Drosophila TTK protein is involved in the processes of cell differentiation and is represented by two isoforms, TTK69 and TTK88, which have a common N-terminal BTB domain and different C-terminal sequences. Earlier, it was shown that TTK69 represses the activity of enhancers and promoters by recruiting a conserved among higher eukaryotes NURD complex to chromatin. The Mep-1 protein was found in the NURD-complex of Drosophila, and this protein can interact with the C-terminal region of TTK69. In the present study, using the yeast two-hybrid system, the interacting regions of the TTK and Mep-1 proteins were mapped. Regions in the unique C-terminal regions of TTK isoforms were identified that can interact simultaneously with two regions of the Mep-1 protein. The results show that, despite the low homology of the C-terminal regions, the TTK isoform retains the ability to interact with two conserved regions of the Mep-1 protein, which suggests the functional significance of this interaction.

Thursday, August 26th - Behavior

Schumann, I., Berger, M., Nowag, N., Schafer, Y., Saumweber, J., Scholz, H. and Thum, A. S. (2021). Ethanol-guided behavior in Drosophila larvae. Sci Rep 11(1): 12307. PubMed ID: 34112872
Summary:
Chemosensory signals allow vertebrates and invertebrates not only to orient in its environment toward energy-rich food sources to maintain nutrition but also to avoid unpleasant or even poisonous substrates. Ethanol is a substance found in the natural environment of Drosophila melanogaster. Accordingly, D. melanogaster has evolved specific sensory systems, physiological adaptations, and associated behaviors at its larval and adult stage to perceive and process ethanol. To systematically analyze how D. melanogaster larvae respond to naturally occurring ethanol, ethanol-induced behavior was examined in great detail by reevaluating existing approaches and comparing them with new experiments. Using behavioral assays, it was confirmed that larvae are attracted to different concentrations of ethanol in their environment. This behavior is controlled by olfactory and other environmental cues. It is independent of previous exposure to ethanol in their food. Moreover, moderate, naturally occurring ethanol concentration of 4% results in increased larval fitness. On the contrary, higher concentrations of 10% and 20% ethanol, which rarely or never appear in nature, increase larval mortality. Finally, ethanol also serves as a positive teaching signal in learning and memory and updates valence associated with simultaneously processed odor information. Since information on how larvae perceive and process ethanol at the genetic and neuronal level is limited, the establishment of standardized assays described here is an important step towards their discovery.
Arya, H., Toltesi, R., Eng, M., Garg, D., Merritt, T. J. S. and Rajpurohit, S. (2021). No water, no mating: Connecting dots from behaviour to pathways. PLoS One 16(6): e0252920. PubMed ID: 34111165
Summary:
Insects hold considerable ecological and agricultural importance making it vital to understand the factors impacting their reproductive output. Environmental stressors are examples of such factors which have a substantial and significant influence on insect reproductive fitness. Insects are also ectothermic and small in size which makes them even more susceptible to environmental stresses. The present study assesses the consequence of desiccation on the mating latency and copulations duration in tropical Drosophila melanogaster. Flies were tested for these reproductive behavioral parameters at varying body water levels and with whole metabolome analysis in order to gain a further understanding of the physiological response to desiccation. The results showed that the duration of desiccation is positively correlated with mating latency and mating failure, while having no influence on the copulation duration. The metabolomic analysis revealed three biological pathways highly affected by desiccation: starch and sucrose metabolism, galactose metabolism, and phenylalanine, tyrosine and tryptophan biosynthesis. These results are consistent with carbohydrate metabolism providing an energy source in desiccated flies and also suggests that the phenylalanine biosynthesis pathway plays a role in the reproductive fitness of the flies. Desiccation is a common issue with smaller insects, like Drosophila and other tropical insects, and these findings indicate that this lack of ambient water can immediately and drastically affect the insect reproductive behaviour, which becomes more crucial because of unpredictable and dynamic weather conditions.
Kohlmeier, P., Zhang, Y., Gorter, J. A., Su, C. Y. and Billeter, J. C. (2021). Mating increases Drosophila melanogaster females' choosiness by reducing olfactory sensitivity to a male pheromone. Nat Ecol Evol. PubMed ID: 34155384
Summary:
Females that are highly selective when choosing a mate run the risk of remaining unmated or delaying commencing reproduction. Therefore, low female choosiness would be beneficial when males are rare but it would be maladaptive if males become more frequent. How can females resolve this issue? Polyandry would allow mating-status-dependent choosiness, with virgin females selecting their first mate with little selectivity and becoming choosier thereafter. This plasticity in choosiness would ensure timely acquisition of sperm and enable females to increase offspring quality during later mating. This study shows that Drosophila melanogaster females display such mating-status-dependent choosiness by becoming more selective once mated and identifies the underlying neurohormonal mechanism. Mating releases juvenile hormone, which desensitizes Or47b olfactory neurons to a pheromone produced by males, resulting in increased preference for pheromone-rich males. Besides providing a mechanism to a long-standing evolutionary prediction, these data suggest that intersexual selection in D. melanogaster, and possibly in all polyandrous, sperm-storing species, is mainly the domain of mated females since virgin females are less selective. Juvenile hormone influences behaviour by changing cue responsiveness across insects; the neurohormonal modulation of olfactory neurons uncovered in D. melanogaster provides an explicit mechanism for how this hormone modulates behavioural plasticity.
McKelvey, E. G. Z., Gyles, J. P., Michie, K., Barquín Pancorbo, V., Sober, L., Kruszewski, L. E., Chan, A. and Fabre, C. C. G. (2021). Drosophila females receive male substrate-borne signals through specific leg neurons during courtship. Curr Biol. PubMed ID: 34174209
Summary:
Substrate-borne vibratory signals are thought to be one of the most ancient and taxonomically widespread communication signals among animal species, including Drosophila flies. During courtship, the male Drosophila abdomen tremulates to generate vibrations in the courting substrate. These vibrations coincide with nearby females becoming immobile, a behavior that facilitates mounting and copulation. It was unknown how the Drosophila female detects these substrate-borne vibratory signals. This study confirmed that the immobility response of the female to the tremulations is not dependent on any air-borne cue. Substrate-borne communication is used by wild Drosophila and the vibrations propagate through those natural substrates (e.g., fruits) where flies feed and court. Transmission of the signals through a variety of substrates was examined, and how each of these substrates modifies the vibratory signal during propagation and affects the female response is described. Moreover, the main sensory structures and neurons that receive the vibrations were identified in the female legs; the mechanically gated ion channels Nanchung and Piezo (but not Trpγ) that mediate sensitivity to the vibrations. Together, these results show that Drosophila flies, like many other arthropods, use substrate-borne communication as a natural means of communication, strengthening the idea that this mode of signal transfer is heavily used and reliable in the wild. These findings also reveal the cellular and molecular mechanisms underlying the vibration-sensing modality necessary for this communication.
Himmel, N. J., Letcher, J. M., Sakurai, A., Gray, T. R., Benson, M. N., Donaldson, K. J. and Cox, D. N. (2021). Identification of a neural basis for cold acclimation in Drosophila larvae. iScience 24(6): 102657. PubMed ID: 34151240
Summary:
Low temperatures can be fatal to insects, but many species have evolved the ability to cold acclimate, thereby increasing their cold tolerance. It has been previously shown that Drosophila melanogaster larvae perform cold-evoked behaviors under the control of noxious cold-sensing neurons (nociceptors), but it is unknown how the nervous system might participate in cold tolerance. This study describes cold-nociceptive behavior among 11 drosophilid species; the predominant cold-evoked larval response was found to be a head-to-tail contraction behavior, which is likely inherited from a common ancestor, but is unlikely to be protective. Therefore the hypothesis that cold nociception functions to protect larvae by triggering cold acclimation was tested. Drosophila melanogaster Class III nociceptors were found to be sensitized by and critical to cold acclimation and that cold acclimation can be optogenetically evoked, sans cold. Collectively, these findings demonstrate that cold nociception constitutes a peripheral neural basis for Drosophila larval cold acclimation.
Leonte, M. B., Leonhardt, A., Borst, A. and Mauss, A. S. (2021). Aerial course stabilization is impaired in motion-blind flies. J Exp Biol. PubMed ID: 34180515
Summary:
Visual motion detection is among the best understood neuronal computations. As extensively investigated in tethered flies, visual motion signals are assumed to be crucial to detect and counteract involuntary course deviations. During free flight, however, course changes are also signalled by other sensory systems. Therefore, it is yet unclear to what extent motion vision contributes to course control. To address this question, flies were genetically rendered motion-blind by blocking their primary motion-sensitive neurons, and their free-flight performance was quantified. Such flies were found to have difficulties maintaining a straight flight trajectory, much like unimpaired flies in the dark. By unilateral wing clipping, an asymmetry was generated in propulsive force, and the ability of flies to compensate for this perturbation was tested. While wild-type flies showed a remarkable level of compensation, motion-blind animals exhibited pronounced circling behaviour. These results therefore directly confirm that motion vision is necessary to fly straight under realistic conditions.

Wednesday, August 25th - Disease Models

Bonilla, M., McPherson, M., Coreas, J., Boulos, M., Chavol, P., Alrabadi, R. I. and Loza-Coll, M. (2021). Repeated ethanol intoxications of Drosophila melanogaster adults increases the resistance to ethanol of their progeny. Alcohol Clin Exp Res 45(7): 1370-1382. PubMed ID: 34120365
Summary:
For decades, Drosophila has been used as a model organism to understand the genetics and neurobiology of ethanol intoxication and tolerance. Previous research has shown that acute and chronic pre-exposures to ethanol can trigger the development of functional ethanol tolerance in flies and has unveiled some of the genetic pathways involved in the process. No previous work has systematically explored whether repeated intoxications of adult flies can affect the ethanol tolerance of their progeny. Adult flies were intoxicated several times (once daily, over several days), and their F1 and F2 progeny were subjected to a functional tolerance test in which flies are exposed to ethanol and video recorded twice within 5 hr. Their behavior was subsequently analyzed to determine how long it took them to become sedated during the first and second exposures. One- and 2-way ANOVAs were used to determine whether parental treatment had an effect on their progeny's baseline resistance and/or acquired functional tolerance to ethanol. Parental flies that were intoxicated several times produced F1 and F2 progeny with a significantly higher resistance to ethanol than progeny from unexposed controls. Further, parental intoxications inconsistently increased the progeny's capacity to develop rapid functional tolerance upon re-exposure to ethanol. The transmission of increased ethanol resistance to progeny lasted several days after the last parental intoxication. This is the first demonstration that repeated parental daily intoxications affect the progeny's response to ethanol in fruit flies. These findings support the use of D. melanogaster to explore conserved pathways underlying the transmission of ethanol tolerance and can help in the identification of novel strategies for managing alcohol use disorder.
Loreto, J. S., Ferreira, S. A., Ardisson-Araujo, D. M. and Barbosa, N. V. (2021). Human type 2 diabetes mellitus-associated transcriptional disturbances in a high-sugar diet long-term exposed Drosophila melanogaster. Comp Biochem Physiol Part D Genomics Proteomics 39: 100866. PubMed ID: 34192612
Summary:
Type 2 Diabetes mellitus (T2DM) is a multifactorial and polygenic disorder with the molecular bases still idiopathic. Interestingly, high-sugar diet (HSD) induces delayed time for pupation and reduced viability in fruit fly larvae hatched from a 30% sucrose-containing medium (HSD-30%). An mRNA-deep sequencing study was carried out to identify differentially transcribed genes in adult fruit fly hatched and reared from an HSD-30%. Seven days after hatching, flies reared on control and HSD-30% were used to glucose and triglyceride level measurements and RNA extraction for sequencing. Remarkably, glucose levels were about 2-fold higher than the control group in fruit flies exposed to HSD-30%, whereas triglycerides levels increased 1.7-fold. After RNA-sequencing, it was found that 13.5% of the genes were differentially transcribed in the dyslipidemic and hyperglycaemic insects. HSD-30% up-regulated genes involved in ribosomal biogenesis (e.g. dTOR, ERK and dS6K) and down-regulated genes involved in energetic process (e.g. Pfk, Gapdh1, and Pyk from pyruvate metabolism; kdn, Idh and Mdh2 from the citric acid cycle; ATPsynC and ATPsynβ from ATP synthesis) and insect development. A remarkable down-regulation for Actin (Act88F) likely impairs muscle development. Moreover, HSD-30% up-regulated both the insulin-like peptides 7 and 8 and down-regulated the insulin receptor substrate p53, isoform A and insulin-like peptide 6 genes, whose functional products are insulin signaling markers. All these features pointed together to a tightly correlation of the T2DM-like phenotype modeled by the D. melanogaster and an intricate array of phenomena, which includes energetic processes, muscle development, and ribosomal synthesis as that observed for the human pathology.
Ghosh, B., Karmakar, S., Prasad, M. and Mandal, A. K. (2021). Praja1 ubiquitin ligase facilitates degradation of polyglutamine proteins and suppresses polyglutamine-mediated toxicity. Mol Biol Cell: mbcE20110747. PubMed ID: 34161122
Summary:
A network of chaperones and ubiquitin ligases sustain intracellular proteostasis, and is integral in preventing aggregation of misfolded proteins associated with various neurodegenerative diseases. Using cell-based studies of polyglutamine (polyQ) diseases: Spinocerebellar ataxia Type 3 (SCA3) and Huntington's disease (HD), this study aimed to identify crucial ubiquitin ligases that protect against polyQ aggregation. Praja1 (PJA1), a Ring-H2 ubiquitin ligase abundantly expressed in the brain is diminished when polyQ repeat proteins (Ataxin-3/Huntingtin) are expressed in cells. PJA1 interacts with polyQ proteins and enhances their degradation resulting in reduced aggregate formation. Down-regulation of PJA1 in neuronal cells increases polyQ protein levels vis-a-vis their aggregates rendering the cells vulnerable to cytotoxic stress. Finally, PJA1 suppresses polyQ toxicity in yeast and rescues eye degeneration in transgenic Drosophila model of SCA3. Thus, these findings establish PJA1 as a robust ubiquitin ligase of polyQ proteins and induction of which might serve as an alternative therapeutic strategy in handling cytotoxic polyglutamine aggregates.
Katsinelos, T., McEwan, W. A., Jahn, T. R. and Nickel, W. (2021). Identification of cis-acting determinants mediating the unconventional secretion of tau. Sci Rep 11(1): 12946. PubMed ID: 34155306
Summary:
The deposition of tau aggregates throughout the brain is a pathological characteristic within a group of neurodegenerative diseases collectively termed tauopathies, which includes Alzheimer's disease. While recent findings suggest the involvement of unconventional secretory pathways driving tau into the extracellular space and mediating the propagation of the disease-associated pathology, many of the mechanistic details governing this process remain elusive. Using Drosophila models of tauopathy, the hyperphosphorylation and aggregation state of tau was correlated with the disease-related neurotoxicity. These newly established systems recapitulate all the previously identified hallmarks of tau secretion, including the contribution of tau hyperphosphorylation as well as the requirement for PI(4,5)P(2) triggering the direct translocation of tau. Using a series of cellular assays, this study demonstrated that both the sulfated proteoglycans on the cell surface and the correct orientation of the protein at the inner plasma membrane leaflet are critical determinants of this process. Finally, two cysteine residues within the microtubule binding repeat domain were identified as novel cis-elements that are important for both unconventional secretion and trans-cellular propagation of tau.
Hofer, S. J., Liang, Y., Zimmermann, A., Schroeder, S., Dengjel, J., Kroemer, G., Eisenberg, T., Sigrist, S. J. and Madeo, F. (2021). Spermidine-induced hypusination preserves mitochondrial and cognitive function during aging. Autophagy: 1-3. PubMed ID: 34105442
Summary:
Spermidine is a natural polyamine, central to cellular homeostasis and growth, that promotes macroautophagy/autophagy. The polyamine pathway is highly conserved from bacteria to mammals and spermidine (prominently found in some kinds of aged cheese, wheat germs, nuts, soybeans, and fermented products thereof, among others) is an intrinsic part of the human diet. Apart from nutrition, spermidine is available to mammalian organisms from intracellular biosynthesis and microbial production in the gut. Importantly, externally supplied spermidine (via drinking water or food) prolongs lifespan, activates autophagy, improves mitochondrial function, and refills polyamine pools that decline during aging in various tissues of model organisms, including mice. In two adjacent studies, how dietary spermidine supplementation enhances eEF5/EIF5A hypusination, cerebral mitochondrial function and cognition in aging Drosophila melanogaster and mice was explored.
Lyu, Y., Promislow, D. E. L. and Pletcher, S. D. (2021). Serotonin signaling modulates aging-associated metabolic network integrity in response to nutrient choice in Drosophila melanogaster. Commun Biol 4(1): 740. PubMed ID: 34131274
Summary:
Aging arises from complex interactions among multiple biochemical products. Systems-level analyses of biological networks may provide insights into the causes and consequences of aging that evade single-gene studies. Previous studies found that dietary choice is sufficient to modulate aging in the vinegar fly, Drosophila melanogaster. This study showed that nutrient choice influenced several measures of metabolic network integrity, including connectivity, community structure, and robustness. Importantly, these effects are mediated by serotonin signaling, as a mutation in serotonin receptor 2A (5-HT2A) eliminated the effects of nutrient choice. Changes in network structure were associated with organism resilience and increased susceptibility to genetic perturbation. These data suggest that the behavioral or perceptual consequences of exposure to individual macronutrients, involving serotonin signaling through 5-HT2A, qualitatively change the state of metabolic networks throughout the organism from one that is highly connected and robust to one that is fragmented, fragile, and vulnerable to perturbations.

Tuesday August 24th - Larval and Adult Physiology

Huang, H., Possidente, D. R. and Vecsey, C. G. (2021). Optogenetic activation of SIFamide (SIFa) neurons induces a complex sleep-promoting effect in the fruit fly Drosophila melanogaster. Physiol Behav 239: 113507. PubMed ID: 34175361
Summary:
Sleep is a universal and extremely complicated function. Sleep is regulated by two systems-sleep homeostasis and circadian rhythms. In a wide range of species, neuropeptides have been found to play a crucial role in the communication and synchronization between different components of both systems. In the fruit fly Drosophila melanogaster, SIFamide (SIFa) is a neuropeptide that has been reported to be expressed in 4 neurons in the pars intercerebralis (PI) area of the brain. Previous work has shown that transgenic ablation of SIFa neurons, mutation of SIFa itself, or knockdown of SIFa receptors reduces sleep, suggesting that SIFa is sleep-promoting. However, those were all constitutive manipulations that could have affected development or resulted in compensation, so the role of SIFa signaling in sleep regulation during adulthood remains unclear. This study, examined the sleep-promoting effect of SIFa through an optogenetic approach, which allowed for neuronal activation with high temporal resolution, while leaving development unaffected. Activation of the red-light sensor Chrimson in SIFa neurons was found to promote sleep in flies in a sexually dimorphic manner, where the magnitude of the sleep effect was greater in females than in males. Because neuropeptidergic neurons often also release other transmitters, RNA interference was used to knock down SIFa while also optogenetically activating SIFa neurons. SIFa knockdown only partially reduced the magnitude of the sleep effect, suggesting that release of other transmitters may contribute to the sleep induction when SIFa neurons are activated. Video-based analysis showed that activation of SIFa neurons for as brief a period as 1 second was able to decrease walking behavior for minutes after the stimulus. Future studies should aim to identify the transmitters that are utilized by SIFa neurons and characterize their upstream activators and downstream targets. It would also be of interest to determine how acute optogenetic activation of SIFa neurons alters other behaviors that have been linked to SIFa, such as mating and feeding.
Weston, R. M., Schmitt, R. E., Grotewiel, M. and Miles, M. F. (2021). Transcriptome analysis of chloride intracellular channel knockdown in Drosophila identifies oxidation-reduction function as possible mechanism of altered sensitivity to ethanol sedation. PLoS One 16(7): e0246224. PubMed ID: 34228751
Summary:
Chloride intracellular channels (CLICs) are a unique family of evolutionarily conserved metamorphic proteins, switching between stable conformations based on redox conditions. CLICs have been implicated in a wide variety biological processes including ion channel activity, apoptosis, membrane trafficking, and enzymatic oxidoreductase activity. Understanding the molecular mechanisms by which CLICs engage in these activities is an area of active research. This study targeted for RNAi knockdown the sole Drosophila melanogaster ortholog, Clic, to identify genes and biological processes associated with Clic expression. Clic knockdown had a substantial impact on global transcription, altering expression of over 7% of transcribed Drosophila genes. Overrepresentation analysis of differentially expressed genes identified enrichment of Gene Ontology terms including Cytoplasmic Translation, Oxidation-Reduction Process, Heme Binding, Membrane, Cell Junction, and Nucleolus. The top term, Cytoplasmic Translation, was enriched almost exclusively with downregulated genes. Drosophila Clic and vertebrate ortholog Clic4 have previously been tied to ethanol sensitivity and ethanol-regulated expression. Clic knockdown-responsive genes from the present study were found to overlap significantly with gene sets from 4 independently published studies related to ethanol exposure and sensitivity in Drosophila. Bioinformatic analysis of genes shared between these studies revealed an enrichment of genes related to amino acid metabolism, protein processing, oxidation-reduction processes, and lipid particles among others. To determine whether the modulation of ethanol sensitivity by Clic may be related to co-regulated oxidation-reduction processes, the effect of hyperoxia on ethanol sedation was evaluated in Clic knockdown flies. Consistent with previous findings, Clic knockdown reduced acute ethanol sedation sensitivity in flies housed under normoxia. However, this effect was reversed by exposure to hyperoxia, suggesting a common set of molecular-genetic mechanism may modulate each of these processes. This study suggests that Drosophila Clic has a major influence on regulation of oxidative stress signaling and that this function overlaps with the molecular mechanisms of acute ethanol sensitivity in the fly.
Aggarwal, D. D., Rybnikov, S., Sapielkin, S., Rashkovetsky, E., Frenkel, Z., Singh, M., Michalak, P. and Korol, A. B. (2021). Seasonal changes in recombination characteristics in a natural population of Drosophila melanogaster. Heredity (Edinb). PubMed ID: 34163036
Summary:
Environmental seasonality is a potent evolutionary force, capable of maintaining polymorphism, promoting phenotypic plasticity and causing bet-hedging. In Drosophila, environmental seasonality has been reported to affect life-history traits, tolerance to abiotic stressors and immunity. Oscillations in frequencies of alleles underlying fitness-related traits were also documented alongside SNPs across the genome. This study tested for seasonal changes in two recombination characteristics, crossover rate and crossover interference, in a natural D. melanogaster population from India using morphological markers of the three major chromosomes. Winter flies, collected after the dry season, have significantly higher desiccation tolerance than their autumn counterparts. This difference proved to hold also for hybrids with three independent marker stocks, suggesting its genetic rather than plastic nature. Significant between-season changes are documented for crossover rate (in 9 of 13 studied intervals) and crossover interference (in four of eight studied pairs of intervals); both single and double crossovers were usually more frequent in the winter cohort. The winter flies also display weaker plasticity of both recombination characteristics to desiccation. The observed differences are ascribed to indirect selection on recombination caused by directional selection on desiccation tolerance. These findings suggest that changes in recombination characteristics can arise even after a short period of seasonal adaptation (~8-10 generations).
Ohhara, Y., Hoshino, G., Imahori, K., Matsuyuki, T. and Yamakawa-Kobayashi, K. (2021). The Nutrient-Responsive Molecular Chaperone Hsp90 Supports Growth and Development in Drosophila. Front Physiol 12: 690564. PubMed ID: 34239451
Summary:
Animals can sense internal nutrients, such as amino acids/proteins, and are able to modify their developmental programs in accordance with their nutrient status. In the fruit fly, Drosophila melanogaster, amino acid/protein is sensed by the fat body, an insect adipose tissue, through a nutrient sensor, target of rapamycin (TOR) complex 1 (TORC1). TORC1 promotes the secretion of various peptide hormones from the fat body in an amino acid/protein-dependent manner. Fat-body-derived peptide hormones stimulate the release of insulin-like peptides, which are essential growth-promoting anabolic hormones, from neuroendocrine cells called insulin-producing cells (IPCs). Although the importance of TORC1 and the fat body-IPC axis has been elucidated, the mechanism by which TORC1 regulates the expression of insulinotropic signal peptides remains unclear. This study shows that an evolutionarily conserved molecular chaperone, heat shock protein 90 (Hsp90), promotes the expression of insulinotropic signal peptides. Fat-body-selective Hsp90 knockdown caused the transcriptional downregulation of insulinotropic signal peptides. IPC activity and systemic growth were also impaired in fat-body-selective Hsp90 knockdown animals. Furthermore, Hsp90 expression depended on protein/amino acid availability and TORC1 signaling. These results strongly suggest that Hsp90 serves as a nutrient-responsive gene that upregulates the fat body-IPC axis and systemic growth. It is proposed that Hsp90 is induced in a nutrient-dependent manner to support anabolic metabolism during the juvenile growth period.
Zappia, M. P., Guarner, A., Kellie-Smith, N., Rogers, A., Morris, R., Nicolay, B., Boukhali, M., Haas, W., Dyson, N. J. and Frolov, M. V. (2021). E2F/Dp inactivation in fat body cells triggers systemic metabolic changes. Elife 10. PubMed ID: 34251339
Summary:
The E2F transcription factors play a critical role in controlling cell fate. In Drosophila, the inactivation of E2F in either muscle or fat body results in lethality, suggesting an essential function for E2F in these tissues. However, the cellular and organismal consequences of inactivating E2F in these tissues are not fully understood. This study shows that the E2F loss exerts both tissue-intrinsic and systemic effects. The proteomic profiling of E2F-deficient muscle and fat body revealed that E2F regulates carbohydrate metabolism, a conclusion further supported by metabolomic profiling. Intriguingly, animals with E2F-deficient fat body had a lower level of circulating trehalose and reduced storage of fat. Strikingly, a sugar supplement was sufficient to restore both trehalose and fat levels, and subsequently rescued animal lethality. Collectively, these data highlight the unexpected complexity of E2F mutant phenotype, which is a result of combining both tissue-specific and systemic changes that contribute to animal development.
Liu, W., Cao, H., Kimari, M., Maronitis, G., Williams, M. J. and Schioth, H. B. (2021). Multidrug Resistance Like Protein 1 Activity in Malpighian Tubules Regulates Lipid Homeostasis in Drosophila. Membranes (Basel) 11(6). PubMed ID: 34201304
Summary:
Multidrug resistance proteins (MRPs), members of the ATP-binding cassette transporter (ABC transporter) family, are pivotal for transporting endo- and xenobiotics, which confer resistance to anticancer agents and contribute to the clearance of oxidative products. However, their function in many biological processes is still unclear. This study investigated the role of an evolutionarily conserved MRP in metabolic homeostasis by knocking down the expression of Drosophila multidrug-resistance like protein 1 (MRP) in several tissues involved in regulating metabolism, including the gut, fat body, and Malpighian tubules. Interestingly, only suppression of MRP in the Malpighian tubules, the functional equivalent to the human kidney, was sufficient to cause abnormal lipid accumulation and disrupt feeding behavior. Furthermore, reduced Malpighian tubule MRP expression resulted in increased Hr96 (homolog of human pregnane X receptor) expression. Hr96 is known to play a role in detoxification and lipid metabolism processes. Reduced expression of MRP in the Malpighian tubules also conveyed resistance to oxidative stress, as well as reduced normal levels of reactive oxygen species in adult flies. This study reveals that an evolutionarily conserved MRP is required in Drosophila Malpighian tubules for proper metabolic homeostasis.

Monday, August 23th - RNA and Transposons

Corgiat, E. B., List, S. M., Rounds, J. C., Corbett, A. H. and Moberg, K. H. (2021). The RNA-binding protein Nab2 regulates the proteome of the developing Drosophila brain. J Biol Chem 297(1): 100877. PubMed ID: 34139237
Summary:
The human ZC3H14 gene, which encodes a ubiquitously expressed polyadenosine zinc finger RNA-binding protein, is mutated in an inherited form of autosomal recessive, nonsyndromic intellectual disability. To gain insight into neurological functions of ZC3H14, a Drosophila melanogaster model was developed of ZC3H14 loss by deleting the fly ortholog, Nab2. Studies in this invertebrate model revealed that Nab2 controls final patterns of neuron projection within fully developed adult brains, but the role of Nab2 during development of the Drosophila brain is not known. This study identified roles for Nab2 in controlling the dynamic growth of axons in the developing brain mushroom bodies, which support olfactory learning and memory, and regulating abundance of a small fraction of the total brain proteome. The group of Nab2-regulated brain proteins, identified by quantitative proteomic analysis, includes the microtubule-binding protein Futsch, the neuronal Ig-family transmembrane protein turtle, the glial:neuron adhesion protein contactin, the Rac GTPase-activating protein tumbleweed, and the planar cell polarity factor Van Gogh, which collectively link Nab2 to the processes of brain morphogenesis, neuroblast proliferation, circadian sleep/wake cycles, and synaptic development. Overall, these data indicate that Nab2 controls the abundance of a subset of brain proteins during the active process of wiring the pupal brain mushroom body and thus provide a window into potentially conserved functions of the Nab2/ZC3H14 RNA-binding proteins in neurodevelopment.
Vaishali, Dimitrova-Paternoga, L., Haubrich, K., Sun, M., Ephrussi, A. and Hennig, J. (2021). Validation and classification of RNA binding proteins identified by mRNA interactome capture. RNA. PubMed ID: 34215685
Summary:
RNA binding proteins (RBPs) take part in all steps of the RNA life cycle and are often essential for cell viability. Most RBPs have a modular organization and comprise a set of canonical RNA binding domains. However, in recent years a number of high-throughput mRNA interactome studies on yeast, mammalian cell lines and whole organisms have uncovered a multitude of novel mRNA interacting proteins that lack classical RNA binding domains. Whereas a few have been confirmed to be direct and functionally relevant RNA binders, biochemical and functional validation of RNA binding of most others is lacking. This study employed a combination of NMR spectroscopy and biochemical studies to test the RNA binding properties of six putative RNA binding proteins. Half of the analysed proteins showed no interaction, whereas the other half displayed weak chemical shift perturbations upon titration with RNA. One of the candidates that was found to interact weakly with RNA in vitro is Drosophila melanogaster End binding protein 1 (EB1), a master regulator of microtubule plus-end dynamics. Further analysis showed that EB1's RNA binding occurs on the same surface as that with which EB1 interacts with microtubules. RNA immunoprecipitation and colocalization experiments suggest that EB1 is a rather non-specific, opportunistic RNA binder. These data suggest that care should be taken when embarking on an RNA binding study involving these unconventional, novel RBPs, and initial and simple in vitro RNA binding experiments are recommended.
Alecki, C. and Francis, N. J. (2021). Identification of R-loop-forming Sequences in Drosophila melanogaster Embryos and Tissue Culture Cells Using DRIP-seq. Bio Protoc 11(9): e4011. PubMed ID: 34124311
Summary:
R-loops are non-canonical nucleic structures composed of an RNA-DNA hybrid and a displaced ssDNA. Originally identified as a source of genomic instability, R-loops have been shown over the last decade to be involved in the targeting of proteins and to be associated with different histone modifications, suggesting a regulatory function. In addition, R-loops have been demonstrated to form differentially during the development of different tissues in plants and to be associated with diseases in mammals. This study provided a single-strand DRIP-seq protocol to identify R-loop-forming sequences in Drosophila melanogaster embryos and tissue culture cells. This protocol differs from earlier DRIP protocols in the fragmentation step. Sonication, unlike restriction enzymes, generates a homogeneous and highly reproducible nucleic acid fragment pool. In addition, it allows the use of this protocol in any organism with minimal optimization. This protocol integrates several steps from published protocols to identify R-loop-forming sequences with high stringency, suitable for de novo characterization.
Behari, J., Borkar, P., Vindu, A., Dandewad, V., Upadrasta, S., Shanmugam, D. and Seshadri, V. (2021). Conserved RNA Binding Activity of Phosphatidyl Inositol 5-Phosphate 4-Kinase (PIP4K2A). Front Mol Biosci 8: 631281. PubMed ID: 34124142
Summary:
Plasmodium falciparum is a causative agent for malaria and has a complex life cycle in human and mosquito hosts. Due to relative paucity of the transcription factors in Plasmodium, it is postulated that posttranscriptional regulation plays an important role in stage-specific gene expression. A conserved element present in the 3'UTR of some transcripts of specific set of mRNAs in gametocyte stages has been identified. Phosphatidylinositol 5-phosphate 4-kinase (PIP4K2A) associates with these RNA. The RNA binding activity of PIP4K2A is independent of its kinase activity. The RNA binding activity of PIP4K2A seems to be conserved across species from Drosophila and C. elegans to humans, suggesting that the RNA binding activity of PIP4K may be important, and there may be host transcripts that may be regulated by PIP4K2A. These results identify a novel RNA binding role for PIP4K2A that may not only play a role in Plasmodium propagation but may also function in regulating gene expression in multicellular organisms.
Naganuma, M., Tadakuma, H. and Tomari, Y. (2021). Single-molecule analysis of processive double-stranded RNA cleavage by Drosophila Dicer-2. Nat Commun 12(1): 4268. PubMed ID: 34257295
Summary:
Drosophila Dicer-2 (Dcr-2) produces small interfering RNAs from long double-stranded RNAs (dsRNAs), playing an essential role in antiviral RNA interference. The dicing reaction by Dcr-2 is enhanced by Loquacious-PD (Loqs-PD), a dsRNA-binding protein that partners with Dcr-2. Previous biochemical analyses have proposed that Dcr-2 uses two distinct-processive or distributive-modes of cleavage by distinguishing the terminal structures of dsRNAs and that Loqs-PD alters the terminal dependence of Dcr-2. However, the direct evidence for this model is lacking, as the dynamic movement of Dcr-2 along dsRNAs has not been traced. Utilizing single-molecule imaging, this study shows that the terminal structures of long dsRNAs and the presence or absence of Loqs-PD do not essentially change Dcr-2's cleavage mode between processive and distributive, but rather simply affect the probability for Dcr-2 to undergo the cleavage reaction. These results provide a refined model for how the dicing reaction by Dcr-2 is regulated.
Bawankar, P., Lence, T., Paolantoni, C., Haussmann, I. U., Kazlauskiene, M., Jacob, D., Heidelberger, J. B., Richter, F. M., Nallasivan, M. P., Morin, V., Kreim, N., Beli, P., Helm, M., Jinek, M., Soller, M. and Roignant, J. Y. (2021). Hakai is required for stabilization of core components of the m(6)A mRNA methylation machinery. Nat Commun 12(1): 3778. PubMed ID: 34145251
Summary:
N(6)-methyladenosine (m(6)A) is the most abundant internal modification on mRNA which influences most steps of mRNA metabolism and is involved in several biological functions. The E3 ubiquitin ligase Hakai was previously found in complex with components of the m(6)A methylation machinery in plants and mammalian cells but its precise function remained to be investigated. This study shows that Hakai is a conserved component of the methyltransferase complex in Drosophila and human cells. In Drosophila, its depletion results in reduced m(6)A levels and altered m(6)A-dependent functions including sex determination. Its ubiquitination domain is required for dimerization and interaction with other members of the m(6)A machinery, while its catalytic activity is dispensable. Finally, this study demonstrates that the loss of Hakai destabilizes several subunits of the methyltransferase complex, resulting in impaired m(6)A deposition. This work adds functional and molecular insights into the mechanism of the m(6)A mRNA writer complex

Friday, August 20th - Immune Response

Slavik, K. M., Morehouse, B. R., Ragucci, A. E., Zhou, W., Ai, X., Chen, Y., Li, L., Wei, Z., Bahre, H., Konig, M., Seifert, R., Lee, A. S. Y., Cai, H., Imler, J. L. and Kranzusch, P. J. (2021). cGAS-like receptors sense RNA and control 3'2'-cGAMP signaling in Drosophila. Nature. PubMed ID: 34261127
Summary:
Cyclic GMP-AMP synthase (cGAS) is a cytosolic DNA sensor that produces the second messenger 2'3'-cGAMP and controls activation of innate immunity in mammalian cells. Animal genomes typically encode multiple proteins with predicted homology to cGAS (CG7194 in Drosophila), but the function of these uncharacterized enzymes is unknown. This study shows that cGAS-like receptors (cGLRs) are innate immune sensors capable of recognizing divergent molecular patterns and catalyzing synthesis of distinct nucleotide second messenger signals. Crystal structures of human and insect cGLRs reveal a nucleotidyltransferase signaling core shared with cGAS and a diversified primary ligand-binding surface modified with significant insertions and deletions. cGLR surface remodeling enables altered ligand specificity and use a forward biochemical screen to identify cGLR1 as a double-stranded RNA sensor in the model organism Drosophila melanogaster. Surprisingly, RNA recognition activates Drosophila cGLR1 to synthesize the novel product cG[3'-5']pA[2'-5']p (3'2'-cGAMP). A crystal structure of Drosophila Stimulator of Interferon Genes (cGLR1) in complex with 3'2'-cGAMP explains selective isomer recognition and this study demonstrates that 3'2'-cGAMP induces an enhanced antiviral state in vivo that protects from viral infection. Similar to radiation of Toll-like receptors in pathogen immunity, these results establish cGLRs as a diverse family of metazoan pattern recognition receptors.
Zhou, H., Ni, J., Wu, S., Ma, F., Jin, P. and Li, S. (2021). lncRNA-CR46018 positively regulates the Drosophila Toll immune response by interacting with Dif/Dorsal. Dev Comp Immunol 124: 104183. PubMed ID: 34174242
Summary:
The Toll signaling pathway is highly conserved from insects to mammals. Drosophila is a model species that is commonly used to study innate immunity. Although many studies have assessed protein-coding genes that regulate the Toll pathway, it is unclear whether long noncoding RNAs (lncRNAs) play regulatory roles in the Toll pathway. This study evaluated the expression of the lncRNA CR46018 in Drosophila. These results showed that this lncRNA was significantly overexpressed after infection of Drosophila with Micrococcus luteus. A CR46018-overexpressing Drosophila strain was then constructed; it was expected that CR46018 overexpression would enhance the expression of various antimicrobial peptides downstream of the Toll pathway, regardless of infection with M. luteus. RNA-seq analysis of CR46018-overexpressing Drosophila after infection with M. luteus showed that upregulated genes were mainly enriched in Toll and Imd signaling pathways. Moreover, bioinformatics predictions and RNA-immunoprecipitation experiments showed that CR46018 interacted with the transcription factors Dif and Dorsal to enhance the Toll pathway. During gram-positive bacterial infection, flies overexpressing CR46018 showed favorable survival compared with flies in the control group. Overall, this current work not only reveals a new immune regulatory factor, lncRNA-CR46018, and explores its potential regulatory model, but also provides a new perspective for the effect of immune disorders on the survival of Drosophila melanogaster.
Yamashita, K., Oi, A., Kosakamoto, H., Yamauchi, T., Kadoguchi, H., Kuraishi, T., Miura, M. and Obata, F. (2021). Activation of innate immunity during development induces unresolved dysbiotic inflammatory gut and shortens lifespan. Dis Model Mech. PubMed ID: 34213567
Summary:
Early-life inflammatory response is associated with risks of age-related pathologies. How transient immune signalling activity during animal development influences life-long fitness is not well understood. Using Drosophila as a model, this study found that activation of innate immune pathway IMD signalling in the developing larvae increases adult starvation resistance, decreases food intake, and shortens organismal lifespan. Interestingly, lifespan is shortened by the IMD activation in the larval gut and fat body, while starvation resistance and food intake are altered by that in neurons. The adult flies developed with IMD activation show sustained IMD activity in the gut, despite complete tissue renewal during metamorphosis. The larval IMD activation increases an immuno-stimulative bacterial species Gluconobacter sp. in the gut microbiome, and this dysbiosis is persistent to adulthood. Removing gut microbiota by antibiotics in adult mitigates intestinal immune activation and rescues the shortened lifespan. This study demonstrates that early-life immune activation triggers long-term physiological changes as highlighted as an irreversible gut microbiota alteration, prolonged inflammatory intestine, and concomitant shortening of the organismal lifespan.
Marra, A., Hanson, M. A., Kondo, S., Erkosar, B. and Lemaitre, B. (2021). Drosophila Antimicrobial Peptides and Lysozymes Regulate Gut Microbiota Composition and Abundance. mBio: e0082421. PubMed ID: 34253067
Summary:
The gut microbiota affects the physiology and metabolism of animals and its alteration can lead to diseases such as gut dysplasia or metabolic disorders. Several reports have shown that the immune system plays an important role in shaping both bacterial community composition and abundance in Drosophila, and that immune deficit, especially during aging, negatively affects microbiota richness and diversity. A key set of Drosophila immune effectors are the antimicrobial peptides (AMPs), which confer defense upon systemic infection. AMPs and lysozymes, a group of digestive enzymes with antimicrobial properties, are expressed in the gut and are good candidates for microbiota regulation. This study took advantage of Drosophila to investigate the role of AMPs and lysozymes in regulation of gut microbiota structure and diversity. Using flies lacking AMPs and newly generated lysozyme mutants, gnotobiotic flies were colonized with a defined set of commensal bacteria. This study study shows that AMPs and, to a lesser extent, lysozymes are necessary to regulate the total and relative abundance of bacteria in the gut microbiota. The direct function of AMPs was decoupled from the immune deficiency (IMD) signaling pathway that regulates AMPs but also many other processes, more narrowly defining the role of these effectors in the microbial dysbiosis observed in IMD-deficient flies upon aging. This work shows that immune effectors, typically associated with resistance to pathogenic infections, also help shape the beneficial gut community, consistent with the idea that host-symbiont interactions use the same "language" typically associated with pathogenesis.
Wagner, C., Uliczka, K., Bossen, J., Niu, X., Fink, C., Thiedmann, M., Knop, M., Vock, C., Abdelsadik, A., Zissler, U. M., Isermann, K., Garn, H., Pieper, M., Wegmann, M., Koczulla, A. R., Vogelmeier, C. F., Schmidt-Weber, C. B., Fehrenbach, H., Konig, P., Silverman, N., Renz, H., Pfefferle, P., Heine, H. and Roeder, T. (2021). Constitutive immune activity promotes JNK- and FoxO-dependent remodeling of Drosophila airways. Cell Rep 35(1): 108956. PubMed ID: 33826881
Summary:
Extensive remodeling of the airways is a major characteristic of chronic inflammatory lung diseases such as asthma or chronic obstructive pulmonary disease (COPD). To elucidate the importance of a deregulated immune response in the airways for remodeling processes, a matching Drosophila model was established. Here, triggering the Imd (immune deficiency) pathway in tracheal cells induced organ-wide remodeling. This structural remodeling comprises disorganization of epithelial structures and comprehensive epithelial thickening. These structural changes do not depend on the Imd pathway's canonical branch terminating on nuclear factor κB (NF-κB) activation. Instead, activation of a different segment of the Imd pathway that branches off downstream of Tak1 and comprises activation of c-Jun N-terminal kinase (JNK) and forkhead transcription factor of the O subgroup (FoxO) signaling is necessary and sufficient to mediate the observed structural changes of the airways. These findings imply that targeting JNK and FoxO signaling in the airways could be a promising strategy to interfere with disease-associated airway remodeling processes.
Holleufer, A., Winther, K. G., Gad, H. H., Ai, X., Chen, Y., Li, L., Wei, Z., Deng, H., Liu, J., Frederiksen, N. A., Simonsen, B., Andersen, L. L., Kleigrewe, K., Dalskov, L., Pichlmair, A., Cai, H., Imler, J. L. and Hartmann, R. (2021). Two cGAS-like receptors induce antiviral immunity in Drosophila. Nature. PubMed ID: 34261128
Summary:
In mammals, cyclic GMP-AMP (cGAMP) synthase (cGAS) produces the cyclic dinucleotide (CDN) 2'3'-cGAMP in response to cytosolic DNA and this triggers an antiviral immune response. cGAS belongs to a large family of cGAS/DncV-like nucleotidyltransferases, present in both prokaryotes and eukaryotes. In bacteria, these enzymes synthesize a range of cyclic oligonucleotide and have recently emerged as important regulators of phage infections. This study identified two novel cGAS-like receptors (cGLRs) in the insect Drosophila melanogaster. cGLR1 and cGLR2 activate Sting and NF-κB dependent antiviral immunity in response to infection with RNA or DNA viruses. cGLR1 is activated by dsRNA to produce the novel CDN 3'2'-cGAMP whereas cGLR2 produces a combination of 2'3'-cGAMP and 3'2' cGAMP in response to a yet unidentified stimulus. These data establish cGAS as the founding member of a family of receptors sensing different types of nucleic acids and triggering immunity through production of CDNs beyond 2'3'-cGAMP.

Friday, August 19th - Adult Development

Mark, B., Bustos-Gonzalez, L., Cascallares, G., Conejera, F. and Ewer, J. (2021). The circadian clock gates Drosophila adult emergence by controlling the timecourse of metamorphosis. Proc Natl Acad Sci U S A 118(27). PubMed ID: 34183412
Summary:
The daily rhythm of adult emergence of holometabolous insects is one of the first circadian rhythms to be studied. In these insects, the circadian clock imposes a daily pattern of emergence by allowing or stimulating eclosion during certain windows of time and inhibiting emergence during others, a process that has been described as "gating." Although the circadian rhythm of insect emergence provided many of the key concepts of chronobiology, little progress has been made in understanding the bases of the gating process itself, although the term "gating" suggests that it is separate from the developmental process of metamorphosis. This study followed the progression through the final stages of Drosophila adult development with single-animal resolution and showed that the circadian clock imposes a daily rhythmicity to the pattern of emergence by controlling when the insect initiates the final steps of metamorphosis itself. Circadian rhythmicity of emergence depends on the coupling between the central clock located in the brain and a peripheral clock located in the prothoracic gland (PG), an endocrine gland whose only known function is the production of the molting hormone, ecdysone. This study shows that the clock exerts its action by regulating not the levels of ecdysone but that of its actions mediated by the ecdysone receptor. These findings may also provide insights for understanding the mechanisms by which the daily rhythms of glucocorticoids are produced in mammals, which result from the coupling between the central clock in the suprachiasmatic nucleus and a peripheral clock located in the suprarenal gland.
Karunendiran, A., Nguyen, C. T., Barzda, V. and Stewart, B. A. (2021). Disruption of Drosophila larval muscle structure and function by UNC45 knockdown. BMC Mol Cell Biol 22(1): 38. PubMed ID: 34256704
Summary:
Proper muscle function is heavily dependent on highly ordered protein complexes. UNC45 is a USC (named since this region is shared by three proteins UNC45/CRO1/She4P) chaperone that is necessary for myosin incorporation into the thick filaments. UNC45 is expressed throughout the entire Drosophila life cycle and it has been shown to be important during late embryogenesis when initial muscle development occurs. However, the effects of UNC45 manipulation at later developmental times, after muscle development, have not yet been studied. UNC45 was knocked down with RNAi in a manner that permitted survival to the pupal stage, allowing for characterization of sarcomere organization in the well-studied third instar larvae. Second harmonic generation (SHG) microscopy revealed changes in the striated pattern of body wall muscles as well as a reduction of signal intensity. This observation was confirmed with immunofluorescence and electron microscopy imaging, showing diminished UNC45 signal and disorganization of myosin and z-disk proteins. Concomitant alterations in both synaptic physiology and locomotor function were also found. Both nerve-stimulated response and spontaneous vesicle release were negatively affected, while larval movement was impaired. This study highlights the dependency of normal sarcomere structure on UNC45 expression. The known role of UNC45 for myosin localization was confirmed, and it was further shown the I-Z-I complex is also disrupted. This suggests a broad need for UNC45 to maintain sarcomere integrity. Newly discovered changes in synaptic physiology reveal the likely presence of a homeostatic response to partially maintain synaptic strength and muscle function.
Wood, B. M., Baena, V., Huang, H., Jorgens, D. M., Terasaki, M. and Kornberg, T. B. (2021). Cytonemes with complex geometries and composition extend into invaginations of target cells. J Cell Biol 220(5). PubMed ID: 33734293
Summary:
Cytonemes are specialized filopodia that mediate paracrine signaling in Drosophila and other animals. Studies using fluorescence confocal microscopy (CM) established their general paths, cell targets, and essential roles in signaling. To investigate details unresolvable by CM, this study used high-pressure freezing and EM to visualize cytoneme structures, paths, contents, and contacts. Cytonemes were previously seen by CM in the Drosophila wing imaginal disc system, including disc, tracheal air sac primordium (ASP), and myoblast cytonemes, and cytonemes were observed extending into invaginations of target cells, and cytonemes were observed connecting ASP cells and connecting myoblasts. Diameters of cytoneme shafts vary between repeating wide (206 ± 51.8 nm) and thin (55.9 ± 16.2 nm) segments. Actin, ribosomes, and membranous compartments are present throughout; rough ER and mitochondria are in wider proximal sections. These results reveal novel structural features of filopodia and provide a basis for understanding cytoneme cell biology and function.
Ordway, A. J., Teeters, G. M., Weasner, B. M., Weasner, B. P., Policastro, R. and Kumar, J. P. (2021). A multi-gene knockdown approach reveals a new role for Pax6 in controlling organ number in Drosophila. Development 148(9). PubMed ID: 33982759
Summary:
Genetic screens are designed to target individual genes for the practical reason of establishing a clear association between a mutant phenotype and a single genetic locus. This allows for a developmental or physiological role to be assigned to the wild-type gene. It has been observed that the concurrent loss of Pax6 and Polycomb epigenetic repressors in Drosophila leads the eye to transform into a wing. This fate change is not seen when either factor is disrupted separately. An implication of this finding is that standard screens may miss the roles that combinations of genes play in development. This study shows that this phenomenon is not limited to Pax6 and Polycomb but rather applies more generally. In the Drosophila eye-antennal disc, the simultaneous downregulation of Pax6 with either the NURF nucleosome remodeling complex or the Pointed transcription factor transforms the head epidermis into an antenna. This is a previously unidentified fate change that is also not observed with the loss of individual genes. It is proposed that the use of multi-gene knockdowns is an essential tool for unraveling the complexity of development.
Dondi, C., Bertin, B., Daponte, J. P., Wojtowicz, I., Jagla, K. and Junion, G. (2021). A polarized nucleus-cytoskeleton-ECM connection in migrating cardioblasts controls heart tube formation. Development. PubMed ID: 34228803
Summary:
The formation of the cardiac tube is a remarkable example of complex morphogenetic processes conserved from invertebrates to humans. It involves coordinated collective migration of contralateral rows of cardiac cells. The molecular processes underlying the specification of cardioblasts (CBs) prior to migration are well established and significant advances have been made in understanding the process of lumen formation. However, the mechanisms of collective cardiac cells migration remain elusive. This study identified CAP and MSP300 as novel actors involved during CBs migration. They both exhibit highly similar temporal and spatial expression patterns in migrating cardiac cells and are necessary for the correct number and alignment of CBs, a prerequisite for the coordination of their collective migration. These data suggest that CAP and MSP300 are part of a protein complex linking focal adhesion sites to nuclei via the actin cytoskeleton that maintains post-mitotic state and correct alignment of CBs.
Mitra, S. and Ryoo, H. D. (2021). The role of Ire1 in Drosophila eye pigmentation revealed by an RNase dead allele. Dev Biol. PubMed ID: 34265355
Summary:
Ire1 is an endoplasmic reticulum (ER) transmembrane RNase that cleaves substrate mRNAs to help cells adapt to ER stress. Because there are cell types with physiological ER stress, loss of Ire1 results in metabolic and developmental defects in diverse organisms. In Drosophila, Ire1 mutants show developmental defects at early larval stages and in pupal eye photoreceptor differentiation. These Drosophila studies relied on a single Ire1 loss of function allele with a Piggybac insertion in the coding sequence. This study reports that an Ire1 allele with a specific impairment in the RNase domain, H890A, unmasks previously unrecognized Ire1 phenotypes in Drosophila eye pigmentation. Specifically, it was found that the adult eye pigmentation is altered, and the pigment granules are compromised in Ire1(H890A) homozygous mosaic eyes. Furthermore, the Ire1(H890A) mutant eyes had dramatically reduced Rhodopsin-1 protein levels. Drosophila eye pigment granules are most notably associated with late endosome/lysosomal defects. These results indicate that the loss of Ire1, which would impair ER homeostasis, also results in altered adult eye pigmentation.

Wednesday, August 18th - Behavior

Vesterberg, A., Rizkalla, R. and Fitzpatrick, M. J. (2021). Environmental influences on for-mediated oviposition decisions in Drosophila melanogaster. J Neurogenet: 1-12. PubMed ID: 34259125
Summary:
Deciding whether or not to lay an egg on a given substrate is an important task undertaken by females of many arthropods. It involves perceiving the environment (e.g. quality of the substrate, temperature, and humidity), formulating a decision, and then conducting the appropriate behaviours to oviposit. This oviposition site selection (OSS) provides a useful system for studying simple decision-making. OSS in fruit flies, Drosophila melanogaster, is influenced by both genetic and environmental variation. Naturally occurring allelic variation in the foraging gene (for) is known to affect OSS. Given a choice of high- and low-nutrient oviposition substrates, groups of rovers (for(R)) are known to lay significantly more of their eggs on low-nutrient sites than sitters (for(s)) and sitter mutants (for(s2)). This study asked three questions: (1) Is the role of for in OSS affected by the availability of alternate oviposition sites? (2) Is the role of for in OSS sensitive to the density of ovipositing females? and (3) Does the gustatory sensation of yeast play a role in for-mediated variation in OSS? This study found a role of choice and female density in rover/sitter differences in OSS, as well as a role of for in response to glycerol, an indicator of yeast. The role of for in OSS decision-making is complex and multi-faceted and should prove fertile ground for further research into the factors affecting decision-making behaviours.
Alwash, N., Allen, A. M., Sokolowski, M. B.. and Levine, J. D. (2021). The Drosophila melanogaster foraging gene affects social networks. J Neurogenet: 1-13. PubMed ID: 34121597
Summary:
Drosophila melanogaster displays social behaviors including courtship, mating, aggression, and group foraging. Recent studies employed social network analyses (SNAs) to show that D. melanogaster strains differ in their group behavior, suggesting that genes influence social network phenotypes. Aside from genes associated with sensory function, few studies address the genetic underpinnings of these networks. The foraging gene (for) is a well-established example of a pleiotropic gene that regulates multiple behavioral phenotypes and their plasticity. In D. melanogaster, there are two naturally occurring alleles of for called rover and sitter that differ in their larval and adult food-search behavior as well as other behavioral phenotypes. It was hypothesized that for affects behavioral elements required to form social networks and the social networks themselves. These effects are evident when gene dosage was manipulated. Flies of the rover and sitter strains were found to exhibit differences in duration, frequency, and reciprocity of pairwise interactions, and they form social networks with differences in assortativity and global efficiency. Consistent with other adult phenotypes influenced by for, rover-sitter heterozygotes show intermediate patterns of dominance in many of these characteristics. Multiple generations of backcrossing a rover allele into a sitter strain showed that many but not all of these rover-sitter differences may be attributed to allelic variation at for. These findings reveal the significant role that for plays in affecting social network properties and their behavioral elements in Drosophila melanogaster.
Ucpunar, H. K. and Grunwald Kadow, I. C. (2021). Flies Avoid Current Atmospheric CO(2) Concentrations. Front Physiol 12: 646401. PubMed ID: 33927640
Summary:
CO(2) differs from most other odors by being ubiquitously present in the air animals inhale. CO(2) levels of the atmosphere, however, are subject to change. Depending on the landscape, temperature, and time of the year, CO(2) levels can change even on shortest time scales. In addition, since the 18th century the CO(2) baseline keeps increasing due to the intensive fossil fuel usage. However, it is not known whether this change is significant for animals, and if yes whether and how animals adapt to this change. Most insects possess olfactory receptors to detect the gaseous molecule, and CO(2) is one of the key odorants for insects such as the vinegar fly Drosophila melanogaster to find food sources and to warn con-specifics. So far, CO(2) and its sensory system have been studied in the context of rotting fruit and other CO(2)-emitting sources to investigate flies' response to significantly elevated levels of CO(2). However, it has not been addressed whether flies detect and potentially react to atmospheric levels of CO(2). By using behavioral experiments, this study shows that flies can detect atmospheric CO(2) concentrations and, if given the choice, prefer air with sub-atmospheric levels of the molecule. Blocking the synaptic release from CO(2) receptor neurons abolishes this choice. Based on electrophysiological recordings, it is hypothesized that CO(2) receptors, similar to ambient temperature receptors, actively sample environmental CO(2) concentrations close to atmospheric levels. Based on recent findings and these data, it is hypothesized that Gr-dependent CO(2) receptors do not primarily serve as a cue detector to find food sources or avoid danger, instead they function as sensors for preferred environmental conditions.
Breugel, F. V. (2021). Correlated decision making across multiple phases of olfactory guided search in Drosophila improves search efficiency. J Exp Biol. PubMed ID: 34286337
Summary:
Nearly all motile organisms must search for food, often requiring multiple phases of exploration across heterogeneous environments. The fruit fly, Drosophila, has emerged as an effective model system for studying this behavior, however, little is known about the extent to which experiences at one point in their search might influence decisions in another. To investigate whether prior experiences impact flies' search behavior after landing, individually labelled fruit flies were tracked as they explored three odor emitting but food-barren objects. Two features of their behavior correlated with the distance they travel on foot. First, flies walked larger distances when they approached the odor source, which they were almost twice as likely to do when landing on the patch farthest downwind. Computational fluid dynamics simulations suggest this patch may have had a stronger baseline odor, but only ∼15% higher than the other two. This small increase, together with flies' high olfactory sensitivity, suggests that perhaps their flight trajectory used to approach the patches plays a role. Second, flies also walked larger distances when the time elapsed since their last visit was longer. However, the correlation is subtle and subject to a large degree of variability. Using agent-based models, it was shown that this small correlation can increase search efficiency by 25-50% across many scenarios. Furthermore, the models developed in this study provide mechanistic hypotheses explaining the variability through either a noisy or straightforward decision-making process. Surprisingly, these stochastic decision-making algorithms enhance search efficiency in challenging but realistic search scenarios compared to deterministic strategies.
Xu, X., Yang, W., Tian, B., Sui, X., Chi, W., Rao, Y. and Tang, C. (2021). Quantitative investigation reveals distinct phases in Drosophila sleep. Commun Biol 4(1): 364. PubMed ID: 33742082
Summary:
The fruit fly, Drosophila melanogaster, has been used as a model organism for the molecular and genetic dissection of sleeping behaviors. However, most previous studies were based on qualitative or semi-quantitative characterizations. This study quantified sleep in flies. An assay was set up to continuously track the activity of flies using infrared camera, which monitored the movement of tens of flies simultaneously with high spatial and temporal resolution. Accurate statistics were obtained regarding the rest and sleep patterns of single flies. Analysis of the data has revealed a general pattern of rest and sleep: the rest statistics obeyed a power law distribution and the sleep statistics obeyed an exponential distribution. Thus, a resting fly would start to move again with a probability that decreased with the time it has rested, whereas a sleeping fly would wake up with a probability independent of how long it had slept. Resting transits to sleeping at time scales of minutes. This method allows quantitative investigations of resting and sleeping behaviors and these results provide insights for mechanisms of falling into and waking up from sleep.
Edmunds, D., Wigby, S. and Perry, J. C. (2021). 'Hangry' Drosophila: food deprivation increases male aggression. Anim Behav 177: 183-190. PubMed ID: 34290451
Summary:
Aggressive interactions are costly, such that individuals should display modified aggression in response to environmental stress. Many organisms experience frequent periods of food deprivation, which can influence an individual's capacity and motivation to engage in aggression. However, because food deprivation can simultaneously decrease an individual's resource-holding potential and increase its valuation of food resources, its net impact on aggression is unclear. This study tested the influence of increasingly prolonged periods of adult food deprivation on inter-male aggression in pairs of fruit flies, Drosophila melanogaster. Males displayed increased aggression following periods of food deprivation longer than a day. Increased aggression in food-deprived flies occurred despite their reduced mass. This result is probably explained by an increased attraction to food resources, as food deprivation increased male occupancy of central food patches, and food patch occupancy was positively associated with aggression. These findings demonstrate that aggressive strategies in male D. melanogaster are influenced by nutritional experience, highlighting the need to consider past nutritional stresses to understand variation in aggression.

Tuesday, August 17th - Disease Models

Maravat, M., Bertrand, M., Landon, C., Fayon, F., Morisset-Lopez, S., Sarou-Kanian, V. and Decoville, M. (2021). Complementary Nuclear Magnetic Resonance-Based Metabolomics Approaches for Glioma Biomarker Identification in a Drosophila melanogaster Model. J Proteome Res. PubMed ID: 34286978
Summary:
Human malignant gliomas are the most common type of primary brain tumor. Composed of glial cells and their precursors, they are aggressive and highly invasive, leading to a poor prognosis. Due to the difficulty of surgically removing tumors and their resistance to treatments, novel therapeutic approaches are needed to improve patient life expectancy and comfort. Glioma has been induced in Drosophila by co-activating the epidermal growth factor receptor and the phosphatidyl-inositol-3 kinase signaling pathways. Complementary nuclear magnetic resonance (NMR) techniques were used to obtain metabolic profiles in the third instar larvae brains. Fresh organs were directly studied by (1)H high resolution-magic angle spinning (HR-MAS) NMR, and brain extracts were analyzed by solution-state (1)H-NMR. Statistical analyses revealed differential metabolic signatures, impacted metabolic pathways, and glioma biomarkers. Each method was efficient to determine biomarkers. The highlighted metabolites including glucose, myo-inositol, sarcosine, glycine, alanine, and pyruvate for solution-state NMR and proline, myo-inositol, acetate, and glucose for HR-MAS show very good performances in discriminating samples according to their nature with data mining based on receiver operating characteristic curves. Combining results allows for a more complete view of induced disturbances and opens the possibility of deciphering the biochemical mechanisms of these tumors. The identified biomarkers provide a means to rebalance specific pathways through targeted metabolic therapy and to study the effects of pharmacological treatments using Drosophila as a model organism.
Reiszadeh Jahromi, S., Ramesh, S. R., Finkelstein, D. I. and Haddadi, M. (2021). alpha-Synuclein E46K Mutation and Involvement of Oxidative Stress in a Drosophila Model of Parkinson's Disease. Parkinsons Dis 2021: 6621507. PubMed ID: 34285796
Summary:
Parkinson's disease (PD) is an age-associated neurodegenerative condition in which some genetic variants are known to increase disease susceptibility on interaction with environmental factors inducing oxidative stress. Different mutations in the SNCA gene (synuclein α) are reported as the major genetic contributors to PD. E46K mutation pathogenicity has not been investigated as intensive as other SNCA gene mutations including A30P and A53T. In this study, based on the GAL4-UAS binary genetic tool, transgenic Drosophila melanogaster flies expressing wild-type and E46K-mutated copies of the human SNCA gene were constructed. Overexpression of human α-synuclein in the central nervous system of these transgenic flies led to disorganized ommatidia structures and loss of dopaminergic neurons. E46K α-synuclein caused remarkable climbing defects, reduced survivorship, higher ethanol sensitivity, and increased PQ-mediated mortality. A noticeable decline in activity of catalase and superoxide dismutase enzymes besides considerable increase in the levels of lipid peroxidation and reactive oxygen species was observed in head capsule homogenates of α-synuclein-expressing flies, which indicates obvious involvement of oxidative stress as a causal factor in SNCA (E46K) neurotoxicity. In all the investigations, E46K copy of the SNCA gene was found to impose more severe defects when compared to wild-type SNCA. It can be concluded that the constructed Drosophila models developed PD-like symptoms that facilitate comparative studies of molecular and cellular pathways implicated in the pathogenicity of different α-synuclein mutations.
Wall, J. M., Basu, A., Zunica, E. R. M., Dubuisson, O. S., Pergola, K., Broussard, J. P., Kirwan, J. P., Axelrod, C. L. and Johnson, A. E. (2021). CRISPR/Cas9-engineered Drosophila knock-in models to study VCP diseases. Dis Model Mech 14(7). PubMed ID: 34296742
Summary:
Mutations in Valosin Containing Protein (VCP) are associated with several degenerative diseases, including multisystem proteinopathy (MSP-1) and amyotrophic lateral sclerosis. However, patients with VCP mutations vary widely in their pathology and clinical penetrance, making it difficult to devise effective treatment strategies. A deeper understanding of how each mutation affects VCP function could enhance the prediction of clinical outcomes and design of personalized treatment options. The power of a genetically tractable model organism coupled with well-established in vivo assays and a relatively short life cycle make Drosophila an attractive system to study VCP disease pathogenesis. Using CRISPR/Cas9, this study generated individual Drosophila knock-in mutants that include nine hereditary VCP disease mutations. These models display many hallmarks of VCP-mediated degeneration, including progressive decline in mobility, protein aggregate accumulation and defects in lysosomal and mitochondrial function. Some novel and unexpected findings were found, including nuclear morphology defects and sex-specific phenotypic differences in several mutants. Taken together, the Drosophila VCP disease models generated in this study will be useful for studying the etiology of individual VCP patient mutations and testing potential genetic and/or pharmacological therapies.
Le, T. D. and Inoue, Y. H. (2021). Sesamin Activates Nrf2/Cnc-Dependent Transcription in the Absence of Oxidative Stress in Drosophila Adult Brains. Antioxidants (Basel) 10(6). PubMed ID: 34200419
Summary:
Sesamin, a major lignin in sesame seeds, possesses health-promoting properties. Sesamin feeding suppresses several aging-related phenotypes such as age-dependent accumulation of damaged proteins in the muscles and neuronal loss in the brains of Drosophila adults with high levels of reactive oxygen species. Sesamin promotes the transcription of several genes that are responsible for oxidative stress, although the underlying mechanism remains unclear. This study aimed to demonstrate that sesamin mediates its action through activation of a transcription factor, Nrf2 (Cnc in Drosophila), essential for anti-aging oxidative stress response. Nrf2/Cnc activation was determined using the antioxidant response element, Green Fluorescence Protein reporter, that can monitor Nrf2/Cnc-dependent transcription. Strong fluorescence was observed in the entire bodies, particularly in the abdomens and brains, of adult flies fed sesamin. Interestingly, Nrf2/Cnc was strongly activated in neuronal cells, especially in several neuron types, including glutamatergic and cholinergic, and some dopaminergic and/or serotonergic neurons but not in GABAergic neurons or the mushroom bodies of flies fed sesamin. These results indicate that the anti-aging effects of sesamin are exerted via activation of Nrf2/Cnc-dependent transcription to circumvent oxidative stress accumulation in several types of neurons of adult brains. Sesamin could be explored as a potential dietary supplement for preventing neurodegeneration associated with accumulation of oxidative stress.
Shukla, A. K., Johnson, K. and Giniger, E. (2021). Common features of aging fail to occur in Drosophila raised without a bacterial microbiome. iScience 24(7): 102703. PubMed ID: 34235409
Summary:
Lifespan is limited both by intrinsic decline in vigor with age and by accumulation of external insults. There exists a general picture of the deficits of aging, one that is reflected in a pattern of age-correlated changes in gene expression conserved across species. By comparing gene expression profiling of Drosophila raised either conventionally, or free of bacteria, this study shows that ∼70% of these conserved, age-associated changes in gene expression fail to occur in germ-free flies. Among the processes that fail to show time-dependent change under germ-free conditions are two aging features that are observed across phylogeny, declining expression of stress response genes and increasing expression of innate immune genes. These comprise adaptive strategies the organism uses to respond to bacteria, rather than being inevitable components of age-dependent decline. Changes in other processes are independent of the microbiome and can serve as autonomous markers of aging of the individual.
Lika, J., Katzenberger, R. J., Ganetzky, B. and Wassarman, D. A. (2021). Effects of blunt force injuries in third-instar Drosophila larvae persist through metamorphosis and reduce adult lifespan. MicroPubl Biol 2021. PubMed ID: 34278243
Summary:
Blunt force injuries are a significant cause of disability and death worldwide. This study describes a Drosophila melanogaster model of blunt force injury that can be used to investigate cellular and molecular mechanisms that underlie the short-term and long-term effects of injuries sustained at a juvenile stage of development. Injuries inflicted in late third-instar larvae using the spring-based High-Impact Trauma (HIT) device robustly activated the humoral defense response process of melanization and caused larval and pupal lethality. Additionally, adults that developed from injured larvae had reduced lifespans, indicating that cellular and molecular mechanisms activated by blunt force injuries in larvae persist through metamorphosis and adult development. Previously, the HIT device has been used to investigate genetic and environmental factors underlying mechanisms that contribute to consequences of blunt force injuries incurred in adult flies. This work expands use of the HIT device to a juvenile stage of development, offering the opportunity to investigate whether the consequences of blunt force injuries involve different factors and mechanisms at different stages of development.

Monday, August 16th - Embryonic Development

Le, T. P. and Chung, S. (2021). Regulation of apical constriction via microtubule- and Rab11-dependent apical transport during tissue invagination. Mol Biol Cell 32(10): 1033-1047. PubMed ID: 33788621
Summary:
The formation of an epithelial tube is a fundamental process for organogenesis. During Drosophila embryonic salivary gland (SG) invagination, Folded gastrulation (Fog)-dependent Rho-associated kinase (Rok) promotes contractile apical myosin formation to drive apical constriction. Microtubules (MTs) are also crucial for this process and are required for forming and maintaining apicomedial myosin. However, the underlying mechanism that coordinates actomyosin and MT networks still remains elusive. This study shows that MT-dependent intracellular trafficking regulates apical constriction during SG invagination. Key components involved in protein trafficking, such as Rab11 and Nuclear fallout (Nuf), are apically enriched near the SG invagination pit in a MT-dependent manner. Disruption of the MT networks or knockdown of Rab11 impairs apicomedial myosin formation and apical constriction. MTs and Rab11 are required for apical enrichment of the Fog ligand and the continuous distribution of the apical determinant protein Crumbs (Crb) and the key adherens junction protein E-Cadherin (E-Cad) along junctions. Targeted knockdown of crb or E-Cad in the SG disrupts apical myosin networks and results in apical constriction defects. These data suggest a role of MT- and Rab11-dependent intracellular trafficking in regulating actomyosin networks and cell junctions to coordinate cell behaviors during tubular organ formation.
Yadav, V., Tolwinski, N. and Saunders, T. E. (2021). Spatiotemporal sensitivity of mesoderm specification to FGFR signalling in the Drosophila embryo. Sci Rep 11(1): 14091. PubMed ID: 34238963
Summary:
Development of the Drosophila embryonic mesoderm is controlled through both internal and external inputs to the mesoderm. One such factor is Heartless (Htl), a Fibroblast Growth Factor Receptor (FGFR) expressed in the mesoderm. Although Htl has been extensively studied, the dynamics of its action are poorly understood after the initial phases of mesoderm formation and spreading. To begin to address this challenge, an optogenetic version of the FGFR Heartless was developed in Drosophila (Opto-htl). Opto-htl enables activation of the FGFR pathway in selective spatial (~ 35 μ section from one of the lateral sides of the embryo) and in temporal domains (ranging from 40 min to 14 h) during embryogenesis. Importantly, the effects can be tuned by the intensity of light-activation, making this approach significantly more flexible than other genetic approaches. Controlled perturbations to the FGFR pathway were performed to define the contribution of Htl signalling to the formation of the developing embryonic heart and somatic muscles. A direct correlation was found between Htl signalling dosage and number of Tinman-positive heart cells specified. Opto-htl activation favours the specification of Tinman positive cardioblasts and eliminates Eve-positive DA1 muscles. This effect is seen to increase progressively with increasing light intensity. Therefore, fine tuning of phenotypic responses to varied Htl signalling dosage can be achieved more conveniently than with other genetic approaches. Overall, Opto-htl is a powerful new tool for dissecting the role of FGFR signalling during development.
Holcomb, M. C., Gao, G. J., Servati, M., Schneider, D., McNeely, P. K., Thomas, J. H. and Blawzdziewicz, J. (2021). Mechanical feedback and robustness of apical constrictions in Drosophila embryo ventral furrow formation. PLoS Comput Biol 17(7): e1009173. PubMed ID: 34228708
Summary:
Formation of the ventral furrow in the Drosophila embryo relies on the apical constriction of cells in the ventral region to produce bending forces that drive tissue invagination. In a recent paper it was observed that apical constrictions during the initial phase of ventral furrow formation produce elongated patterns of cellular constriction chains prior to invagination; it was argued that these are indicative of tensile stress feedback. This study quantitatively analyzed the constriction patterns preceding ventral furrow formation and found that they are consistent with the predictions of an active-granular-fluid model of a monolayer of mechanically coupled stress-sensitive constricting particles. The model shows that tensile feedback causes constriction chains to develop along underlying precursor tensile stress chains that gradually strengthen with subsequent cellular constrictions. As seen in both this model and available optogenetic experiments, this mechanism allows constriction chains to penetrate or circumvent zones of reduced cell contractility, thus increasing the robustness of ventral furrow formation to spatial variation of cell contractility by rescuing cellular constrictions in the disrupted regions.
Yoo, B., Kim, H. Y., Chen, X., Shen, W., Jang, J. S., Stein, S. N., Cormier, O., Pereira, L., Shih, C. R. Y., Krieger, C., Reed, B., Harden, N. and Wang, S. J. H. (2021). 20-hydroxyecdysone (20E) signaling regulates amnioserosa morphogenesis during Drosophila dorsal closure: EcR modulates gene expression in a complex with the AP-1 subunit, Jun. Biol Open. PubMed ID: 34296248
Summary:
Steroid hormones influence diverse biological processes throughout the animal life cycle, including metabolism, stress resistance, reproduction, and lifespan. In insects, the steroid hormone, 20-hydroxyecdysone (20E), is the central hormone regulator of molting and metamorphosis, and plays roles in tissue morphogenesis. For example, amnioserosa contraction, which is a major driving force in Drosophila dorsal closure (DC), is defective in embryos mutant for 20E biosynthesis. This study shows that 20E signaling modulates the transcription of several DC participants in the amnioserosa and other dorsal tissues during late embryonic development, including zipper, which encodes for non-muscle myosin. Canonical ecdysone signaling typically involves the binding of Ecdysone receptor (EcR) and Ultraspiracle heterodimers to ecdysone-response elements (EcREs) within the promoters of responsive genes to drive expression. Evidence that 20E signaling instead acts in parallel to the JNK cascade via a direct interaction between EcR and the AP-1 transcription factor subunit, Jun, which together binds to genomic regions containing AP-1 binding sites but no EcREs to control gene expression. This work demonstrates a novel mode of action for 20E signaling in Drosophila that likely functions beyond DC, and may provide further insights into mammalian steroid hormone receptor interactions with AP-1.
Duan, J., Rieder, L., Colonnetta, M. M., Huang, A., McKenney, M., Watters, S., Deshpande, G., Jordan, W., Fawzi, N. and Larschan, E. (2021). CLAMP and Zelda function together to promote Drosophila zygotic genome activation. Elife 10. PubMed ID: 34342574
Summary:
During the essential and conserved process of zygotic genome activation (ZGA), chromatin accessibility must increase to promote transcription. Drosophila is a well-established model for defining mechanisms that drive ZGA. Zelda (ZLD) is a key pioneer transcription factor (TF) that promotes ZGA in the Drosophila embryo. However, many genomic loci that contain GA-rich motifs become accessible during ZGA independent of ZLD. Therefore, it was hypothesized that other early TFs that function with ZLD have not yet been identified, especially those that are capable of binding to GA-rich motifs such as CLAMP. This study demonstrated that Drosophila embryonic development requires maternal CLAMP to: 1) activate zygotic transcription; 2) increase chromatin accessibility at promoters of specific genes that often encode other essential TFs; 3) enhance chromatin accessibility and facilitate ZLD occupancy at a subset of key embryonic promoters. Thus, CLAMP functions as a pioneer factor which plays a targeted yet essential role in ZGA.
Sharma, S. and Rikhy, R. (2021). Spatiotemporal recruitment of RhoGTPase protein GRAF inhibits actomyosin ring constriction in Drosophila cellularization. Elife 10. PubMed ID: 33835025
Summary:
Actomyosin contractility is regulated by Rho-GTP in cell migration, cytokinesis and morphogenesis in embryo development. Whereas Rho activation by Rho-GTP exchange factor (GEF), RhoGEF2, is well known in actomyosin contractility during cytokinesis at the base of invaginating membranes in Drosophila cellularization, Rho inhibition by RhoGTPase-activating proteins (GAPs) remains to be studied. This study found that the RhoGAP, GRAF, inhibits actomyosin contractility during cellularization. GRAF is enriched at the cleavage furrow tip during actomyosin assembly and initiation of ring constriction. Graf depletion shows increased Rho-GTP, increased Myosin II and ring hyper-constriction dependent upon the loss of the RhoGTPase domain. GRAF and RhoGEF2 are present in a balance for appropriate activation of actomyosin ring constriction. RhoGEF2 depletion and abrogation of Myosin II activation in Rho kinase mutants suppress the Graf hyper-constriction defect. Therefore, GRAF recruitment restricts Rho-GTP levels in a spatiotemporal manner for inhibiting actomyosin contractility during cellularization.

Friday, August 13th - Signaling

Le, T. P. and Chung, S. (2021). Regulation of apical constriction via microtubule- and Rab11-dependent apical transport during tissue invagination. Mol Biol Cell 32(10): 1033-1047. PubMed ID: 33788621
Summary:
The formation of an epithelial tube is a fundamental process for organogenesis. During Drosophila embryonic salivary gland (SG) invagination, Folded gastrulation (Fog)-dependent Rho-associated kinase (Rok) promotes contractile apical myosin formation to drive apical constriction. Microtubules (MTs) are also crucial for this process and are required for forming and maintaining apicomedial myosin. However, the underlying mechanism that coordinates actomyosin and MT networks still remains elusive. This study shows that MT-dependent intracellular trafficking regulates apical constriction during SG invagination. Key components involved in protein trafficking, such as Rab11 and Nuclear fallout (Nuf), are apically enriched near the SG invagination pit in a MT-dependent manner. Disruption of the MT networks or knockdown of Rab11 impairs apicomedial myosin formation and apical constriction. MTs and Rab11 are required for apical enrichment of the Fog ligand and the continuous distribution of the apical determinant protein Crumbs (Crb) and the key adherens junction protein E-Cadherin (E-Cad) along junctions. Targeted knockdown of crb or E-Cad in the SG disrupts apical myosin networks and results in apical constriction defects. These data suggest a role of MT- and Rab11-dependent intracellular trafficking in regulating actomyosin networks and cell junctions to coordinate cell behaviors during tubular organ formation.
Yadav, V., Tolwinski, N. and Saunders, T. E. (2021). Spatiotemporal sensitivity of mesoderm specification to FGFR signalling in the Drosophila embryo. Sci Rep 11(1): 14091. PubMed ID: 34238963
Summary:
Development of the Drosophila embryonic mesoderm is controlled through both internal and external inputs to the mesoderm. One such factor is Heartless (Htl), a Fibroblast Growth Factor Receptor (FGFR) expressed in the mesoderm. Although Htl has been extensively studied, the dynamics of its action are poorly understood after the initial phases of mesoderm formation and spreading. To begin to address this challenge, an optogenetic version of the FGFR Heartless was developed in Drosophila (Opto-htl). Opto-htl enables activation of the FGFR pathway in selective spatial (~ 35 μ section from one of the lateral sides of the embryo) and in temporal domains (ranging from 40 min to 14 h) during embryogenesis. Importantly, the effects can be tuned by the intensity of light-activation, making this approach significantly more flexible than other genetic approaches. Controlled perturbations to the FGFR pathway were performed to define the contribution of Htl signalling to the formation of the developing embryonic heart and somatic muscles. A direct correlation was found between Htl signalling dosage and number of Tinman-positive heart cells specified. Opto-htl activation favours the specification of Tinman positive cardioblasts and eliminates Eve-positive DA1 muscles. This effect is seen to increase progressively with increasing light intensity. Therefore, fine tuning of phenotypic responses to varied Htl signalling dosage can be achieved more conveniently than with other genetic approaches. Overall, Opto-htl is a powerful new tool for dissecting the role of FGFR signalling during development.
Holcomb, M. C., Gao, G. J., Servati, M., Schneider, D., McNeely, P. K., Thomas, J. H. and Blawzdziewicz, J. (2021). Mechanical feedback and robustness of apical constrictions in Drosophila embryo ventral furrow formation. PLoS Comput Biol 17(7): e1009173. PubMed ID: 34228708
Summary:
Formation of the ventral furrow in the Drosophila embryo relies on the apical constriction of cells in the ventral region to produce bending forces that drive tissue invagination. In a recent paper it was observed that apical constrictions during the initial phase of ventral furrow formation produce elongated patterns of cellular constriction chains prior to invagination; it was argued that these are indicative of tensile stress feedback. This study quantitatively analyzed the constriction patterns preceding ventral furrow formation and found that they are consistent with the predictions of an active-granular-fluid model of a monolayer of mechanically coupled stress-sensitive constricting particles. The model shows that tensile feedback causes constriction chains to develop along underlying precursor tensile stress chains that gradually strengthen with subsequent cellular constrictions. As seen in both this model and available optogenetic experiments, this mechanism allows constriction chains to penetrate or circumvent zones of reduced cell contractility, thus increasing the robustness of ventral furrow formation to spatial variation of cell contractility by rescuing cellular constrictions in the disrupted regions.
Yoo, B., Kim, H. Y., Chen, X., Shen, W., Jang, J. S., Stein, S. N., Cormier, O., Pereira, L., Shih, C. R. Y., Krieger, C., Reed, B., Harden, N. and Wang, S. J. H. (2021). 20-hydroxyecdysone (20E) signaling regulates amnioserosa morphogenesis during Drosophila dorsal closure: EcR modulates gene expression in a complex with the AP-1 subunit, Jun. Biol Open. PubMed ID: 34296248
Summary:
Steroid hormones influence diverse biological processes throughout the animal life cycle, including metabolism, stress resistance, reproduction, and lifespan. In insects, the steroid hormone, 20-hydroxyecdysone (20E), is the central hormone regulator of molting and metamorphosis, and plays roles in tissue morphogenesis. For example, amnioserosa contraction, which is a major driving force in Drosophila dorsal closure (DC), is defective in embryos mutant for 20E biosynthesis. This study showd that 20E signaling modulates the transcription of several DC participants in the amnioserosa and other dorsal tissues during late embryonic development, including zipper, which encodes for non-muscle myosin. Canonical ecdysone signaling typically involves the binding of Ecdysone receptor (EcR) and Ultraspiracle heterodimers to ecdysone-response elements (EcREs) within the promoters of responsive genes to drive expression. During DC, however, evidence is provided that 20E signaling instead acts in parallel to the JNK cascade via a direct interaction between EcR and the AP-1 transcription factor subunit, Jun, which together binds to genomic regions containing AP-1 binding sites but no EcREs to control gene expression. This work demonstrates a novel mode of action for 20E signaling in Drosophila that likely functions beyond DC, and may provide further insights into mammalian steroid hormone receptor interactions with AP-1.
Duan, J., Rieder, L., Colonnetta, M. M., Huang, A., McKenney, M., Watters, S., Deshpande, G., Jordan, W., Fawzi, N. and Larschan, E. (2021). CLAMP and Zelda function together to promote Drosophila zygotic genome activation. Elife 10. PubMed ID: 34342574
Summary:
During the essential and conserved process of zygotic genome activation (ZGA), chromatin accessibility must increase to promote transcription. Drosophila is a well-established model for defining mechanisms that drive ZGA. Zelda (ZLD) is a key pioneer transcription factor (TF) that promotes ZGA in the Drosophila embryo. However, many genomic loci that contain GA-rich motifs become accessible during ZGA independent of ZLD. Therefore, it was hypothesized that other early TFs that function with ZLD have not yet been identified, especially those that are capable of binding to GA-rich motifs such as CLAMP. This study demonstrated that Drosophila embryonic development requires maternal CLAMP to: 1) activate zygotic transcription; 2) increase chromatin accessibility at promoters of specific genes that often encode other essential TFs; 3) enhance chromatin accessibility and facilitate ZLD occupancy at a subset of key embryonic promoters. Thus, CLAMP functions as a pioneer factor which plays a targeted yet essential role in ZGA.
Sharma, S. and Rikhy, R. (2021). Spatiotemporal recruitment of RhoGTPase protein GRAF inhibits actomyosin ring constriction in Drosophila cellularization. Elife 10. PubMed ID: 33835025
Summary:
Actomyosin contractility is regulated by Rho-GTP in cell migration, cytokinesis and morphogenesis in embryo development. Whereas Rho activation by Rho-GTP exchange factor (GEF), RhoGEF2, is well known in actomyosin contractility during cytokinesis at the base of invaginating membranes in Drosophila cellularization, Rho inhibition by RhoGTPase-activating proteins (GAPs) remains to be studied. This study found that the RhoGAP, GRAF, inhibits actomyosin contractility during cellularization. GRAF is enriched at the cleavage furrow tip during actomyosin assembly and initiation of ring constriction. Graf depletion shows increased Rho-GTP, increased Myosin II and ring hyper-constriction dependent upon the loss of the RhoGTPase domain. GRAF and RhoGEF2 are present in a balance for appropriate activation of actomyosin ring constriction. RhoGEF2 depletion and abrogation of Myosin II activation in Rho kinase mutants suppress the Graf hyper-constriction defect. Therefore, GRAF recruitment restricts Rho-GTP levels in a spatiotemporal manner for inhibiting actomyosin contractility during cellularization.

Friday, August 13th - Signaling

Witte, L., Linnemannstons, K., Honemann-Capito, M. and Gross, J. C. (2021). Visualization and Quantitation of Wg trafficking in the Drosophila Wing Imaginal Epithelium. Bio Protoc 11(11): e4040. PubMed ID: 34250206
Summary:
Secretory Wnt trafficking can be studied in the polarized epithelial monolayer of Drosophila wing imaginal discs (WID). In this tissue, Wg (Drosophila Wnt-I) is presented on the apical surface of its source cells before being internalized into the endosomal pathway. Long-range Wg secretion and spread depend on secondary secretion from endosomal compartments, but the exact post-endocytic fate of Wg is poorly understood. This paper summarizes and presents three protocols for the immunofluorescence-based visualization and quantitation of different pools of intracellular and extracellular Wg in WID: (1) steady-state extracellular Wg; (2) dynamic Wg trafficking inside endosomal compartments; and (3) dynamic Wg release to the cell surface. Using a genetic driver system for gene manipulation specifically at the posterior part of the WID (EnGal4) provides a robust internal control that allows for direct comparison of signal intensities of control and manipulated compartments of the same WID. Therefore, it also circumvents the high degree of staining variability usually associated with whole-tissue samples. In combination with the genetic manipulation of Wg pathway components that is easily feasible in Drosophila, these methods provide a tool-set for the dissection of secretory Wg trafficking and can help understanding of how Wnt proteins travel along endosomal compartments for short- and long-range signal secretion.
Xiao, Y., Yuan, Y., Jimenez, M., Soni, N. and Yadlapalli, S. (2021). Clock proteins regulate spatiotemporal organization of clock genes to control circadian rhythms. Proc Natl Acad Sci U S A 118(28). PubMed ID: 34234015
Summary:
Circadian clocks regulate ∼24-h oscillations in gene expression, behavior, and physiology. This study has elucidated how spatiotemporal organization and dynamics of core clock proteins and genes affect circadian rhythms in Drosophila clock neurons. Using high-resolution imaging and DNA-fluorescence in situ hybridization techniques, it was demonstrate that Drosophila clock proteins (PERIOD and CLOCK) are organized into a few discrete foci at the nuclear envelope during the circadian repression phase and play an important role in the subnuclear localization of core clock genes to control circadian rhythms. Specifically, this study showed that core clock genes, period and timeless, are positioned close to the nuclear periphery by the PERIOD protein specifically during the repression phase, suggesting that subnuclear localization of core clock genes might play a key role in their rhythmic gene expression. Finally, loss of Lamin B receptor, a nuclear envelope protein, was shown to leads to disruption of PER foci and per gene peripheral localization and results in circadian rhythm defects. These results demonstrate that clock proteins play a hitherto unexpected role in the subnuclear reorganization of core clock genes to control circadian rhythms, revealing how clocks function at the subcellular level. The results further suggest that clock protein foci might regulate dynamic clustering and spatial reorganization of clock-regulated genes over the repression phase to control circadian rhythms in behavior and physiology.
Wada, Y., Ohsawa, S. and Igaki, T. (2021). Yorkie ensures robust tissue growth in Drosophila ribosomal protein mutants. Development. PubMed ID: 34228792
Summary:
Heterozygosity of a ribosomal protein gene causes a variety of developmental abnormalities in humans, which are collectively known as ribosomopathies, yet the underlying mechanisms remain elusive. This study analyzed Drosophila mutants heterozygous for a ribosomal protein gene, called Minute (M)/+ mutants. While M/+ flies develop essentially normal wings, simultaneous deletion of one copy of the Hippo pathway effector yki resulted in severe wing growth defects. These defects were caused by JNK-mediated cell death in the wing pouch via Eiger/TNF signaling. The JNK activation in M/+, yki/+ wing discs required a caspase Dronc, which is normally blocked by DIAP. Notably, heterozygosity of yki reduced DIAP1 expression in the wing pouch, leading to elevation of Dronc activity. Dronc and JNK formed a positive feedback loop that amplifies Dronc activation, leading to apoptosis. These observations suggest a novel mechanism of robust tissue growth whereby tissues with reduced ribosomal protein prevent ectopic apoptosis via Yki activity.
Kietz, C., Mohan, A. K., Pollari, V., Tuominen, I. E., Ribeiro, P. S., Meier, P. and Meinander, A. (2021). Drice restrains Diap2-mediated inflammatory signalling and intestinal inflammation. Cell Death Differ. PubMed ID: 34262145
Summary:
The Drosophila IAP protein, Diap2, is a key mediator of NF-κB signalling and innate immune responses. Diap2 is required for both local immune activation, taking place in the epithelial cells of the gut and trachea, and for mounting systemic immune responses in the cells of the fat body. This study has found that transgenic expression of Diap2 leads to a spontaneous induction of NF-κB target genes, inducing chronic inflammation in the Drosophila midgut, but not in the fat body. Drice is a Drosophila effector caspase known to interact and form a stable complex with Diap2. This complex formation was found to induce its subsequent degradation, thereby regulating the amount of Diap2 driving NF-κB signalling in the intestine. Concordantly, loss of Drice activity leads to accumulation of Diap2 and to chronic intestinal inflammation. Interestingly, Drice does not interfere with pathogen-induced signalling, suggesting that it protects from immune responses induced by resident microbes. Accordingly, no inflammation was detected in transgenic Diap2 flies and Drice-mutant flies reared in axenic conditions. Hence, this study shows that Drice, by restraining Diap2, halts unwanted inflammatory signalling in the intestine.
O'Connor, J. T., Stevens, A. C., Shannon, E. K., Akbar, F. B., LaFever, K. S., Narayanan, N. P., Gailey, C. D., Hutson, M. S. and Page-McCaw, A. (2021). Proteolytic activation of Growth-blocking peptides triggers calcium responses through the GPCR Mthl10 during epithelial wound detection. Dev Cell. PubMed ID: 34273275
Summary:
The presence of a wound triggers surrounding cells to initiate repair mechanisms, but it is not clear how cells initially detect wounds. In epithelial cells, the earliest known wound response, occurring within seconds, is a dramatic increase in cytosolic calcium. This study shows that wounds in the Drosophila notum trigger cytoplasmic calcium increase by activating extracellular cytokines, Growth-blocking peptides (Gbps), which initiate signaling in surrounding epithelial cells through the G-protein-coupled receptor Methuselah-like 10 (Mthl10). Latent Gbps are present in unwounded tissue and are activated by proteolytic cleavage. Using wing discs, this study showed that multiple protease families can activate Gbps, suggesting that they act as a generalized protease-detector system. Experimental and computational evidence is presented that proteases released during wound-induced cell damage and lysis serve as the instructive signal: these proteases liberate Gbp ligands, which bind to Mthl10 receptors on surrounding epithelial cells, and activate downstream release of calcium.
Liu, X., Blazenovix, I., Contreras, A. J., Pham, T. M., Tabuloc, C. A., Li, Y. H., Ji, J., Fiehn, O. and Chiu, J. C. (2021). Hexosamine biosynthetic pathway and O-GlcNAc-processing enzymes regulate daily rhythms in protein O-GlcNAcylation. Nat Commun 12(1): 4173. PubMed ID: 34234137
Summary:
The integration of circadian and metabolic signals is essential for maintaining robust circadian rhythms and ensuring efficient metabolism and energy use. Using Drosophila as an animal model, this study shows that cellular protein O-GlcNAcylation exhibits robust 24-hour rhythm and represents a key post-translational mechanism that regulates circadian physiology. Strong correlation was observed between protein O-GlcNAcylation rhythms and clock-controlled feeding-fasting cycles, suggesting that O-GlcNAcylation rhythms are primarily driven by nutrient input. Interestingly, daily O-GlcNAcylation rhythms are severely dampened when flies are subjected to time-restricted feeding at unnatural feeding time. This suggests the presence of clock-regulated buffering mechanisms that prevent excessive O-GlcNAcylation at non-optimal times of the day-night cycle. This buffering mechanism is mediated by the expression and activity of GFAT, OGT, and OGA, which are regulated through integration of circadian and metabolic signals. Finally, a mathematical model was generated to describe the key factors that regulate daily O-GlcNAcylation rhythm.

Wednesday, August 12th - Evolution

Xia, S., VanKuren, N. W., Chen, C., Zhang, L., Kemkemer, C., Shao, Y., Jia, H., Lee, U., Advani, A. S., Gschwend, A., Vibranovski, M. D., Chen, S., Zhang, Y. E. and Long, M. (2021). Genomic analyses of new genes and their phenotypic effects reveal rapid evolution of essential functions in Drosophila development. PLoS Genet 17(7): e1009654. PubMed ID: 34242211
Summary:
It is a conventionally held dogma that the genetic basis underlying development is conserved in a long evolutionary time scale. Ample experiments based on mutational, biochemical, functional, and complementary knockdown/knockout approaches have revealed the unexpectedly important role of recently evolved new genes in the development of Drosophila. The recent progress in the genome-wide experimental testing of gene effects and improvements in the computational identification of new genes (< 40 million years ago, Mya) open the door to investigate the evolution of gene essentiality with a phylogenetically high resolution. These advancements also raised interesting issues in techniques and concepts related to phenotypic effect analyses of genes, particularly of those that recently originated. This study reports analyses of these issues, including reproducibility and efficiency of knockdown experiment and difference between RNAi libraries in the knockdown efficiency and testing of phenotypic effects. This study reports a large data from knockdowns of 11,354 genes (~75% of the Drosophila melanogaster total genes), including 702 new genes (~66% of the species total new genes that aged < 40 Mya), revealing a similarly high proportion (~32.2%) of essential genes that originated in various Sophophora subgenus lineages and distant ancestors beyond the Drosophila genus. The transcriptional compensation effect from CRISPR knockout were detected for highly similar duplicate copies. Knockout of a few young genes detected analogous essentiality in various functions in development. Taken together, this experimental and computational analyses provide valuable data for detection of phenotypic effects of genes in general and further strong evidence for the concept that new genes in Drosophila quickly evolved essential functions in viability during development.
Lima, L. F., Torres, A. Q., Jardim, R., Mesquita, R. D. and Schama, R. (2021). Evolution of Toll, Spatzle and MyD88 in insects: the problem of the Diptera bias. BMC Genomics 22(1): 562. PubMed ID: 34289811
Summary:
Arthropoda, the most numerous and diverse metazoan phylum, has species in many habitats where they encounter various microorganisms and, as a result, mechanisms for pathogen recognition and elimination have evolved. The Toll pathway, involved in the innate immune system, was first described as part of the developmental pathway for dorsal-ventral differentiation in Drosophila. This study evaluated the diversity of Toll pathway gene families in 39 Arthropod genomes, encompassing 13 different Insect Orders. Through computational methods, this study sheds some light into the evolution and functional annotation of protein families involved in the Toll pathway innate immune response. The data indicates that: 1) intracellular proteins of the Toll pathway show mostly species-specific expansions; 2) the different Toll subfamilies seem to have distinct evolutionary backgrounds; 3) patterns of gene expansion observed in the Toll phylogenetic tree indicate that homology based methods of functional inference might not be accurate for some subfamilies; 4) Spatzle subfamilies are highly divergent and also pose a problem for homology based inference; 5) Spatzle subfamilies should not be analyzed together in the same phylogenetic framework; 6) network analyses seem to be a good first step in inferring functional groups in these cases. It was specifically shown that understanding Drosophila's Toll functions might not indicate the same function in other species. These results show the importance of using species representing the different orders to better understand insect gene content, origin and evolution. More specifically, in intracellular Toll pathway gene families the presence of orthologues has important implications for homology based functional inference. Also, the different evolutionary backgrounds of Toll gene subfamilies should be taken into consideration when functional studies are performed, especially for TOLL9, TOLL, TOLL2_7, and the new TOLL10 clade. The presence of Diptera specific clades or the ones lacking Diptera species show the importance of overcoming the Diptera bias when performing functional characterization of Toll pathways.
Otte, K. A., Nolte, V., Mallard, F. and Schlotterer, C. (2021). The genetic architecture of temperature adaptation is shaped by population ancestry and not by selection regime. Genome Biol 22(1): 211. PubMed ID: 34271951
Summary:
Understanding the genetic architecture of temperature adaptation is key for characterizing and predicting the effect of climate change on natural populations. One particularly promising approach is Evolve and Resequence, which combines advantages of experimental evolution such as time series, replicate populations, and controlled environmental conditions, with whole genome sequencing. Recent analysis of replicate populations from two different Drosophila simulans founder populations, which were adapting to the same novel hot environment, uncovered very different architectures-either many selection targets with large heterogeneity among replicates or fewer selection targets with a consistent response among replicates. This study exposed the founder population from Portugal to a cold temperature regime. Although almost no selection targets are shared between the hot and cold selection regime, the adaptive architecture was similar. A moderate number was detected of targets under strong selection (19 selection targets, mean selection coefficient = 0.072) and parallel responses in the cold evolved replicates. This similarity across different environments indicates that the adaptive architecture depends more on the ancestry of the founder population than the specific selection regime. These observations will have broad implications for the correct interpretation of the genomic responses to a changing climate in natural populations.
Huang, Y., Lack, e. B., Hoppel, G. T. and Pool, J. E. (2021). Parallel and Population-specific Gene Regulatory Evolution in Cold-Adapted Fly Populations. Genetics. PubMed ID: 33989401
Summary:
hanges in gene regulation at multiple levels may comprise an important share of the molecular changes underlying adaptive evolution in nature. However, few studies have assayed within- and between-population variation in gene regulatory traits at a transcriptomic scale, and therefore inferences about the characteristics of adaptive regulatory changes have been elusive. This study assessed quantitative trait differentiation in gene expression levels and alternative splicing (intron usage) between three closely-related pairs of natural populations of Drosophila melanogaster from contrasting thermal environments that reflect three separate instances of cold tolerance evolution. The cold-adapted populations were known to show population genetic evidence for parallel evolution at the SNP level, and this study found evidence for parallel expression evolution between them, with stronger parallelism at larval and adult stages than for pupae. A flexible method was implemented to estimate cis- versus trans-encoded contributions to expression or splicing differences at the adult stage. The apparent contributions of cis- versus trans-regulation to adaptive evolution vary substantially among population pairs. While two of three population pairs show a greater enrichment of cis-regulatory differences among adaptation candidates, trans-regulatory differences are more likely to be implicated in parallel expression changes between population pairs. Genes with significant cis-effects are enriched for signals of elevated genetic differentiation between cold- and warm-adapted populations, suggesting that they are potential targets of local adaptation. These findings expand knowledge of adaptive gene regulatory evolution and the ability to make inferences about this important and widespread process.
Kim, B. Y., Wang, J., Miller, D. E., ..., Kopp, A., Matute, D. R. and Petrov, D. A. (2021). Highly contiguous assemblies of 101 drosophilid genomes. Elife 10. PubMed ID: 34279216
Summary:
Over 100 years of studies in Drosophila melanogaster and related species in the genus Drosophila have facilitated key discoveries in genetics, genomics, and evolution. While high-quality genome assemblies exist for several species in this group, they only encompass a small fraction of the genus. Recent advances in long-read sequencing allow high-quality genome assemblies for tens or even hundreds of species to be efficiently generated. This study utilized Oxford Nanopore sequencing to build an open community resource of genome assemblies for 101 lines of 93 drosophilid species encompassing 14 species groups and 35 sub-groups. The genomes are highly contiguous and complete, with an average contig N50 of 10.5 Mb and greater than 97% BUSCO completeness in 97/101 assemblies. This study shows that Nanopore-based assemblies are highly accurate in coding regions, particularly with respect to coding insertions and deletions. These assemblies, along with a detailed laboratory protocol and assembly pipelines, are released as a public resource and will serve as a starting point for addressing broad questions of genetics, ecology, and evolution at the scale of hundreds of species.
Liu, J., Viales, R. R., Khoueiry, P., Reddington, J. P., Girardot, C., Furlong, E. and Robinson-Rechavi, M. (2021). The hourglass model of evolutionary conservation during embryogenesis extends to developmental enhancers with signatures of positive selection. Genome Res. PubMed ID: 34266978
Summary:
Inter-species comparisons of both morphology and gene expression within a phylum have revealed a period in the middle of embryogenesis with more similarity between species compared to earlier and later time-points. This "developmental hourglass" pattern has been observed in many phyla, yet the evolutionary constraints on gene expression, and underlying mechanisms of how this is regulated, remains elusive. Moreover, the role of positive selection on gene regulation in the more diverged earlier and later stages of embryogenesis remains unknown. Using DNase-seq to identify regulatory regions in two distant Drosophila species (D. melanogaster and D. virilis), this study assessed the evolutionary conservation and adaptive evolution of enhancers throughout multiple stages of embryogenesis. This revealed a higher proportion of conserved enhancers at the phylotypic period, providing a regulatory basis for the hourglass expression pattern. Using an in silico mutagenesis approach, signatures of positive selection on developmental enhancers were detected at early and late stages of embryogenesis, with a depletion at the phylotypic period, suggesting positive selection as one evolutionary mechanism underlying the hourglass pattern of animal evolution.

Tuesday, August 10th - Adult neural development and function

Wiggin, T. D., Hsiao, Y., Liu, J. B., Huber, R. and Griffith, L. C. (2021). Rest Is Required to Learn an Appetitively-Reinforced Operant Task in Drosophila. Front Behav Neurosci 15: 681593. PubMed ID: 34220464
Summary:
Maladaptive operant conditioning contributes to development of neuropsychiatric disorders. Candidate genes have been identified that contribute to this maladaptive plasticity, but the neural basis of operant conditioning in genetic model organisms remains poorly understood. The fruit fly Drosophila melanogaster is a versatile genetic model organism that readily forms operant associations with punishment stimuli. However, operant conditioning with a food reward has not been demonstrated in flies, limiting the types of neural circuits that can be studied. This study presents the first sucrose-reinforced operant conditioning paradigm for flies. In the paradigm, flies walk along a Y-shaped track with reward locations at the terminus of each hallway. When flies turn in the reinforced direction at the center of the track, they receive a sucrose reward at the end of the hallway. Only flies that rest early in training learn the reward contingency normally. Flies rewarded independently of their behavior do not form a learned association but have the same amount of rest as trained flies, showing that rest is not driven by learning. Optogenetically-induced sleep does not promote learning, indicating that sleep itself is not sufficient for learning the operant task. The sensitivity of this assay to detect the effect of genetic manipulations was validated by testing the classic learning mutant dunce. Dunce flies are learning-impaired in the Y-Track task, indicating a likely role for cAMP in the operant coincidence detector. This novel training paradigm will provide valuable insight into the molecular mechanisms of disease and the link between sleep and learning.
Crocker, K. L., Marischuk, K., Rimkus, S. A., Zhou, H., Yin, J. C. P. and Boekhoff-Falk, G. (2021). Neurogenesis in the adult Drosophila brain. Genetics. PubMed ID: 34117750
Summary:
Neurodegenerative diseases such as Alzheimer's and Parkinson's currently affect ∼25 million people worldwide. The global incidence of traumatic brain injury (TBI) is estimated at ∼70 million/year. Both neurodegenerative diseases and TBI remain without effective treatments. This study utilized adult Drosophila melanogaster to investigate the mechanisms of brain regeneration with the long term goal of identifying targets for neural regenerative therapies. This study specifically focused on neurogenesis, i.e. the generation of new cells, as opposed to the regrowth of specific subcellular structures such as axons. Like mammals, Drosophila have few proliferating cells in the adult brain. Nonetheless, within 24 hours of a Penetrating Traumatic Brain Injury (PTBI) to the central brain, there is a significant increase in the number of proliferating cells. Both new glia and new neurons were detected, along with the formation of new axon tracts that target appropriate brain regions. Glial cells divide rapidly upon injury to give rise to new glial cells. Other cells near the injury site upregulate neural progenitor genes including asense and deadpan and later give rise to the new neurons. Locomotor abnormalities observed after PTBI are reversed within two weeks of injury, supporting the idea that there is functional recovery. Together, these data indicate that adult Drosophila brains are capable of neuronal repair. It is anticipated that this paradigm will facilitate the dissection of the mechanisms of neural regeneration and that these processes will be relevant to human brain repair.
Lee, K. and Doe, C. Q. (2021). A locomotor neural circuit persists and functions similarly in larvae and adult Drosophila. Elife 10. PubMed ID: 34259633
Summary:
Individual neurons can undergo drastic structural changes, known as neuronal remodeling or structural plasticity. One example of this is in response to hormones, such as during puberty in mammals or metamorphosis in insects. However, in each of these examples, it remains unclear whether the remodeled neuron resumes prior patterns of connectivity, and if so, whether the persistent circuits drive similar behaviors. This study utilized a well-characterized neural circuit in the Drosophila larva: the moonwalker descending neuron (MDN) circuit. Previously work showed that larval MDN induces backward crawling, and synapses onto the Pair1 interneuron to inhibit forward crawling. MDN is remodeled during metamorphosis and regulates backward walking in the adult fly. This study investigated whether Pair1 is remodeled during metamorphosis and functions within the MDN circuit during adulthood. Morphology and molecular markers were assayed to demonstrate that Pair1 is remodeled during metamorphosis and persists in the adult fly. MDN-Pair1 connectivity is lost during early pupal stages, when both neurons are severely pruned back, but connectivity is re-established at mid-pupal stages and persist into the adult. In the adult, optogenetic activation of Pair1 resulted in arrest of forward locomotion, similar to what is observed in larvae. Thus, the MDN-Pair1 neurons are an interneuronal circuit - a pair of synaptically connected interneurons - that is re-established during metamorphosis, yet generates similar locomotor behavior at both larval and adult stages.
Liao, M., Liang, X. and Howard, J. (2021). The narrowing of dendrite branches across nodes follows a well-defined scaling law. Proc Natl Acad Sci U S A 118(27). PubMed ID: 34215693
Summary:
The systematic variation of diameters in branched networks has tantalized biologists since the discovery of da Vinci's rule for trees. Da Vinci's rule can be formulated as a power law with exponent two: The square of the mother branch's diameter is equal to the sum of the squares of those of the daughters. Power laws, with different exponents, have been proposed for branching in circulatory systems (Murray's law with exponent 3) and in neurons (Rall's law with exponent 3/2). The laws have been derived theoretically, based on optimality arguments, but, for the most part, have not been tested rigorously. Using superresolution methods to measure the diameters of dendrites in highly branched Drosophila class IV sensory neurons, this study has found that these types of power laws do not hold. In their place, a different diameter-scaling law was developed: The cross-sectional area is proportional to the number of dendrite tips supported by the branch plus a constant, corresponding to a minimum diameter of the terminal dendrites. The area proportionality accords with a requirement for microtubules to transport materials and nutrients for dendrite tip growth. The minimum diameter may be set by the force, on the order of a few piconewtons, required to bend membrane into the highly curved surfaces of terminal dendrites. Because the observed scaling differs from Rall's law, it is proposed that cell biological constraints, such as intracellular transport and protrusive forces generated by the cytoskeleton, are important in determining the branched morphology of these cells.
Sareen, P. F., McCurdy, L. Y. and Nitabach, M. N. (2021). A neuronal ensemble encoding adaptive choice during sensory conflict in Drosophila. Nat Commun. 12(1):4131. PubMed ID: 34226544
Summary:
Feeding decisions are fundamental to survival, and decision making is often disrupted in disease. This study shows that neural activity in a small population of neurons projecting to the fan-shaped body higher-order central brain region of Drosophila represents food choice during sensory conflict. Food deprived flies made tradeoffs between appetitive and aversive values of food. An upstream neuropeptidergic and dopaminergic network was identified that relays internal state and other decision-relevant information to a specific subset of fan-shaped body neurons. These neurons were strongly inhibited by the taste of the rejected food choice, suggesting that they encode behavioral food choice. These findings reveal that fan-shaped body taste responses to food choices are determined not only by taste quality, but also by previous experience (including choice outcome) and hunger state, which are integrated in the fan-shaped body to encode the decision before relay to downstream motor circuits for behavioral implementation.
Hindmarsh Sten, T., Li, R., Otopalik, A. and Ruta, V. (2021). Sexual arousal gates visual processing during Drosophila courtship. Nature 595(7868): 549-553. PubMed ID: 34234348
Summary:
Long-lasting internal arousal states motivate and pattern ongoing behaviour, enabling the temporary emergence of innate behavioural programs that serve the needs of an animal, such as fighting, feeding, and mating. However, how internal states shape sensory processing or behaviour remains unclear. In Drosophila, male flies perform a lengthy and elaborate courtship ritual that is triggered by the activation of sexually dimorphic P1 neurons, during which they faithfully follow and sing to a female. By recording from males as they court a virtual 'female', this study gained insight into how the salience of visual cues is transformed by a male's internal arousal state to give rise to persistent courtship pursuit. The gain of LC10a visual projection neurons is selectively increased during courtship, enhancing their sensitivity to moving targets. A concise network model indicates that visual signalling through the LC10a circuit, once amplified by P1-mediated arousal, almost fully specifies a male's tracking of a female. Furthermore, P1 neuron activity correlates with ongoing fluctuations in the intensity of a male's pursuit to continuously tune the gain of the LC10a pathway. Together, these results reveal how a male's internal state can dynamically modulate the propagation of visual signals through a high-fidelity visuomotor circuit to guide his moment-to-moment performance of courtship.

Monday, August 9th - Disease models

Gumeni, S., Papanagnou, E. D., Manola, M. S. and Trougakos, I. P. (2021). Nrf2 activation induces mitophagy and reverses Parkin/Pink1 knock down-mediated neuronal and muscle degeneration phenotypes. Cell Death Dis 12(7): 671. PubMed ID: 34218254
Summary:
The balanced functionality of cellular proteostatic modules is central to both proteome stability and mitochondrial physiology; thus, the age-related decline of proteostasis also triggers mitochondrial dysfunction, which marks multiple degenerative disorders. Non-functional mitochondria are removed by mitophagy, including Parkin/Pink1-mediated mitophagy. A common feature of neuronal or muscle degenerative diseases, is the accumulation of damaged mitochondria due to disrupted mitophagy rates. This study exploited Drosophila as a model organism to investigate the functional role of Parkin/Pink1 in regulating mitophagy and proteostatic responses, as well as in suppressing degenerative phenotypes at the whole organism level. Parkin or Pink1 knock down in young flies modulated proteostatic components in a tissue-dependent manner, increased cell oxidative load, and suppressed mitophagy in neuronal and muscle tissues, causing mitochondrial aggregation and neuromuscular degeneration. Concomitant to Parkin or Pink1 knock down cncC/Nrf2 overexpression, induced the proteostasis network, suppressed oxidative stress, restored mitochondrial function, and elevated mitophagy rates in flies' tissues; it also, largely rescued Parkin or Pink1 knock down-mediated neuromuscular degenerative phenotypes. These in vivo findings highlight the critical role of the Parkin/Pink1 pathway in mitophagy, and support the therapeutic potency of Nrf2 (a druggable pathway) activation in age-related degenerative diseases.
Iyengar, A. and Wu, C. F. (2021). Fly seizure EEG: field potential activity in the Drosophila brain. J Neurogenet: 1-11. PubMed ID: 34278939
Summary:
Hypersynchronous neural activity is a characteristic feature of seizures. Although many Drosophila mutants of epilepsy-related genes display clear behavioral spasms and motor unit hyperexcitability, field potential measurements of aberrant hypersynchronous activity across brain regions during seizures have yet to be described. This study reports a straightforward method to observe local field potentials (LFPs) from the Drosophila brain to monitor ensemble neural activity during seizures in behaving tethered flies. High frequency stimulation across the brain reliably triggers a stereotypic sequence of electroconvulsive seizure (ECS) spike discharges readily detectable in the dorsal longitudinal muscle (DLM) and coupled with behavioral spasms. During seizure episodes, the LFP signal displayed characteristic large-amplitude oscillations with a stereotypic temporal correlation to DLM flight muscle spiking. ECS-related LFP events were clearly distinct from rest- and flight-associated LFP patterns. The LFP activity was further characterized during different types of seizures originating from genetic and pharmacological manipulations. In the 'bang-sensitive' sodium channel mutant bangsenseless (bss), the LFP pattern was prolonged, and the temporal correlation between LFP oscillations and DLM discharges was altered. Following administration of the pro-convulsant GABA(A) blocker picrotoxin, a qualitatively different LFP activity pattern was uncovered that consisted of a slow (1-Hz), repetitive, waveform, closely coupled with DLM bursting and behavioral spasms. This approach to record brain LFPs presents an initial framework for electrophysiological analysis of the complex brain-wide activity patterns in the large collection of Drosophila excitability mutants.
Inoue, E., Suzuki, T., Shimizu, Y., Sudo, K., Kawasaki, H. and Ishida, N. (2021). Saffron ameliorated motor symptoms, short life span and retinal degeneration in Parkinson's disease fly models. Gene 799: 145811. PubMed ID: 34224829
Summary:
Parkinson's disease (PD) is a common neurodegenerative disorder with motor symptoms linked to the loss of dopaminergic neurons in the brain. α-Synuclein is an aggregation-prone neural protein that plays a role in the pathogenesis of PD. In a previous paper, it was found that saffron; the stigma of Crocus sativus Linne (Iridaceae), and its constituents (crocin and crocetin) suppressed aggregation of α-synuclein and promoted the dissociation of α-synuclein fibrils in vitro. This study investigated the effect of dietary saffron and its constituent, crocetin, in vivo on a fly PD model overexpressing several mutant α-synuclein in a tissue-specific manner. Saffron and crocetin significantly suppressed the decrease of climbing ability in the Drosophila overexpressing A30P (A30P fly PD model) or G51D (G51D fly PD model) mutated α-synuclein in neurons. Saffron and crocetin extended the life span in the G51D fly PD model. Saffron suppressed the rough-eyed phenotype and the dispersion of the size histogram of the ocular long axis in the eye of A30P fly PD model. Saffron had a cytoprotective effect on a human neuronal cell line with α-synuclein fibrils. These data showed that saffron and its constituent crocetin have protective effects on the progression of PD disease in animals in vivo and suggest that saffron and crocetin can be used to treat PD.
Ham, S. J., Lee, D., Xu, W. J., Cho, E., Choi, S., Min, S., Park, S. and Chung, J. (2021). Loss of UCHL1 rescues the defects related to Parkinson's disease by suppressing glycolysis. Sci Adv 7(28). PubMed ID: 34244144
Summary:
The role of ubiquitin carboxyl-terminal hydrolase L1 (UCHL1; also called PARK5) in the pathogenesis of Parkinson's disease (PD) has been controversial. This study finds that the loss of UCHL1 destabilizes pyruvate kinase (PKM) and mitigates the PD-related phenotypes induced by PTEN-induced kinase 1 (PINK1) or Parkin loss-of-function mutations in Drosophila and mammalian cells. In UCHL1 knockout cells, cellular pyruvate production and ATP levels are diminished, and the activity of AMP-activated protein kinase (AMPK) is highly induced. Consequently, the activated AMPK promotes the mitophagy mediated by Unc-51-like kinase 1 (ULK1) and FUN14 domain-containing 1 (FUNDC1), which underlies the effects of UCHL1 deficiency in rescuing PD-related defects. Furthermore, this study identified tripartite motif-containing 63 (TRIM63) as a previously unknown E3 ligase of PKM and demonstrate its antagonistic interaction with UCHL1 to regulate PD-related pathologies. These results suggest that UCHL1 is an integrative factor for connecting glycolysis and PD pathology.
Kim, A. K., Kwon, D. W., Yeom, E., Lee, K. P., Kwon, K. S., Yu, K. and Lee, K. S. (2021). Lipophorin receptor 1 (LpR1) in Drosophila muscle influences life span by regulating mitochondrial aging. Biochem Biophys Res Commun 568: 95-102. PubMed ID: 34217014
Summary:
Sarcopenia is a syndrome characterized by progressive loss of muscle mass and function during aging. Although mitochondrial dysfunction and related metabolic defects precede age-related changes in muscle, their contributions to muscle aging are still not well known. This study used a Drosophila model to investigate the role of lipophorin receptors (LpRs), a Drosophila homologue of the mammalian very low-density lipoprotein receptor (VLDLR), in mitochondrial dynamics and muscle aging. Muscle-specific knockdown of LpR1 or LpR2 resulted in mitochondrial dysfunction and reduced proteostasis, which contributed to muscle aging. Activation of AMP-activated protein kinase (AMPK) ameliorated muscle dysfunction induced by LpR1 knockdown. These results suggest that LpR1/VLDLR is a novel key target that modulates age-dependent lipid remodeling and muscle homeostasis.
Gokhale, A., Lee, C. E., Zlatic, S. A., ...., Glausier, J. R., Lewis, D. A. and Faundez, V. (2021). Mitochondrial Proteostasis Requires Genes Encoded in a Neurodevelopmental Syndrome Locus. J Neurosci. PubMed ID: 34261699
Summary:
Eukaryotic cells maintain proteostasis, the balance between protein synthesis, folding, re-folding and degradation, through mechanisms that require cytoplasmic and mitochondrial translation. Genetic defects affecting cytoplasmic translation perturb synapse development, neurotransmission, and are causative of neurodevelopmental disorders such as Fragile X syndrome. In contrast, there is little indication that mitochondrial proteostasis, either in the form of mitochondrial protein translation and/or degradation, is required for synapse development and function. This study focused on two genes deleted in a recurrent copy number variation causing neurodevelopmental disorders, the 22q11.2 microdeletion syndrome. SLC25A1 and MRPL40, two genes present in the microdeleted segment and whose products localize to mitochondria, interact and are necessary for mitochondrial ribosomal integrity and proteostasis. Drosophila studies show that mitochondrial ribosome function is necessary for synapse neurodevelopment, function, and behavior. It is proposed that mitochondrial proteostasis perturbations, either by genetic or environmental factors, are a pathogenic mechanism for neurodevelopmental disorders.

Friday August 6th - RNA and Transposons

Aksoy, E. and Raikhel, A. S. (2021). Juvenile hormone regulation of microRNAs is mediated by E75 in the Dengue vector mosquito Aedes aegypti. Proc Natl Acad Sci U S A 118(29). PubMed ID: 34266957
Summary:
MicroRNAs (miRNAs) are small noncoding RNAs that play critical roles in controlling posttranscriptional gene regulation and have a profound effect on mosquito reproduction and metabolism. Juvenile hormone (JH) is critical for achieving reproductive competence in the main vector of human arboviral diseases, Aedes aegypti. This study reports a JH-mediated mechanism governing miRNA expression. Using a transcription factor screen with multiple primary miRNA (pri-miRNA) promoters, it was identified that the Ecdysone-induced protein E75 (E75) isoform (E75-RD) induced miRNA gene promoter activity. E75 binding sites were determined in miRNA promoters by means of cell transfection assay. E75-RD was found to be up-regulated by JH, as shown by the JH application and RNA interference (RNAi) of the JH receptor Methoprene-tolerant (Met). Small RNA sequencing from RNAi of Met and E75 displayed an overlapping miRNA cohort, suggesting E75 to be an intermediate component within the JH hierarchical network controlling miRNAs. Further experiments confirmed that E75-RD positively regulates several miRNAs including miR-2940. Reducing miR-2940 resulted in the arrest of follicle development and number of eggs laid. Performing miRNA target predictions and RT-qPCR from antagomir Ant-2940-3p-treated fat body tissues identified the mRNA target Clumsy (AAEL002518). The molecular interaction between this gene target and miR-2940 was confirmed using an in vitro dual luciferase assay was performed in Drosophila S2 cells and in Ae. aegypti Aag2 cell lines. Finally, a phenotypic rescue experiment to demonstrate that miR-2940/Clumsy is responsible for the disruption in egg development. Collectively, these results established the role of JH-mediated E75-RD in regulation of miRNA gene expression during the mosquito reproductive cycle.
Gonzalez, L. E., Tang, X. and Lin, H. (2021). Maternal Piwi Regulates Primordial Germ Cell Development to Ensure the Fertility of Female Progeny in Drosophila. Genetics. PubMed ID: 34142134
Summary:
In many animals, germline development is initiated by proteins and RNAs that are expressed maternally. PIWI proteins and their associated small noncoding PIWI-interacting RNAs (piRNAs), which guide PIWI to target RNAs by base-pairing, are among the maternal components deposited into the germline of the Drosophila early embryo. Piwi has been extensively studied in the adult ovary and testis, where it is required for transposon suppression, germline stem cell self-renewal, and fertility. Consequently, loss of Piwi in the adult ovary using piwi-null alleles or knockdown from early oogenesis results in complete sterility, limiting investigation into possible embryonic functions of maternal Piwi. This study shows that the maternal Piwi protein persists in the embryonic germline through gonad coalescence, suggesting that maternal Piwi can regulate germline development beyond early embryogenesis. Using a maternal knockdown strategy, this study found that maternal Piwi is required for the fertility and normal gonad morphology of female, but not male, progeny. Following maternal piwi knockdown, transposons were mildly derepressed in the early embryo but were fully repressed in the ovaries of adult progeny. Furthermore, the maternal piRNA pool was diminished, reducing the capacity of the PIWI/piRNA complex to target zygotic genes during embryogenesis. Examination of embryonic germ cell proliferation and ovarian gene expression showed that the germline of female progeny was partially masculinized by maternal piwi knockdown. This study reveals a novel role for maternal Piwi in the germline development of female progeny and suggests that the PIWI/piRNA pathway is involved in germline sex determination in Drosophila.
Lowe, D. D. and Montell, D. J. (2021). Unconventional translation initiation factor EIF2A is required for Drosophila spermatogenesis. Dev Dyn. PubMed ID: 34278643
Summary:
EIF2A is an unconventional translation factor required for initiation of protein synthesis from non-AUG codons from a variety of transcripts, including oncogenes and stress related transcripts in mammalian cells. Its function in multicellular organisms has not been reported. This study identified and characterize mutant alleles of the CG7414 gene, which encodes the Drosophila EIF2A ortholog. CG7414 was shown to undergo sex-specific splicing that regulates its male-specific expression. A Mi{Mic} transposon insertion was identified that disrupts the coding regions of all predicted isoforms and is a likely null allele, and a PBac transposon insertion into an intron was identified that is a hypomorph. The Mi{Mic} allele is homozygous lethal, while the viable progeny from the hypomorphic PiggyBac allele are male sterile and female fertile. In dEIF2A mutant flies, sperm failed to individualize due to defects in F-actin cones and failure to form and maintain cystic bulges, ultimately leading to sterility. These results demonstrate that EIF2A is essential in a multicellular organism, both for normal development and spermatogenesis, and provide an entrée into the elucidation of the role of EIF2A and unconventional translation in vivo.
Huang, X., Hu, H., Webster, A., Zou, F., Du, J., Patel, D. J., Sachidanandam, R., Toth, K. F., Aravin, A. A. and Li, S. (2021). Binding of guide piRNA triggers methylation of the unstructured N-terminal region of Aub leading to assembly of the piRNA amplification complex. Nat Commun 12(1): 4061. PubMed ID: 34210982
Summary:
PIWI proteins use guide piRNAs to repress selfish genomic elements, protecting the genomic integrity of gametes and ensuring the fertility of animal species. Efficient transposon repression depends on amplification of piRNA guides in the ping-pong cycle, which in Drosophila entails tight cooperation between two PIWI proteins, Aub and Ago3. This study shows that post-translational modification, symmetric dimethylarginine (sDMA), of Aub is essential for piRNA biogenesis, transposon silencing and fertility. Methylation is triggered by loading of a piRNA guide into Aub, which exposes its unstructured N-terminal region to the PRMT5 methylosome complex. Thus, sDMA modification is a signal that Aub is loaded with piRNA guide. Amplification of piRNA in the ping-pong cycle requires assembly of a tertiary complex scaffolded by Krimper, which simultaneously binds the N-terminal regions of Aub and Ago3. To promote generation of new piRNA, Krimper uses its two Tudor domains to bind Aub and Ago3 in opposite modification and piRNA-loading states. These results reveal that post-translational modifications in unstructured regions of PIWI proteins and their binding by Tudor domains that are capable of discriminating between modification states is essential for piRNA biogenesis and silencing.
Hong, W., Zhang, J., Dong, H., Shi, Y., Ma, H., Zhou, F., Xu, B., Fu, Y., Zhang, S., Hou, S., Li, G., Wu, Y., Chen, S., Zhu, X., You, W., Shi, F., Yang, X., Gong, Z., Huang, J. and Jin, Y. (2021). Intron-targeted mutagenesis reveals roles for Dscam1 RNA pairing architecture-driven splicing bias in neuronal wiring. Cell Rep 36(2): 109373. PubMed ID: 34260933
Summary:
Drosophila melanogaster Down syndrome cell adhesion molecule (Dscam1) can generate 38,016 different isoforms through largely stochastic, yet highly biased, alternative splicing. These isoforms are required for nervous functions. However, the functional significance of splicing bias remains unknown. This study provides evidence that Dscam1 splicing bias is required for mushroom body (MB) axonal wiring. Mutant flies were generated with normal overall protein levels and an identical number but global changes in exon 4 and 9 isoform bias (DscamΔ4D(-/-) and DscamΔ9D(-/-)), respectively. In contrast to DscamΔ4D(-/-), DscamΔ9D(-/-) exhibits remarkable MB defects, suggesting a variable domain-specific requirement for isoform bias. Importantly, changes in isoform bias cause axonal defects but do not influence the self-avoidance of axonal branches. It is concluded that, in contrast to the isoform number that provides the molecular basis for neurite self-avoidance, isoform bias may play a role in MB axonal wiring by influencing non-repulsive signaling.
Hopes, T., Norris, K., Agapiou, M., McCarthy, C. G. P., Lewis, P. A., O'Connell, M. J., Fontana, J. and Aspden, J. L. (2021). Ribosome heterogeneity in Drosophila melanogaster gonads through paralog-switching. Nucleic Acids Res. PubMed ID: 34283226
Summary:
Ribosomes have long been thought of as homogeneous macromolecular machines, but recent evidence suggests they are heterogeneous and could be specialised to regulate translation. This study characterised ribosomal protein heterogeneity across 4 tissues of Drosophila melanogaster. Testes and ovaries were found to contain the most heterogeneous ribosome populations, which occurs through a combination of paralog-enrichment and paralog-switching. Structures were solved of ribosomes purified from in vivo tissues by cryo-EM, revealing differences in precise ribosomal arrangement for testis and ovary 80S ribosomes. Differences in the amino acid composition of paralog pairs and their localisation on the ribosome exterior indicate paralog-switching could alter the ribosome surface, enabling different proteins to regulate translation. One testis-specific paralog-switching pair is also found in humans, suggesting this is a conserved site of ribosome heterogeneity. Overall, this work leads to a proposal that mRNA translation might be regulated in the gonads through ribosome heterogeneity, providing a potential means of ribosome specialisation.

Thursday August 5th - Evolution

Glaser-Schmitt, A., Wittmann, M. J., Ramnarine, T. J. S. and Parsch, J. (2021). Sexual antagonism, temporally fluctuating selection, and variable dominance affect a regulatory polymorphism in Drosophila melanogaster. Mol Biol Evol. PubMed ID: 34289067
Summary:
Understanding how genetic variation is maintained within species is a major goal of evolutionary genetics that can shed light on the preservation of biodiversity. This study examined the maintenance of a regulatory single-nucleotide polymorphism (SNP) of the X-linked Drosophila melanogaster gene fezzik. The derived variant at this site is at intermediate frequency in many worldwide populations, but absent in populations from the ancestral species range in sub-Saharan Africa. Wild-caught individuals from a single European population were collected and genotyped biannually over a period of five years, revealing an overall difference in allele frequency between the sexes and a consistent change in allele frequency across seasons in females but not in males. Modelling based on the observed allele and genotype frequencies suggested that both sexually antagonistic and temporally fluctuating selection may help maintain variation at this site. The derived variant is predicted to be female-beneficial and mostly recessive; however, there was uncertainty surrounding the dominance estimates, and long-term modelling projections suggest that it is more likely to be dominant. By examining gene expression phenotypes, it was found that phenotypic dominance was variable and dependent upon developmental stage and genetic background, suggesting that dominance may be variable at this locus. It was further determined that fezzik expression and genotype are associated with starvation resistance in a sex-dependent manner, suggesting a potential phenotypic target of selection. By characterizing the mechanisms of selection acting on this SNP, the results improve understanding of how selection maintains genetic and phenotypic variation in natural populations.
Hughes, J. T., Williams, M. E., Rebeiz, M. and Williams, T. M. (2021). Widespread cis- and trans-regulatory evolution underlies the origin, diversification, and loss of a sexually dimorphic fruit fly pigmentation trait. J Exp Zool B Mol Dev Evol. PubMed ID: 34254440
Summary:
Changes in gene expression are a prominent feature of morphological evolution. These changes occur to hierarchical gene regulatory networks (GRNs) of transcription factor genes that regulate the expression of trait-building differentiation genes. While changes in the expression of differentiation genes are essential to phenotypic evolution, they can be caused by mutations within cis-regulatory elements (CREs) that drive their expression (cis-evolution) or within genes for CRE-interacting transcription factors (trans-evolution). Locating these mutations remains a challenge, especially when experiments are limited to one species that possesses the ancestral or derived phenotype. This study investigated CREs that control the expression of the differentiation genes tan and yellow, the expression of which evolved during the gain, modification, and loss of dimorphic pigmentation among Sophophora fruit flies. These CREs were shown to be necessary components of a pigmentation GRN, as deletion from Drosophila melanogaster (derived dimorphic phenotype) resulted in lost expression and lost male-specific pigmentation. This study evaluated the ability of orthologous CRE sequences to drive reporter gene expression in species with modified (Drosophila auraria), secondarily lost (Drosophila ananassae), and ancestrally absent (Drosophila willistoni) pigmentation. The transgene host frequently determines CRE activity, implicating trans-evolution as a significant factor for this trait's diversity. The gain of dimorphic Bab transcription factor expression as a trans-change contributing to the dimorphic trait was evaluated. The findings suggest an amenability to change for the landscape of trans-regulators and begs for an explanation as to why this is so common compared to the evolution of differentiation gene CREs.
Keller Valsecchi, C. I., Marois, E., Basilicata, M. F., Georgiev, P. and Akhtar, A. (2021). Distinct mechanisms mediate X chromosome dosage compensation in Anopheles and Drosophila. Life Sci Alliance 4(9). PubMed ID: 34266874
Summary:
Sex chromosomes induce potentially deleterious gene expression imbalances that are frequently corrected by dosage compensation (DC). Three distinct molecular strategies to achieve DC have been previously described in nematodes, fruit flies, and mammals. Is this a consequence of distinct genomes, functional or ecological constraints, or random initial commitment to an evolutionary trajectory? DC was studied in the malaria mosquito Anopheles gambiae. The Anopheles and Drosophila X chromosomes evolved independently but share a high degree of homology. Anopheles achieves DC by a mechanism distinct from the Drosophila MSL complex-histone H4 lysine 16 acetylation pathway. CRISPR knockout of Anopheles msl-2 leads to embryonic lethality in both sexes. Transcriptome analyses indicate that this phenotype is not a consequence of defective X chromosome DC. By immunofluorescence and ChIP, H4K16ac does not preferentially enrich on the male X. Instead, the mosquito MSL pathway regulates conserved developmental genes. It is concluded that a novel mechanism confers X chromosome up-regulation in Anopheles. These findings highlight the pluralism of gene-dosage buffering mechanisms even under similar genomic and functional constraints.
de Fatima Chaves Rego, N., Chahad-Ehlers, S., Boni Campanini, E., Rafael Torres, F. and Alves de Brito, R. (2021). VRILLE shows high divergence among Higher Diptera flies but may retain role as transcriptional repressor of Clock. J Insect Physiol: 104284. PubMed ID: 34256034
Summary:
In the circadian system, the clock gene vrille (vri) is an essential component of the second feedback loop, being responsible in Drosophila for the rhythmicity of the Clock (Clk) gene transcription by its repression. This study investigated vri in a fruit fly pest, the Tephritidae Anastrepha fraterculus. A combination of strategies was used to sequence and characterize Afravri in male and female head transcriptomes of A. fraterculus; two putative isoforms were detected that may correspond to A and D vri isoforms of Drosophila. Both isoforms produced a full-length sequence that translates to 842 amino acids. While the protein sequence showed significant divergence to orthologous sequences from other organisms, the bZIP domain was highly conserved. Molecular evolutionary analyses showed that vri in Higher Diptera flies has been evolving under positive selection. A more detailed analysis showed positive selection also in Tephritidae with 29 sites evolving under positive selection in comparison with Drosophilidae. Real time expression analysis in LD and DD conditions showed cyclic expression of Afravri mRNA with oscillation opposite to AfraClk, suggesting that VRI may also behave in Anastrepha as a transcriptional repressor of Clk, providing another indication that Higher Diptera might share common interlocked transcript-translation feedback loops (TTFLs) mechanisms that differ from other insects in target genes.
Pechmann, M., Kenny, N. J., Pott, L., Heger, P., Chen, Y. T., Buchta, T., Ozuak, O., Lynch, J. and Roth, S. (2021). Striking parallels between dorsoventral patterning in Drosophila and Gryllus reveal a complex evolutionary history behind a model gene regulatory network. Elife 10. PubMed ID: 33783353
Summary:
Dorsoventral pattering relies on Toll and BMP signalling in all insects studied so far, with variations in the relative contributions of both pathways. Drosophila and the beetle Tribolium share extensive dependence on Toll, while representatives of more distantly related lineages like the wasp Nasonia and bug Oncopeltus rely more strongly on BMP signalling. This study shows that in the cricket Gryllus bimaculatus, an evolutionarily distant outgroup, Toll has, like in Drosophila, a direct patterning role for the ventral half of the embryo. In addition, Toll polarises BMP signalling, although this does not involve the conserved BMP inhibitor Sog/Chordin. Finally, Toll activation relies on ovarian patterning mechanisms with striking similarity to Drosophila. These data suggest two surprising hypotheses: (1) that Toll's patterning function in Gryllus and Drosophila is the result of convergent evolution or (2) a Drosophila-like system arose early in insect evolution and was extensively altered in multiple independent lineages.
Khallaf, M. A., Cui, R., Weissflog, J., Erdogmus, M., Svatos, A., Dweck, H. K. M., Valenzano, D. R., Hansson, B. S. and Knaden, M. (2021). Large-scale characterization of sex pheromone communication systems in Drosophila. Nat Commun 12(1): 4165. PubMed ID: 34230464
Summary:
Insects use sex pheromones as a reproductive isolating mechanism to attract conspecifics and repel heterospecifics. Despite the profound knowledge of sex pheromones, little is known about the coevolutionary mechanisms and constraints on their production and detection. Using whole-genome sequences to infer the kinship among 99 drosophilids, this study investigated how phylogenetic and chemical traits have interacted at a wide evolutionary timescale. Through a series of chemical syntheses and electrophysiological recordings, this study identified 52 sex-specific compounds, many of which are detected via olfaction. Behavioral analyses reveal that many of the 43 male-specific compounds are transferred to the female during copulation and mediate female receptivity and/or male courtship inhibition. Measurement of phylogenetic signals demonstrates that sex pheromones and their cognate olfactory channels evolve rapidly and independently over evolutionary time to guarantee efficient intra- and inter-specific communication systems. These results show how sexual isolation barriers between species can be reinforced by species-specific olfactory signals.

Wednesday August 4th - Synapse and Vesicles

Weiss, J. T. and Donlea, J. M. (2021). Sleep deprivation results in diverse patterns of synaptic scaling across the Drosophila mushroom bodies. Curr Biol. PubMed ID: 34107302
Summary:
Sleep is essential for a variety of plastic processes, including learning and memory. However, the consequences of insufficient sleep on circuit connectivity remain poorly understood. To better appreciate the effects of sleep loss on synaptic connectivity across a memory-encoding circuit, changes were examined in the distribution of synaptic markers in the Drosophila mushroom body (MB). Protein-trap tags for active zone components indicate that recent sleep time is inversely correlated with Bruchpilot (BRP) abundance in the MB lobes; sleep loss elevates BRP while sleep induction reduces BRP across the MB. Overnight sleep deprivation also elevated levels of dSyd-1 and Cacophony, but not other pre-synaptic proteins. Cell-type-specific genetic reporters show that MB-intrinsic Kenyon cells (KCs) exhibit increased pre-synaptic BRP throughout the axonal lobes after sleep deprivation; similar increases were not detected in projections from large interneurons or dopaminergic neurons that innervate the MB. These results indicate that pre-synaptic plasticity in KCs is responsible for elevated levels of BRP in the MB lobes of sleep-deprived flies. Because KCs provide synaptic inputs to several classes of post-synaptic partners, a fluorescent reporter for synaptic contacts was used to test whether each class of KC output connections is scaled uniformly by sleep loss. The KC output synapses that were observed in this study can be divided into three classes: KCs to MB interneurons; KCs to dopaminergic neurons; and KCs to MB output neurons. No single class showed uniform scaling across each constituent member, indicating that different rules may govern plasticity during sleep loss across cell types.
Krick, N., Ryglewski, S., Pichler, A., Bikbaev, A., Gotz, T., Kobler, O., Heine, M., Thomas, U. and Duch, C. (2021). Separation of presynaptic Ca(v)2 and Ca(v)1 channel function in synaptic vesicle exo- and endocytosis by the membrane anchored Ca(2+) pump PMCA. Proc Natl Acad Sci U S A 118(28). PubMed ID: 34244444
Summary:
Synaptic vesicle (SV) release, recycling, and plastic changes of release probability co-occur side by side within nerve terminals and rely on local Ca(2+) signals with different temporal and spatial profiles. The mechanisms that guarantee separate regulation of these vital presynaptic functions during action potential (AP)-triggered presynaptic Ca(2+) entry remain unclear. Combining Drosophila genetics with electrophysiology and imaging reveals the localization of two different voltage-gated calcium channels at the presynaptic terminals of glutamatergic neuromuscular synapses (the Drosophila Ca(v)2 homolog, Dmca1A or cacophony, and the Ca(v)1 homolog, Dmca1D) but with spatial and functional separation. Ca(v)2 within active zones is required for AP-triggered neurotransmitter release. By contrast, Ca(v)1 localizes predominantly around active zones and contributes substantially to AP-evoked Ca(2+) influx but has a small impact on release. Instead, L-type calcium currents through Ca(v)1 fine-tune short-term plasticity and facilitate SV recycling. Separate control of SV exo- and endocytosis by AP-triggered presynaptic Ca(2+) influx through different channels demands efficient measures to protect the neurotransmitter release machinery against Ca(v)1-mediated Ca(2+) influx. The plasma membrane Ca(2+) ATPase (PMCA) resides in between active zones and isolates Ca(v)2-triggered release from Ca(v)1-mediated dynamic regulation of recycling and short-term plasticity, two processes which Ca(v)2 may also contribute to. As L-type Ca(v)1 channels also localize next to PQ-type Ca(v)2 channels within axon terminals of some central mammalian synapses, it is proposed that Ca(v)2, Ca(v)1, and PMCA act as a conserved functional triad that enables separate control of SV release and recycling rates in presynaptic terminals.
Lund, V. K., Lycas, M. D., Schack, A., Andersen, R. C., Gether, U. and Kjaerulff, O. (2021). Rab2 drives axonal transport of dense core vesicles and lysosomal organelles. Cell Rep 35(2): 108973. PubMed ID: 33852866
Summary:
Fast axonal transport of neuropeptide-containing dense core vesicles (DCVs), endolysosomal organelles, and presynaptic components is critical for maintaining neuronal functionality. How the transport of DCVs is orchestrated remains an important unresolved question. The small GTPase Rab2 mediates DCV biogenesis and endosome-lysosome fusion. This study used Drosophila to demonstrate that Rab2 also plays a critical role in bidirectional axonal transport of DCVs, endosomes, and lysosomal organelles, most likely by controlling molecular motors. It was further shown that the lysosomal motility factor Arl8 is required as well for axonal transport of DCVs, but unlike Rab2, it is also critical for DCV exit from cell bodies into axons. Evidence is provided that the upstream regulators of Rab2 and Arl8, Ema and BORC, activate these GTPases during DCV transport. These results uncover the mechanisms underlying axonal transport of DCVs and reveal surprising parallels between the regulation of DCV and lysosomal motility.
Hallin, E. I., Markusson, S., Bottger, L., Torda, A. E., Bramham, C. R. and Kursula, P. (2021). Crystal and solution structures reveal oligomerization of individual capsid homology domains of Drosophila Arc. PLoS One 16(5): e0251459. PubMed ID: 33989344
Summary:
One of the key proteins in long-term synaptic plasticity and memory is the activity-regulated cytoskeleton-associated protein (Arc). Mammalian Arc forms virus-like capsid structures in a process requiring the N-terminal domain and contains two C-terminal lobes that are structural homologues to retroviral capsids. Drosophila has two isoforms of Arc, dArc1 and dArc2, with low sequence similarity to mammalian Arc, but lacking a large N-terminal domain. Both dArc isoforms are related to the Ty3/gypsy retrotransposon capsid, consisting of N- and C-terminal lobes. Structural characterization of the four individual dArc lobe domains was carried out. As opposed to the corresponding mammalian Arc lobe domains, which are monomeric, the dArc lobes were all oligomeric in solution, indicating a strong propensity for homophilic interactions. A truncated N-lobe from dArc2 formed a domain-swapped dimer in the crystal structure, resulting in a novel dimer interaction that could be relevant for capsid assembly or other dArc functions. This domain-swapped structure resembles the dimeric protein C of flavivirus capsids, as well as the structure of histones dimers. The strong oligomerization properties of the isolated dArc lobe domains explain the ability of dArc to form capsids in the absence of any large N-terminal domain, in contrast to the mammalian protein.
Kamalesh, K., Scher, N., Biton, T., Schejter, E. D., Shilo, B. Z. and Avinoam, O. (2021). Exocytosis by vesicle crumpling maintains apical membrane homeostasis during exocrine secretion. Dev Cell 56(11): 1603-1616.e1606. PubMed ID: 34102104
Summary:
Exocrine secretion commonly employs micron-scale vesicles that fuse to a limited apical surface, presenting an extreme challenge for maintaining membrane homeostasis. Using Drosophila melanogaster larval salivary glands, this study shows that the membranes of fused vesicles undergo actomyosin-mediated folding and retention, which prevents them from incorporating into the apical surface. In addition, the diffusion of proteins and lipids between the fused vesicle and the apical surface is limited. Actomyosin contraction and membrane crumpling are essential for recruiting clathrin-mediated endocytosis to clear the retained vesicular membrane. Finally, membrane crumpling was also observed in secretory vesicles of the mouse exocrine pancreas. It is concluded that membrane sequestration by crumpling followed by targeted endocytosis of the vesicular membrane, represents a general mechanism of exocytosis that maintains membrane homeostasis in exocrine tissues that employ large secretory vesicles.
Banerjee, S., Vernon, S., Jiao, W., Choi, B. J., Ruchti, E., Asadzadeh, J., Burri, O., Stowers, R. S. and McCabe, B. D. (2021). Miniature neurotransmission is required to maintain Drosophila synaptic structures during ageing. Nat Commun 12(1): 4399. PubMed ID: 34285221
Summary:
The decline of neuronal synapses is an established feature of ageing accompanied by the diminishment of neuronal function, and in the motor system at least, a reduction of behavioural capacity. This study has investigated Drosophila motor neuron synaptic terminals during ageing. Cumulative fragmentation of presynaptic structures was observed, accompanied by diminishment of both evoked and miniature neurotransmission occurring in tandem with reduced motor ability. Through discrete manipulation of each neurotransmission modality, it was found that miniature but not evoked neurotransmission is required to maintain presynaptic architecture and that increasing miniature events can both preserve synaptic structures and prolong motor ability during ageing. These results establish that miniature neurotransmission, formerly viewed as an epiphenomenon, is necessary for the long-term stability of synaptic connections.

Tuesday, August 3rd - Chromatin

Wei, X., Eickbush, D. G., Speece, I. and Larracuente, A. M. (2021). Heterochromatin-dependent transcription of satellite DNAs in the Drosophila melanogaster female germline. Elife 10. PubMed ID: 34259629
Summary:
Large blocks of tandemly repeated DNAs-satellite DNAs (satDNAs)-play important roles in heterochromatin formation and chromosome segregation. Little is known about how satDNAs are regulated, however their misregulation is associated with genomic instability and human diseases. The Drosophila melanogaster germline was used as a model to study the regulation of satDNA transcription and chromatin. This study shows that complex satDNAs (>100-bp repeat units) are transcribed into long noncoding RNAs and processed into piRNAs (PIWI interacting RNAs). This satDNA piRNA production depends on the Rhino-Deadlock-Cutoff complex and the transcription factor Moonshiner-a previously-described non-canonical pathway that licenses heterochromatin-dependent transcription of dual-strand piRNA clusters. This study shows that this pathway is important for establishing heterochromatin at satDNAs. Therefore, satDNAs are regulated by piRNAs originating from their own genomic loci. This novel mechanism of satDNA regulation provides insight into the role of piRNA pathways in heterochromatin formation and genome stability.
Ahmad, K. and Henikoff, S. (2021). The H3.3K27M oncohistone antagonizes reprogramming in Drosophila. PLoS Genet 17(7): e1009225. PubMed ID: 34280185
Summary:
Development proceeds by the activation of genes by transcription factors and the inactivation of others by chromatin-mediated gene silencing. In certain cases development can be reversed or redirected by mis-expression of master regulator transcription factors. This must involve the activation of previously silenced genes, and such developmental aberrations are thought to underlie a variety of cancers. This study expressed the wing-specific Vestigial master regulator to reprogram the developing eye, and test the role of silencing in reprogramming using an H3.3K27M oncohistone mutation that dominantly inhibits histone H3K27 trimethylation. Production of the oncohistone was found to block eye-to-wing reprogramming. CUT&Tag chromatin profiling of mutant tissues shows that H3K27me3 of domains is generally reduced upon oncohistone production, suggesting that a previous developmental program must be silenced for effective transformation. Strikingly, Vg and H3.3K27M synergize to stimulate overgrowth of eye tissue, a phenotype that resembles that of mutations in Polycomb silencing components. Transcriptome profiling of elongating RNA Polymerase II implicates the mis-regulation of signaling factors in overgrowth. These results demonstrate that growth dysregulation can result from the simple combination of crippled silencing and transcription factor mis-expression, an effect that may explain the origins of oncohistone-bearing cancers.
Eggers, N. and Becker, P. B. (2021). Cell-free genomics reveal intrinsic, cooperative and competitive determinants of chromatin interactions. Nucleic Acids Res 49(13): 7602-7617. PubMed ID: 34181732
Summary:
Metazoan transcription factors distinguish their response elements from a large excess of similar sequences. This study explored underlying principles of DNA shape read-out and factor cooperativity in chromatin using a unique experimental system. Chromatin on Drosophila genomes was reconstructed in extracts of preblastoderm embryos, mimicking the naive state of the zygotic genome prior to developmental transcription activation. The intrinsic binding specificities of three recombinant transcription factors, alone and in combination, were then compared with GA-rich recognition sequences genome-wide. For MSL2, all functional elements reside on the X chromosome, allowing to distinguish physiological elements from non-functional 'decoy' sites. The physiological binding profile of MSL2 is approximated through interaction with other factors: cooperativity with CLAMP and competition with GAF, which sculpts the profile by occluding non-functional sites. An extended DNA shape signature is differentially read out in chromatin. These results reveal novel aspects of target selection in a complex chromatin environment.
Zenk, F., Zhan, Y., Kos, P., Loser, E., Atinbayeva, N., Schachtle, M., Tiana, G., Giorgetti, L. and Iovino, N. (2021). HP1 drives de novo 3D genome reorganization in early Drosophila embryos. Nature 593(7858): 289-293. PubMed ID: 33854237
Summary:
Fundamental features of 3D genome organization are established de novo in the early embryo, including clustering of pericentromeric regions, the folding of chromosome arms and the segregation of chromosomes into active (A-) and inactive (B-) compartments. However, the molecular mechanisms that drive de novo organization remain unknown. In this study, by combining chromosome conformation capture (Hi-C), chromatin immunoprecipitation with high-throughput sequencing (ChIP-seq), 3D DNA fluorescence in situ hybridization (3D DNA FISH) and polymer simulations, this study shows that heterochromatin protein 1a (HP1a) is essential for de novo 3D genome organization during Drosophila early development. The binding of HP1a at pericentromeric heterochromatin is required to establish clustering of pericentromeric regions. Moreover, HP1a binding within chromosome arms is responsible for overall chromosome folding and has an important role in the formation of B-compartment regions. However, depletion of HP1a does not affect the A-compartment, which suggests that a different molecular mechanism segregates active chromosome regions. This work identifies HP1a as an epigenetic regulator that is involved in establishing the global structure of the genome in the early embryo.
Ghotbi, E., Ye, P., Ervin, T., Kum, A., Benes, J. and Jones, R. S. (2021). Polycomb-group recruitment to a Drosophila target gene is the default state that is inhibited by a transcriptional activator. Sci Adv 7(29). PubMed ID: 34272248
Summary:
Polycomb-group (PcG) proteins are epigenetic regulators that maintain the transcriptional repression of target genes following their initial repression by transcription factors. PcG target genes are repressed in some cells, but active in others. Therefore, a mechanism must exist by which PcG proteins distinguish between the repressed and active states and only assemble repressive chromatin environments at target genes that are repressed. This study presents experimental evidence that the repressed state of a Drosophila PcG target gene, giant (gt), is not identified by the presence of a repressor. Rather, de novo establishment of PcG-mediated silencing at gt is the default state that is prevented by the presence of an activator or coactivator, which may inhibit the catalytic activity of Polycomb-repressive complex 2 (PRC2).
Bag, I., Chen, S., Rosin, L. F., Chen, Y., Liu, C. Y., Yu, G. Y. and Lei, E. P. (2021). M1BP cooperates with CP190 to activate transcription at TAD borders and promote chromatin insulator activity. Nat Commun 12(1): 4170. PubMed ID: 34234130
Summary:
Genome organization is driven by forces affecting transcriptional state, but the relationship between transcription and genome architecture remains unclear. This study identified the Drosophila transcription factor Motif 1 Binding Protein (M1BP) in physical association with the gypsy chromatin insulator core complex, including the universal insulator protein CP190. M1BP is required for enhancer-blocking and barrier activities of the gypsy insulator as well as its proper nuclear localization. Genome-wide, M1BP specifically colocalizes with CP190 at Motif 1-containing promoters, which are enriched at topologically associating domain (TAD) borders. M1BP facilitates CP190 chromatin binding at many shared sites and vice versa. Both factors promote Motif 1-dependent gene expression and transcription near TAD borders genome-wide. Finally, loss of M1BP reduces chromatin accessibility and increases both inter- and intra-TAD local genome compaction. These results reveal physical and functional interaction between CP190 and M1BP to activate transcription at TAD borders and mediate chromatin insulator-dependent genome organization.

Monday, August 2nd - Cytoskeleton and Junctions

Chung, S., Le, T. P., Vishwakarma, V., Cheng, Y. L. and Andrew, D. J. (2021). Isoform-specific roles of the Drosophila filamin-type protein Jitterbug (Jbug) during development. Genetics. PubMed ID: 34173831
Summary:
Filamins are highly conserved actin-crosslinking proteins that regulate organization of the actin cytoskeleton. As key components of versatile signaling scaffolds, filamins are implicated in developmental anomalies and cancer. Multiple isoforms of filamins exist, raising the possibility of distinct functions for each isoform during development and in disease. This study provides an initial characterization of jitterbug (jbug), which encodes one of the two filamin-type proteins in Drosophila. Jbug antiserum was generated that recognizes all of the spliced forms and reveals differential expression of different Jbug isoforms during development, and a significant maternal contribution of Jbug protein. To reveal the function of Jbug isoforms, new genetic tools were developed, including a null allele that deletes all isoforms, hypomorphic alleles that affect only a subset, and UAS lines for Gal4-driven expression of the major isoforms. Using these tools, it was demonstrated that Jbug is required for viability and that specific isoforms are required in the formation of actin-rich protrusions including thoracic bristles in adults and ventral denticles in the embryo. Specific isoforms of Jbug show differential localization within epithelia, and maternal and zygotic loss of jbug disrupts Crumbs (Crb) localization in several epithelial cell types.
Herrera-Perez, R. M., Cupo, C., Allan, C., Lin, A. and Kasza, K. E. (2021). Using optogenetics to link myosin patterns to contractile cell behaviors during convergent extension. Biophys J. PubMed ID: 34293302
Summary:
Distinct patterns of actomyosin contractility are often associated with particular epithelial tissue shape changes during development. For example, a planar polarized pattern of myosin II localization regulated by Rho1 signaling during Drosophila body axis elongation is thought to drive cell behaviors that contribute to convergent extension. This study developed optogenetic tools to activate (optoGEF) or deactivate (optoGAP) Rho1 signaling. These tools were used to manipulate myosin patterns at the apical side of the germband epithelium during Drosophila axis elongation, and the effects on contractile cell behaviors were analyzed. Uniform activation or inactivation of Rho1 signaling across the apical surface of the germband is sufficient to disrupt the planar polarized pattern of myosin at cell junctions on the timescale of 3-5 min, leading to distinct changes in junctional and medial myosin patterns in optoGEF and optoGAP embryos. These two perturbations to Rho1 activity both disrupt axis elongation and cell intercalation These studies demonstrate that acute optogenetic perturbations to Rho1 activity are sufficient to rapidly override the endogenous planar polarized myosin pattern in the germband during axis elongation. Moreover, these results reveal that the levels of Rho1 activity and the balance between medial and junctional myosin play key roles, not only in organizing the cell rearrangements that are known to directly contribute to axis elongation, but also in regulating cell area fluctuations and cell packings, which have been proposed to be important factors influencing the mechanics of tissue deformation and flow.
Bonello, T., Aguilar-Aragon, M., Tournier, A. and Thompson, B. J. (2021). A picket fence function for adherens junctions in epithelial cell polarity. Cells Dev: 203719. PubMed ID: 34242843
Summary:
Adherens junctions are a defining feature of all epithelial cells, providing cell-cell adhesion and contractile ring formation that is essential for cell and tissue morphology. In Drosophila, adherens junctions are concentrated between the apical and basolateral plasma membrane domains, defined by aPKC-Par6-Baz and Lgl/Dlg/Scrib, respectively. Whether adherens junctions contribute to apical-basal polarization itself has been unclear because neuroblasts exhibit apical-basal polarization of aPKC-Par6-Baz and Lgl in the absence of adherens junctions. This study shows that, upon disruption of adherens junctions in epithelial cells, apical polarity determinants such as aPKC can still segregate from basolateral Lgl, but lose their sharp boundaries and also overlap with Dlg and Scrib - similar to neuroblasts. In addition, control of apical versus basolateral domain size is lost, along with control of cell shape, in the absence of adherens junctions. Manipulating the levels of apical Par3/Baz or basolateral Lgl polarity determinants in experiments and in computer simulations confirms that adherens junctions provide a 'picket fence' diffusion barrier that restricts the spread of polarity determinants along the membrane to enable precise domain size control. Movement of adherens junctions in response to mechanical forces during morphogenetic change thus enables spontaneous adjustment of apical versus basolateral domain size as an emergent property of the polarising system.
Gillard, G., Girdler, G. and Roper, K. (2021). A release-and-capture mechanism generates an essential non-centrosomal microtubule array during tube budding. Nat Commun 12(1): 4096. PubMed ID: 34215746
Summary:
Non-centrosomal microtubule arrays serve crucial functions in cells, yet the mechanisms of their generation are poorly understood. During budding of the epithelial tubes of the salivary glands in the Drosophila embryo, it has been demonstrated that the activity of pulsatile apical-medial actomyosin depends on a longitudinal non-centrosomal microtubule array. This study uncovered that the exit from the last embryonic division cycle of the epidermal cells of the salivary gland placode leads to one centrosome in the cells losing all microtubule-nucleation capacity. This restriction of nucleation activity to the second, Centrobin-enriched, centrosome is key for proper morphogenesis. Furthermore, the microtubule-severing protein Katanin and the minus-end-binding protein Patronin accumulate in an apical-medial position only in placodal cells. Loss of either in the placode prevents formation of the longitudinal microtubule array and leads to loss of apical-medial actomyosin and impaired apical constriction. A mechanism is proposed whereby Katanin-severing at the single active centrosome releases microtubule minus-ends that are then anchored by apical-medial Patronin to promote formation of the longitudinal microtubule array crucial for apical constriction and tube formation.
Nabais, C., Pessoa, D., de-Carvalho, J., van Zanten, T., Duarte, P., Mayor, S., Carneiro, J., Telley, I. A. and Bettencourt-Dias, M. (2021). Plk4 triggers autonomous de novo centriole biogenesis and maturation. J Cell Biol 220(5). PubMed ID: 33760919
Summary:
Centrioles form centrosomes and cilia. In most proliferating cells, centrioles assemble through canonical duplication, which is spatially, temporally, and numerically regulated by the cell cycle and the presence of mature centrioles. However, in certain cell types, centrioles assemble de novo, yet by poorly understood mechanisms. This study established a controlled system to investigate de novo centriole biogenesis, using Drosophila melanogaster egg explants overexpressing Polo-like kinase 4 (Plk4), a trigger for centriole biogenesis. At a high Plk4 concentration, centrioles form de novo, mature, and duplicate, independently of cell cycle progression and of the presence of other centrioles. Plk4 concentration determines the temporal onset of centriole assembly. Moreover, the results suggest that distinct biochemical kinetics regulate de novo and canonical biogenesis. Finally, which other factors modulate de novo centriole assembly was investigate, and proteins of the pericentriolar material (PCM), and in particular γ-tubulin, were found to promote biogenesis, likely by locally concentrating critical components.
Hahn, I., Voelzmann, A., Parkin, J., Fulle, J. B., Slater, P. G., Lowery, L. A., Sanchez-Soriano, N. and Prokop, A. (2021). Tau, XMAP215/Msps and Eb1 co-operate interdependently to regulate microtubule polymerisation and bundle formation in axons. PLoS Genet 17(7): e1009647. PubMed ID: 34228717
Summary:
The formation and maintenance of microtubules requires their polymerisation, but little is known about how this polymerisation is regulated in cells. Focussing on the essential microtubule bundles in axons of Drosophila and Xenopus neurons, this study showed that the plus-end scaffold Eb1, the polymerase XMAP215/Msps and the lattice-binder Tau co-operate interdependently to promote microtubule polymerisation and bundle organisation during axon development and maintenance. Eb1 and XMAP215/Msps promote each other's localisation at polymerising microtubule plus-ends. Tau outcompetes Eb1-binding along microtubule lattices, thus preventing depletion of Eb1 tip pools. The three factors genetically interact and show shared mutant phenotypes: reductions in axon growth, comet sizes, comet numbers and comet velocities, as well as prominent deterioration of parallel microtubule bundles into disorganised curled conformations. This microtubule curling is caused by Eb1 plus-end depletion which impairs spectraplakin-mediated guidance of extending microtubules into parallel bundles. This demonstration that Eb1, XMAP215/Msps and Tau co-operate during the regulation of microtubule polymerisation and bundle organisation, offers new conceptual explanations for developmental and degenerative axon pathologies. Home page: The Interactive Fly© 2020 Thomas B. Brody, Ph.D.

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