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Wednesday, November 27th 2019 - Adult neural function

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Smith, P., Buhl, E., Tsaneva-Atanasova, K. and Hodge, J. J. L. (2019). Shaw and Shal voltage-gated potassium channels mediate circadian changes in Drosophila clock neuron excitability. J Physiol. PubMed ID: 31612994
Summary:
Like in mammals, Drosophila circadian clock neurons display rhythms of activity with higher action potential firing rates and more positive resting membrane potentials during the day. This rhythmic excitability has been widely observed but, critically, its regulation remains unresolved. This study has characterized and modeled the changes underlying these electrical activity rhythms in the lateral ventral clock neurons (LNvs). Currents mediated by the voltage-gated potassium channels Shaw (Kv3) and Shal (Kv4) oscillate in a circadian manner. Disruption of these channels, by expression of dominant negative (DN) subunits, leads to changes in circadian locomotor activity and shortens lifespan. LNv whole-cell recordings then show that changes in Shaw and Shal currents drive changes in action potential firing rate and that these rhythms are abolished when the circadian molecular clock is stopped. A whole-cell biophysical model using Hodgkin-Huxley equations can recapitulate these changes in electrical activity. Based on this model and by using dynamic clamp to manipulate clock neurons directly, it is possible to rescue the pharmacological block of Shaw and Shal, restore the firing rhythm, and thus demonstrate the critical importance of Shaw and Shal. Together, these findings point to a key role for Shaw and Shal in controlling circadian firing of clock neurons and show that changes in clock neuron currents can account for this. Moreover, with dynamic clamp it is possible to switch the LNvs between morning-like and evening-like states of electrical activity. It is concluded that changes in Shaw and Shal underlie the daily oscillation in LNv firing rate.
Baik, L. S., Au, D. D., Nave, C., Foden, A. J., Enrriquez-Villalva, W. K. and Holmes, T. C. (2019). Distinct mechanisms of Drosophila CRYPTOCHROME-mediated light-evoked membrane depolarization and in vivo clock resetting. Proc Natl Acad Sci U S A 116(46): 23339-23344. PubMed ID: 31659046
Summary:
Drosophila CRYPTOCHROME (dCRY) mediates electrophysiological depolarization and circadian clock resetting in response to blue or ultraviolet (UV) light. Whether electron transfer down a conserved chain of tryptophan residues underlies biological responses following dCRY light activation has been controversial. To examine these issues in in vivo and in ex vivo whole-brain preparations, transgenic flies were generated expressing tryptophan mutant dCRYs in the conserved electron transfer chain, and then neuronal electrophysiological phototransduction and behavioral responses to light were measured. Electrophysiological-evoked potential analysis shows that dCRY mediates UV and blue-light-evoked depolarizations that are long lasting, persisting for nearly a minute. Surprisingly, dCRY appears to mediate red-light-evoked depolarization in wild-type flies, absent in both cry-null flies, and following acute treatment with the flavin-specific inhibitor diphenyleneiodonium in wild-type flies. This suggests a previously unsuspected functional signaling role for a neutral semiquinone flavin state (FADH(*)) for dCRY. The W420 tryptophan residue located closest to the FAD-dCRY interaction site is critical for blue- and UV-light-evoked electrophysiological responses, while other tryptophan residues within electron transfer distance to W420 do not appear to be required for light-evoked electrophysiological responses. Mutation of the dCRY tryptophan residue W342, more distant from the FAD interaction site, mimics the cry-null behavioral light response to constant light exposure. These data indicate that light-evoked dCRY electrical depolarization and clock resetting are mediated by distinct mechanisms.
Aso, Y., Ray, R. P., Long, X., Bushey, D., Cichewicz, K., Ngo, T. T., Sharp, B., Christoforou, C., Hu, A., Lemire, A. L., Tillberg, P., Hirsh, J., Litwin-Kumar, A. and Rubin, G. M. (2019). Nitric oxide acts as a cotransmitter in a subset of dopaminergic neurons to diversify memory dynamics. Elife 8. PubMed ID: 31724947
Summary:
Animals employ diverse learning rules and synaptic plasticity dynamics to record temporal and statistical information about the world. However, the molecular mechanisms underlying this diversity are poorly understood. The anatomically defined compartments of the insect mushroom body function as parallel units of associative learning, with different learning rates, memory decay dynamics and flexibility. This study shows that nitric oxide (NO) acts as a neurotransmitter in a subset of dopaminergic neurons in Drosophila. NO's effects develop more slowly than those of dopamine and depend on soluble guanylate cyclase in postsynaptic Kenyon cells. NO acts antagonistically to dopamine; it shortens memory retention and facilitates the rapid updating of memories. The interplay of NO and dopamine enables memories stored in local domains along Kenyon cell axons to be specialized for predicting the value of odors based only on recent events. These results provide key mechanistic insights into how diverse memory dynamics are established in parallel memory systems.
Zhang, X., Noyes, N. C., Zeng, J., Li, Y. and Davis, R. L. (2019). Aversive training induces both pre- and postsynaptic suppression in Drosophila. J Neurosci. PubMed ID: 31558620
Summary:
The alpha'beta' subtype of Drosophila mushroom body neurons (MBn) is required for memory acquisition, consolidation and early memory retrieval after aversive olfactory conditioning. However, in vivo functional imaging studies have failed to detect an early forming memory trace in these neurons as reflected by an enhanced G-CaMP signal in response to presentation of the learned odor. This study shows that aversive olfactory conditioning suppresses the calcium responses to the learned odor in both alpha'3 and alpha'2 axon segments of alpha'beta' MBn and in the dendrites of alpha'3 MBOn immediately after conditioning using female flies. Notably, the cellular memory traces in both alpha'3 MBn and alpha'3 MBOn are short-lived and persist for less than 30 min. The suppressed response in alpha'3 MBn is accompanied by a reduction of acetylcholine (ACh) release, suggesting that the memory trace in postsynaptic alpha'3 MBOn may simply reflect the suppression in presynaptic alpha'3 MBn. Furthermore, this study shows that the alpha'3 MBn memory trace does not occur from the inhibition of GABAergic neurons via GABAA receptor activation. Since activation of the alpha'3 MBOn drives approach behavior of adult flies, the results demonstrate that aversive conditioning promotes avoidance behavior through suppression of the alpha'3 MBn-MBOn circuit.
Zhao, B., Sun, J., Zhang, X., Mo, H., Niu, Y., Li, Q., Wang, L. and Zhong, Y. (2019). Long-term memory is formed immediately without the need for protein synthesis-dependent consolidation in Drosophila. Nat Commun 10(1): 4550. PubMed ID: 31591396
Summary:
It is believed that long-term memory (LTM) cannot be formed immediately because it must go through a protein synthesis-dependent consolidation process. However, the current study uses Drosophila aversive olfactory conditioning to show that such processes are dispensable for context-dependent LTM (cLTM). Single-trial conditioning yields cLTM that is formed immediately in a protein-synthesis independent manner and is sustained over 14 days without decay. Unlike retrieval of traditional LTM, which requires only the conditioned odour and is mediated by mushroom-body neurons, cLTM recall requires both the conditioned odour and reinstatement of the training-environmental context. It is mediated through lateral-horn neurons that connect to multiple sensory brain regions. The cLTM cannot be retrieved if synaptic transmission from any one of these centres is blocked, with effects similar to those of altered encoding context during retrieval. The present study provides strong evidence that long-term memory can be formed easily without the need for consolidation.
Babski, H., Jovanic, T., Surel, C., Yoshikawa, S., Zwart, M. F., Valmier, J., Thomas, J. B., Enriquez, J., Carroll, P. and Garces, A. (2019). A GABAergic Maf-expressing interneuron subset regulates the speed of locomotion in Drosophila. Nat Commun 10(1): 4796. PubMed ID: 31641138
Summary:
Interneurons (INs) coordinate motoneuron activity to generate appropriate patterns of muscle contractions, providing animals with the ability to adjust their body posture and to move over a range of speeds. In Drosophila larvae several IN subtypes have been morphologically described and their function well documented. However, the general lack of molecular characterization of those INs prevents the identification of evolutionary counterparts in other animals, limiting the understanding of the principles underlying neuronal circuit organization and function. This study characterized a restricted subset of neurons in the nerve cord expressing the Maf transcription factor Traffic Jam (TJ). TJ(+) neurons were found to be highly diverse and selective activation of these different subtypes disrupts larval body posture and induces specific locomotor behaviors. Finally, this study shows that a small subset of TJ(+) GABAergic INs, singled out by the expression of a unique transcription factors code, controls larval crawling speed.

Tuesday, November 26th - Vesicles and Synapse

Kamimura, K., Odajima, A., Ikegawa, Y., Maru, C. and Maeda, N. (2019). The HSPG glypican regulates experience-dependent synaptic and behavioral plasticity by modulating the non-canonical BMP pathway.. Cell Rep 28(12): 3144-3156.e3144. PubMed ID: 31533037
Summary:
Under food deprivation conditions, Drosophila larvae exhibit increases in locomotor speed and synaptic bouton numbers at neuromuscular junctions (NMJs). Octopamine, the invertebrate counterpart of noradrenaline, plays critical roles in this process; however, the underlying mechanisms remain unclear. This study shows that a glypican (Dlp) negatively regulates type I synaptic bouton formation, postsynaptic expression of GluRIIA, and larval locomotor speed. Starvation-induced octopaminergic signaling decreases Dlp expression, leading to increases in synapse formation and locomotion. Dlp is expressed by postsynaptic muscle cells and suppresses the non-canonical BMP pathway, which is composed of the presynaptic BMP receptor Wit and postsynaptic GluRIIA-containing ionotropic glutamate receptor. During starvation, decreases in Dlp increase non-canonical BMP signaling, leading to increases in GluRIIA expression, type I bouton number, and locomotor speed. These results demonstrate that octopamine controls starvation-induced neural plasticity by regulating Dlp and provides insights into how proteoglycans can influence behavioral and synaptic plasticity.
Cai, X., Fahmy, K. and Baumgartner, S. (2019). bicoid RNA localization requires the trans-Golgi network. Hereditas 156: 30. PubMed ID: 31528161
Summary:
The formation of the bicoid (bcd) mRNA gradient is a crucial step for Bcd protein gradient formation in Drosophila. In the past, a microtubule (MT)-based cortical network had been shown to be indispensable for bcd mRNA transport to the posterior. This study reports the identification of a MT-binding protein CLASP/Chb as the first component associated with this cortical MT network. Since CLASPs in vertebrates were shown to serve as an acentriolar microtubule organization center (aMTOC) in concert with trans-Golgi proteins, this study examined the effect of the Drosophila trans-Golgins on bcd localization and gradient formation. Using a genetic approach, it was demonstrated that the Drosophila trans-Golgins dGCC88, dGolgin97 and dGCC185 indeed affect bcd mRNA localization during oocyte development. Consequently, the bcd mRNA is already mislocalized before the egg is fertilized. The expression domains of genes downstream of the hierarchy of bcd, e.g. of the gap gene empty spiracles or of the pair-rule gene even-skipped are changed, indicating an altered segmental anlagen, due to a faulty bcd gradient. Thus, at the end of embryogenesis, trans-Golgin mutants show bcd-like cuticle phenotypes. These data provides evidence that the Golgi as a cellular member of the secretory pathway exerts control on bcd localization which indicates that bcd gradient formation is probably more intricate than previously presumed.
Arancibia, D., Lira, M., Cruz, Y., Barrera, D. P., Montenegro-Venegas, C., Godoy, J. A., Garner, C. C., Inestrosa, N. C., Gundelfinger, E. D., Zamorano, P. and Torres, V. I. (2019). Serine-arginine protein kinase SRPK2 modulates the assembly of the active zone scaffolding protein CAST1/ERC2. Cells 8(11). PubMed ID: 31671734
Summary:
Neurons release neurotransmitters at a specialized region of the presynaptic membrane, the active zone (AZ), where a complex meshwork of proteins organizes the release apparatus. The formation of this proteinaceous cytomatrix at the AZ (CAZ) depends on precise homo- and hetero-oligomerizations of distinct CAZ proteins. The CAZ protein CAST1/ERC2 contains four coiled-coil (CC) domains that interact with other CAZ proteins, but also promote self-assembly, which is an essential step for its integration during AZ formation. The self-assembly and synaptic recruitment of the Drosophila protein Bruchpilot (BRP), a partial homolog of CAST1/ERC2, is modulated by the serine-arginine protein kinase (SRPK79D). This study demonstrates that overexpression of the vertebrate SRPK2 regulates the self-assembly of CAST1/ERC2 in HEK293T, SH-SY5Y and HT-22 cells and the CC1 and CC4 domains are involved in this process. Moreover, the isoform SRPK2 forms a complex with CAST1/ERC2 when co-expressed in HEK293T and SH-SY5Y cells. More importantly, SRPK2 is present in brain synaptic fractions and synapses, suggesting that this protein kinase might control the level of self-aggregation of CAST1/ERC2 in synapses, and thereby modulate presynaptic assembly.
Hiramatsu, N., Tago, T., Satoh, T. and Satoh, A. K. (2019). ER membrane protein complex is required for the insertions of late-synthesized transmembrane helices of Rh1 in Drosophila photoreceptors. Mol Biol Cell: mbcE19080434. PubMed ID: 31553680
Summary:
Most of membrane proteins are synthesized on and inserted into the membrane of the endoplasmic reticulum (ER), in eukaryote. The widely conserved ER membrane protein complex (EMC) facilitates the biogenesis of a wide range of membrane proteins. This study investigated the EMC function using Drosophila photoreceptor as a model system. The EMC was necessary only for the biogenesis of a subset of multi-pass membrane proteins such as rhodopsin (Rh1), TRP, TRPL, Csat, Cni, SERCA, and Na(+)K(+)ATPase alpha, but not for that of secretory or single-pass membrane proteins. Additionally, in EMC-deficient cells, Rh1 was translated to its C-terminus but degraded independently from ER-associated degradation. Thus, EMC exerted its effect after translation but before or during the membrane integration of transmembrane domains (TMDs). Finally, this study found that EMC was not required for the stable expression of the first three TMDs of Rh1 but was required for that of the fourth and fifth TMDs. These results suggested that EMC is required for the ER membrane insertion of succeeding TMDs of multi-pass membrane proteins.
Kim, J. H. and Chen, E. H. (2019). The fusogenic synapse at a glance. J Cell Sci 132(18). PubMed ID: 31527149
Summary:
Cell-cell fusion is a fundamental process underlying fertilization, development, regeneration and physiology of metazoans. It is a multi-step process involving cell recognition and adhesion, actin cytoskeletal rearrangements, fusogen engagement, lipid mixing and fusion pore formation, ultimately resulting in the integration of two fusion partners. This study focused on the asymmetric actin cytoskeletal rearrangements at the site of fusion, known as the fusogenic synapse, which was first discovered during myoblast fusion in Drosophila embryos and later also found in mammalian muscle and non-muscle cells. At the asymmetric fusogenic synapse, actin-propelled invasive membrane protrusions from an attacking fusion partner trigger actomyosin-based mechanosensory responses in the receiving cell. The interplay between the invasive and resisting forces generated by the two fusion partners puts the fusogenic synapse under high mechanical tension and brings the two cell membranes into close proximity, promoting the engagement of fusogens to initiate fusion pore formation.
Chaudhary, V. and Boutros, M. (2019). Exocyst-mediated apical Wg secretion activates signaling in the Drosophila wing epithelium. PLoS Genet 15(9): e1008351. PubMed ID: 31527874
Summary:
Wnt proteins are secreted signaling factors that regulate cell fate specification and patterning decisions throughout the animal kingdom. In the Drosophila wing epithelium, Wingless (Wg, the homolog of Wnt1) is secreted from a narrow strip of cells at the dorsal-ventral boundary. However, the route of Wg secretion in polarized epithelial cells remains poorly understood and key proteins involved in this process are still unknown. This study performed an in vivo RNAi screen and identified members of the exocyst complex to be required for apical but not basolateral Wg secretion. Specifically blocking the apical Wg secretion leads to reduced downstream signaling. Using an in vivo 'temporal-rescue' assay, these results further indicate that apically secreted Wg activates target genes that require high signaling activity. In conclusion, these results demonstrate that the exocyst is required for an apical route of Wg secretion from polarized wing epithelial cells.

Monday, November 25th - Behavior

Aggarwal, A., Reichert, H. and VijayRaghavan, K. (2019). A locomotor assay reveals deficits in heterozygous Parkinson's disease model and proprioceptive mutants in adult Drosophila. Proc Natl Acad Sci U S A. PubMed ID: 31748267
Summary:
Severe locomotor impairment is a common phenotype of neurodegenerative disorders such as Parkinson's disease (PD). Drosophila models of PD, studied for more than a decade, have helped in understanding the interaction between various genetic factors, such as parkin and PINK1, in this disease. To characterize locomotor behavioral phenotypes for these genes, fly climbing assays have been widely used. While these simple current assays for locomotor defects in Drosophila mutants measure some locomotor phenotypes well, it is possible that detection of subtle changes in behavior is important to understand the manifestation of locomotor disorders. This study introduces a climbing behavior assay which provides such fine-scale behavioral data and tests this proposition for the Drosophila model. This inexpensive, fully automated assay was used to quantitatively characterize the climbing behavior at high parametric resolution in 3 contexts. First, wild-type flies were characterized, and a hitherto unknown sexual dimorphism in climbing behavior was uncovered. Second, climbing behavior was studied of heterozygous mutants of genes implicated in the fly PD model, and previously unreported prominent locomotor defects were revealed in some of these heterozygous fly lines. Finally, locomotor defects were studied in a homozygous proprioceptory mutation (Trp-gamma1) known to affect fine motor control in Drosophila. Moreover, aberrant geotactic behavior was identified in Trp-gamma1 mutants, thereby opening up a finer assay for geotaxis and its genetic basis. This assay is therefore a cost-effective, general tool for measuring locomotor behaviors of wild-type and mutant flies in fine detail and can reveal subtle motor defects.
Troutwine, B., Park, A., Velez-Hernandez, M. E., Lew, L., Mihic, S. J. and Atkinson, N. S. (2019). F654A and K558Q Mutations in NMDA Receptor 1 affect ethanol-induced behaviors in Drosophila. Alcohol Clin Exp Res. PubMed ID: 31593608
Summary:
NMDA receptors regulate synaptic plasticity and modulate a wide variety of behaviors. Mammalian NMDA receptors are inhibited by ethanol even at low concentrations. In mice, the F639A mutation in transmembrane domain 3 of the NR1 subunit reduces ethanol sensitivity of the receptor and in some paradigms, reduces behavioral ethanol sensitivity and increases ethanol consumption. This study tested the fly equivalent of the F639A and K544Q mutations for effects on ethanol sensitivity. Drosophila shows a high degree of behavioral and mechanistic conservation in its responses to ethanol. This study used homologous recombination and CRISPR/Cas9 genome editing to generate amino acid changes in the Drosophila NMDAR1 gene, yielding F654A and K558Q alleles. Animals were tested for the degree of ethanol sensitivity, the ability to acquire tolerance to ethanol, ethanol drinking preference, circadian rhythmicity, learning, and locomotor defects. Mutating the NMDAR1 channel was also found to reduce ethanol sensitivity in adult flies. However, in flies, it was the K558Q mutation (orthologous to K544Q in mice) that reduces ethanol sensitivity in a recovery-from-sedation assay. The effects of the F654A mutation (orthologous to F639A in mice) were substantially different in flies than in mammals. In flies, F654A mutation produces phenotypes opposite those in mammals. In flies, the mutant allele is homozygous viable, does not seem to affect health, and increases ethanol sensitivity. Both mutations increased feeding but did not alter the animal's preference for 5% ethanol food. F654A depressed circadian rhythmicity and the capacity of males to court, but it did not depress the capacity for associative learning. K554Q, on the other hand, has little effect on circadian rhythmicity, only slightly suppresses male courtship, and is a strong learning mutant. It is concluded that mutations in transmembrane domain 3 and in the extracellular vestibule calcium binding site of the NR1 NMDA subunit affect ethanol sensitivity in Drosophila.
Yen, H. H., Han, R. and Lo, C. C. (2019). Quantification of visual fixation behavior and spatial orientation memory in Drosophila melanogaster. Front Behav Neurosci 13: 215. PubMed ID: 31572145
Summary:
Drosophila melanogaster has been shown to exhibit short-term orientation memory by fixating on orientations toward previously displayed visual landmarks. However, the fixation behavior varies and is often mixed with other types of movement. Therefore, carefully designed statistical measures are required in order to properly describe the characteristics of the fixation behavior and to quantify the orientation memory exhibited by the fruit flies. To this end, this paper proposes a set of analytical methods. First, the deviation angle, which is used to quantify the deviation of the fruit fly's heading from the landmark positions, is defined. The deviation angle is defined based on the fruit fly's perspective and is able to reveal more task-relevant movement patterns than the commonly used definition which is based on the "observer's perspective." A temporal deviation angle plot is introduced that visually presents the complex movement pattern as a function of time. Next, a fixation index is defined that tolerates fluctuation in the movement and performs better in quantifying the level of fixation behavior, or the orientation memory, than the conventional method.
Sehdev, A., Mohammed, Y. G., Tafrali, C. and Szyszka, P. (2019). Social foraging extends associative odor-food memory expression in an automated learning assay for Drosophila melanogaster. J Exp Biol 222(Pt 19). PubMed ID: 31527181
Summary:
Animals socially interact during foraging and share information about the quality and location of food sources. The mechanisms of social information transfer during foraging have been mostly studied at the behavioral level, and its underlying neural mechanisms are largely unknown. Fruit flies have become a model for studying the neural bases of social information transfer, because they provide a large genetic toolbox to monitor and manipulate neuronal activity, and they show a rich repertoire of social behaviors. Fruit flies aggregate, they use social information for choosing a suitable mating partner and oviposition site, and they show better aversive learning when in groups. However, the effects of social interactions on associative odor-food learning have not yet been investigated. This paper presents an automated learning and memory assay for walking flies that allows the study of the effect of group size on social interactions and on the formation and expression of associative odor-food memories. Both inter-fly attraction and the duration of odor-food memory expression were found to increase with group size. This study opens up opportunities to investigate how social interactions during foraging are relayed in the neural circuitry of learning and memory expression.
Xie, X., Tabuchi, M., Corver, A., Duan, G., Wu, M. N. and Kolodkin, A. L. (2019). Semaphorin 2b regulates sleep-circuit formation in the Drosophila central brain. Neuron. PubMed ID: 31564592
Summary:
The fan-shaped body (FB) neuropil in the Drosophila brain central complex (CX) controls a variety of adult behaviors, including navigation and sleep. How neuronal processes are organized into precise layers and columns in the FB and how alterations in FB neural-circuit wiring affect animal behaviors are unknown. This study reports that secreted semaphorin 2b (Sema-2b) acts through its transmembrane receptor Plexin B (PlexB) to locally attract neural processes to specific FB laminae. Aberrant Sema-2b/PlexB signaling leads to select disruptions in neural lamination, and these disruptions result in the formation of ectopic inhibitory connections between subsets of FB neurons. These structural alternations and connectivity defects are associated with changes in fly sleep and arousal, emphasizing the importance of lamination-mediated neural wiring in a central brain region critical for normal sleep behavior.
Tsai, K. T. and Chou, Y. H. (2019). Random walk revisited: Quantification and comparative analysis of Drosophila walking trajectories. iScience 19: 1145-1159. PubMed ID: 31541919
Summary:
Walking trajectory is frequently measured to assess animal behavior. Air-supported spherical treadmills have been developed for real-time monitoring of animal walking trajectories. However, current systems for mice mainly employ computer mouse microcameras (chip-on-board sensors) to monitor ball motion, and these detectors exhibit technical issues with focus and rotation scale. In addition, computational methods to analyze and quantify the "random walk" of organisms are under-developed. This work has overcome the hurdle of frame-to-signal translation to develop a treadmill system with camera-based detection. Moreover, a package of mathematical methods were generated to quantify distinct aspects of Drosophila walking trajectories. By extracting and quantifying certain features of walking dynamics with high temporal resolution, it was found that depending on their internal state, flies employ different walking strategies to approach environmental cues. This camera-based treadmill system and method package may also be applicable to monitor the walking trajectories of other diverse animal species.

Friday, November 22nd - Enhancers and gene regulation

Yang, Y., Fang, Q. and Shen, H. B. (2019). Predicting gene regulatory interactions based on spatial gene expression data and deep learning. PLoS Comput Biol 15(9): e1007324. PubMed ID: 31527870
Summary:
Reverse engineering of gene regulatory networks (GRNs) is a central task in systems biology. Most of the existing methods for GRN inference rely on gene co-expression analysis or TF-target binding information, where the determination of co-expression is often unreliable merely based on gene expression levels, and the TF-target binding data from high-throughput experiments may be noisy, leading to a high ratio of false links and missed links, especially for large-scale networks. In recent years, the microscopy images recording spatial gene expression have become a new resource in GRN reconstruction, as the spatial and temporal expression patterns contain much abundant gene interaction information. Till now, the spatial expression resources have been largely underexploited, and only a few traditional image processing methods have been employed in the image-based GRN reconstruction. Moreover, co-expression analysis using conventional measurements based on image similarity may be inaccurate, because it is the local-pattern consistency rather than global-image-similarity that determines gene-gene interactions. This study presents GripDL (Gene regulatory interaction prediction via Deep Learning), which incorporates high-confidence TF-gene regulation knowledge from previous studies, and constructs GRNs for Drosophila eye development based on Drosophila embryonic gene expression images. Benefitting from the powerful representation ability of deep neural networks and the supervision information of known interactions, the new method outperforms traditional methods with a large margin and reveals new intriguing knowledge about Drosophila eye development.
Zhou, J., Schor, I. E., Yao, V., Theesfeld, C. L., Marco-Ferreres, R., Tadych, A., Furlong, E. E. M. and Troyanskaya, O. G. (2019). Accurate genome-wide predictions of spatio-temporal gene expression during embryonic development. PLoS Genet 15(9): e1008382. PubMed ID: 31553718
Summary:
This study applied a machine learning approach to the problem of timing and location of gene expression by training models on all public gene expression and chromatin data, even from whole-organism experiments, to provide genome-wide, quantitative spatio-temporal predictions for all genes. Structured in silico nano-dissection, a computational approach that predicts gene expression in >200 tissue-developmental stages, was developed. The algorithm integrates expression signals from a compendium of 6,378 genome-wide expression and chromatin profiling experiments in a cell lineage-aware fashion. Performance was systematically evaluated via cross-validation and experimentally confirmed 22 new predictions for four different embryonic tissues. The model also predicts complex, multi-tissue expression and developmental regulation with high accuracy. The potential was further shown of applying these genome-wide predictions to extract tissue specificity signals from non-tissue-dissected experiments, and to prioritize tissues and stages for disease modeling. This resource, together with the exploratory tools are freely available at webserver http://find.princeton.edu, which provides a valuable tool for a range of applications, from predicting spatio-temporal expression patterns to recognizing tissue signatures from differential gene expression profiles.
King, T., Johnson, J. E. and Bateman, J. R. (2019). Position effects influence transvection in Drosophila melanogaster. Genetics. PubMed ID: 31611231
Summary:
Transvection is an epigenetic phenomenon wherein regulatory elements communicate between different chromosomes in trans, and is thereby dependent upon the three-dimensional organization of the genome. Transvection is best understood in Drosophila, where homologous chromosomes are closely paired in most somatic nuclei, although similar phenomena have been observed in other species. Previous data have supported that the Drosophila genome is generally permissive to enhancer action in trans, a form of transvection where an enhancer on one homolog activates gene expression from a promoter on a paired homolog. However, the capacity of different genomic positions to influence the quantitative output of transvection has yet to be addressed. To investigate this question, this study employed a transgenic system that assesses and compares enhancer action in cis and in trans at defined chromosomal locations. Using the strong synthetic eye-specific enhancer GMR, this study showed that loci supporting strong cis-expression also support robust enhancer action in trans, whereas locations with weaker cis-expression show greatly reduced transvection in a fluorescent reporter assay. A subsequent analysis is consistent with a model wherein the chromatin state of the transgenic insertion site is the primary determinant of the degree to which enhancer action in trans will be supported, whereas other factors such as locus-specific variation in somatic homolog pairing are of less importance in influencing position effects on transvection.
Barr, K., Reinitz, J. and Radulescu, O. (2019). An in silico analysis of robust but fragile gene regulation links enhancer length to robustness. PLoS Comput Biol 15(11): e1007497. PubMed ID: 31730659
Summary:
Organisms must ensure that expression of genes is directed to the appropriate tissues at the correct times, while simultaneously ensuring that these gene regulatory systems are robust to perturbation. This idea is captured by a mathematical concept called r-robustness, which says that a system is robust to a perturbation in up to r - 1 randomly chosen parameters. r-robustness implies that the biological system has a small number of sensitive parameters and that this number can be used as a robustness measure. This work used this idea to investigate the robustness of gene regulation using a sequence level model of the Drosophila melanogaster gene even-skipped. Robustness is considered with respect to mutations of the enhancer sequence and with respect to changes of the transcription factor concentrations. Gene regulation was found to be r-robust with respect to mutations in the enhancer sequence, and a number of sensitive nucleotides were identified. In both natural and in silico predicted enhancers, the number of nucleotides that are sensitive to mutation correlates negatively with the length of the sequence, meaning that longer sequences are more robust. The exact degree of robustness obtained is dependent not only on DNA sequence, but also on the local concentration of regulatory factors. Gene regulation can be remarkably sensitive to changes in transcription factor concentrations at the boundaries of expression features, while it is robust to perturbation elsewhere.
McDonald, S. I., Beachum, A. N., Hinnant, T. D., Blake, A. J., Bynum, T., Hickman, E. P., Barnes, J., Churchill, K. L., Roberts, T. S., Zangwill, D. E. and Ables, E. T. (2019). Novel cis-regulatory regions in ecdysone responsive genes are sufficient to promote gene expression in Drosophila ovarian cells. Gene Expr Patterns: 119074. PubMed ID: 31563631
Summary:
The insect steroid hormone ecdysone is a key regulator of oogenesis in Drosophila melanogaster and many other species. Despite the diversity of cellular functions of ecdysone in oogenesis, the molecular regulation of most ecdysone-responsive genes in ovarian cells remains largely unexplored. A functional screen was performed using the UAS/Gal4 system to identify non-coding cis-regulatory elements within well-characterized ecdysone-response genes capable of driving transcription of an indelible reporter in ovarian cells. Using two publicly available transgenic collections (the FlyLight and Vienna Tiles resources), 62 Gal4 drivers were tested corresponding to ecdysone-response genes EcR, usp, E75, br, ftz-f1 and Hr3. 31 lines were observed that were sufficient to drive a UAS-lacZ reporter in discrete cell populations in the ovary. Reporter expression was reproducibly observed in both somatic and germ cells at distinct stages of oogenesis, including those previously characterized as critical points of ecdysone regulation. These studies identified several useful new reagents, adding to the UAS/Gal4 toolkit available for genetic analysis of oogenesis in Drosophila. Further, this study provides novel insight into the molecular regulation of ecdysone signaling in oogenesis.
Kyrchanova, O., Wolle, D., Sabirov, M., Kurbidaeva, A., Aoki, T., Maksimenko, O., Kyrchanova, M., Georgiev, P. and Schedl, P. (2019). Distinct elements confer the blocking and bypass functions of the Bithorax Fab-8 boundary. Genetics. PubMed ID: 31551239
Summary:
Boundaries in the Drosophila bithorax complex (BX-C) enable the regulatory domains that drive parasegment specific expression of the three Hox genes to function autonomously. The four regulatory domains (iab-5, iab-6, iab-7 and iab-8) that control the expression of the Abdominal-B (Abd-B) gene are located downstream of the transcription unit and are delimited by the Mcp, Fab-6, Fab-7 and Fab-8 boundaries. These boundaries function to block crosstalk between neighboring regulatory domains. In addition, three of the boundaries (Fab-6, Fab-7 and Fab-8) must also have bypass activity so that regulatory domains distal to the boundaries can contact the Abd-B promoter. In these studies a functional dissection was undertaken of the Fab-8 boundary using a boundary replacement strategy. The studies indicate that the Fab-8 boundary has two separable sub-elements. The distal sub-element blocks crosstalk, but can not support bypass. The proximal sub-element has only minimal blocking activity but is able to mediate bypass. A large multiprotein complex, the LBC, binds to sequences in the proximal sub-element and contributes to its bypass activity. The same LBC complex has been implicated in the bypass activity of the Fab-7 boundary.

Thursday, November 21st - Adult Physiology

Dmitrieva, A. S., Ivnitsky, S. B., Maksimova, I. A., Panchenko, P. L., Kachalkin, A. V. and Markov, A. V. (2019). Yeasts affect tolerance of Drosophila melanogaster to food substrate with high NaCl concentration. PLoS One 14(11): e0224811. PubMed ID: 31693706
Summary:
The ability of model animal species, such as Drosophila melanogaster, to adapt quickly to various adverse conditions has been shown in many experimental evolution studies. It is usually assumed by default that such adaptation is due to changes in the gene pool of the studied population of macroorganisms. At the same time, it is known that microbiome can influence biological processes in macroorganisms. In order to assess the possible impact of microbiome on adaptation, an evolutionary experiment was performed in which some D. melanogaster lines were reared on a food substrate with high NaCl concentration while the others were reared on the standard (favourable) substrate. The reproductive efficiency of experimental lines was evaluated on the high salt substrate three years after the experiment started. These tests confirmed that the lines reared on the salty substrate became more tolerant to high NaCl concentration. Moreover, pre-inoculation of the high salt medium with homogenized salt-tolerant flies tended to improve reproductive efficiency of naive flies on this medium (compared to pre-inoculation with homogenized control flies). The analysis of yeast microbiome in fly homogenates revealed significant differences in number and species richness of yeasts between salt-tolerant and control lines. It was also found that some individual yeast lines extracted from the salt-tolerant flies improved reproductive efficiency of naive flies on salty substrate (compared to baker's yeast and no yeast controls), whereas the effect of the yeast lines extracted from the control flies tended to be smaller. The yeast Starmerella bacillaris extracted from the salt-tolerant flies showed the strongest positive effect. This yeast is abundant in all salt-tolerant lines, and very rare or absent in all control lines. The results are consistent with the hypothesis that some components of the yeast microbiome of D. melanogaster contribute to to flies' tolerance to food substrate with high NaCl concentration.
Salachan, P. V., Burgaud, H. and Sorensen, J. G. (2019). Testing the thermal limits: Non-linear reaction norms drive disparate thermal acclimation responses in Drosophila melanogaster. J Insect Physiol 118: 103946. PubMed ID: 31525352
Summary:
Critical thermal limits are important ecological parameters for studying thermal biology and for modelling species' distributions under current and changing climatic conditions (including predicting the risk of extinction for species from future warming). However, estimates of the critical thermal limits are biased by the choice of assay and assay conditions, which differ among studies. Furthermore, estimates of the potential for phenotypic plasticity (thermal acclimation) to buffer against thermal variability are usually based on single assay conditions and (usually linear) extrapolation from a few acclimation temperatures. This study produced high resolution estimates of adult acclimation capacity for upper tolerance limits at different assay conditions (ramping rates and knock-down temperatures) using CTmax (dynamic) and knock-down (static) thermal assays in the model species Drosophila melanogaster. The reaction norms were found to be highly dependent on assay conditions. Progressively lower ramping rates or higher knock-down temperatures led to overall lower tolerance estimates. More surprisingly, extended assays (lower ramping rates or lower knock-down temperatures) also led to increasingly non-linear reaction norms for upper thermal tolerance across adult acclimation temperatures. These results suggest that the magnitude (capacity) and direction (beneficial or detrimental) of acclimation responses are highly sensitive to assay conditions. The results offer a framework for comparison of acclimation responses between different assay conditions and a potential for explaining disparate acclimation capacity theories. Cautious interpretation of acclimation capacities is advocated along with careful consideration of assay conditions, which should represent realistic environmental conditions based on species' ecological niches.
George, J., Tuomela, T., Kemppainen, E., Nurminen, A., Braun, S., Yalgin, C. and Jacobs, H. T. (2019). Mitochondrial dysfunction generates a growth-restraining signal linked to pyruvate in Drosophila larvae. Fly (Austin): 1-17. PubMed ID: 31526131
Summary:
The Drosophila bang-sensitive mutant tko25t, manifesting a global deficiency in oxidative phosphorylation due to a mitochondrial protein synthesis defect, exhibits a pronounced delay in larval development. Previous work has identified a number of metabolic abnormalities in tko25t larvae, including elevated pyruvate and lactate, and found the larval gut to be a crucial tissue for the regulation of larval growth in the mutant. This study established that expression of wild-type tko in any of several other tissues of tko25t also partially alleviates developmental delay. The effects appeared to be additive, whilst knockdown of tko in a variety of specific tissues phenocopied tko25t, producing developmental delay and bang-sensitivity. These findings imply the existence of a systemic signal regulating growth in response to mitochondrial dysfunction. Drugs and RNAi-targeted on pyruvate metabolism interacted with tko25t in ways that implicated pyruvate or one of its metabolic derivatives in playing a central role in generating such a signal. RNA-seq revealed that dietary pyruvate-induced changes in transcript representation were mostly non-coherent with those produced by tko25t or high-sugar, consistent with the idea that growth regulation operates primarily at the translational and/or metabolic level.
Rudman, S. M., Greenblum, S., Hughes, R. C., Rajpurohit, S., Kiratli, O., Lowder, D. B., Lemmon, S. G., Petrov, D. A., Chaston, J. M. and Schmidt, P. (2019). Microbiome composition shapes rapid genomic adaptation of Drosophila melanogaster. Proc Natl Acad Sci U S A 116(40): 20025-20032. PubMed ID: 31527278
Summary:
Population genomic data has revealed patterns of genetic variation associated with adaptation in many taxa. Yet understanding the adaptive process that drives such patterns is challenging; it requires disentangling the ecological agents of selection, determining the relevant timescales over which evolution occurs, and elucidating the genetic architecture of adaptation. Doing so for the adaptation of hosts to their microbiome is of particular interest with growing recognition of the importance and complexity of host-microbe interactions. This study tracked the pace and genomic architecture of adaptation to an experimental microbiome manipulation in replicate populations of Drosophila melanogaster in field mesocosms. Shifts in microbiome composition altered population dynamics and led to divergence between treatments in allele frequencies, with regions showing strong divergence found on all chromosomes. Moreover, at divergent loci previously associated with adaptation across natural populations, the more common allele was found in fly populations experimentally enriched for a certain microbial group was also more common in natural populations with high relative abundance of that microbial group. These results suggest that microbiomes may be an agent of selection that shapes the pattern and process of adaptation and, more broadly, that variation in a single ecological factor within a complex environment can drive rapid, polygenic adaptation over short timescales.
Dong, W., Dobler, R., Dowling, D. K. and Moussian, B. (2019). The cuticle inward barrier in Drosophila melanogaster is shaped by mitochondrial and nuclear genotypes and a sex-specific effect of diet. PeerJ 7: e7802. PubMed ID: 31592352
Summary:
An important role of the insect cuticle is to prevent wetting (i.e., permeation of water) and also to prevent penetration of potentially harmful substances. This barrier function mainly depends on the hydrophobic cuticle surface composed of lipids including cuticular hydrocarbons (CHCs). This study investigated to what extent the cuticle inward barrier function depends on the genotype, comprising mitochondrial and nuclear genes in the fruit fly Drosophila melanogaster, and investigated the contribution of interactions between mitochondrial and nuclear genotypes (mito-nuclear interactions) on this function. In addition, the effects of nutrition and sex on the cuticle barrier function were assessed. Based on a dye penetration assay, this study found that cuticle barrier function varies across three fly lines that were captured from geographically separated regions in three continents. Testing different combinations of mito-nuclear genotypes, it was shown that the inward barrier efficiency is modulated by the nuclear and mitochondrial genomes independently. An interaction was found between diet and sex. These findings provide new insights into the regulation of cuticle inward barrier function in nature.
Weinrich, T. W., Kam, J. H., Ferrara, B. T., Thompson, E. P., Mitrofanis, J. and Jeffery, G. (2019). A day in the life of mitochondria reveals shifting workloads. Sci Rep 9(1): 13898. PubMed ID: 31554906
Summary:
Mitochondria provide energy for cellular function. This study examined daily changing patterns of mitochondrial function and metabolism in Drosophila in vivo in terms of their complex (I-IV) activity, ATP production, glycolysis, and whole fly respiration in the morning, afternoon and night. Complex activity and respiration showed significant and unexpected variation, peaking in the afternoon. However, ATP levels by contrast are >40% greater in the morning and lowest at night when glycolysis peaks. Complex activity modulation was at the protein level with no evidence for differential transcription over the day. Timing differences between increased ATP production and peaks of complex activity may result from more efficient ATP production early in the day leaving complex activity with spare capacity. Optical stimulation of mitochondria is only possible in the mornings when there is such spare capacity. These results provide first evidence of shifts in cellular energy capacity at the organism level. Understanding their translation may be significant to the chosen timing of energy demanding interventions to improve function and health.

Wednesday, November 20th - Disease Models

Xu, F., Kula-Eversole, E., Iwanaszko, M., Lim, C. and Allada, R. (2019). Ataxin2 functions via CrebA to mediate Huntingtin toxicity in circadian clock neurons. PLoS Genet 15(10): e1008356. PubMed ID: 31593562
Summary:
Disrupted circadian rhythms is a prominent and early feature of neurodegenerative diseases including Huntington's disease (HD). In HD patients and animal models, striatal and hypothalamic neurons expressing molecular circadian clocks are targets of mutant Huntingtin (mHtt) pathogenicity. Yet how mHtt disrupts circadian rhythms remains unclear. In a genetic screen for modifiers of mHtt effects on circadian behavior in Drosophila, this study discovered a role for the neurodegenerative disease gene Ataxin2 (Atx2). Genetic manipulations of Atx2 modify the impact of mHtt on circadian behavior as well as mHtt aggregation and demonstrate a role for Atx2 in promoting mHtt aggregation as well as mHtt-mediated neuronal dysfunction. RNAi knockdown of the Fragile X mental retardation gene, dfmr1, an Atx2 partner, also partially suppresses mHtt effects and Atx2 effects depend on dfmr1. Atx2 knockdown reduces the cAMP response binding protein A (CrebA) transcript at dawn. CrebA transcript level shows a prominent diurnal regulation in clock neurons. Loss of CrebA also partially suppresses mHtt effects on behavior and cell loss and restoration of CrebA can suppress Atx2 effects. These results indicate a prominent role of Atx2 in mediating mHtt pathology, specifically via its regulation of CrebA, defining a novel molecular pathway in HD pathogenesis.
Wen, D. T., Zheng, L., Li, J. X., Cheng, D., Liu, Y., Lu, K. and Hou, W. Q. (2019). Endurance exercise resistance to lipotoxic cardiomyopathy is associated with cardiac NAD(+)/dSIR2/PGC-1alpha pathway activation in old Drosophila. Biol Open 8(10). PubMed ID: 31624074
Summary:
Lipotoxic cardiomyopathy is caused by excessive lipid accumulation in myocardial cells and it is a form of cardiac dysfunction. Cardiac PGC-1alpha overexpression prevents lipotoxic cardiomyopathy induced by a high-fat diet (HFD). The level of NAD(+) and Sir2 expression upregulate the transcriptional activity of PGC-1alpha. Exercise improves cardiac NAD(+) level and PGC-1alpha activity. However, the relationship between exercise, NAD(+)/dSIR2/PGC-1alpha pathway and lipotoxic cardiomyopathy remains unknown. In this study, flies were fed a HFD and exercised. The heart dSir2 gene was specifically expressed or knocked down by UAS/hand-Gal4 system. The results showed that either a HFD or dSir2 knockdown remarkably increased cardiac TG level and dFAS expression, reduced heart fractional shortening and diastolic diameter, increased arrhythmia index, and decreased heart NAD(+) level, dSIR2 protein, dSir2 and PGC-1alpha expression levels. Contrarily, either exercise or dSir2 overexpression remarkably reduced heart TG level, dFAS expression and arrhythmia index, and notably increased heart fractional shortening, diastolic diameter, NAD(+) level, dSIR2 level, and heart dSir2 and PGC-1alpha expression. Therefore, exercise training could improve lipotoxic cardiomyopathy induced by a HFD or cardiac dSir2 knockdown in old Drosophila. The NAD(+)/dSIR2/PGC-1alpha pathway activation was an important molecular mechanism of exercise resistance against lipotoxic cardiomyopathy.
Schroeder, A. M., Allahyari, M., Vogler, G., Missinato, M. A., Nielsen, T., Yu, M. S., Theis, J. L., Larsen, L. A., Goyal, P., Rosenfeld, J., Nelson, T. J., Olson, T. M., Colas, A. R., Grossfeld, P. and Bodmer, R. (2019). Model system identification of novel congenital heart disease gene candidates: focus on RPL13. Hum Mol Genet. PubMed ID: 31625562
Summary:
Genetics is a significant factor contributing to congenital heart disease (CHD), but understanding of the genetic players and networks involved in CHD pathogenesis is limited. This study searched for de novo Copy Number Variations (CNVs) in a cohort of 167 CHD patients to identify DNA segments containing potential pathogenic genes. This search focused on new candidate disease genes within 19 deleted de novo CNVs that did not cover known CHD genes. For this study, an integrated high-throughput phenotypical platform was developed to probe for defects in cardiogenesis and cardiac output in human iPSC-derived multipotent cardiac progenitor cells (MCPs) and, in parallel, in the Drosophila in vivo heart model. Notably, knockdown in MCPs of RPL13, a ribosomal gene and SON, an RNA splicing cofactor, reduced proliferation and differentiation of cardiomyocytes, while increasing fibroblasts. In the fly, heart-specific RpL13 knockdown, predominantly at embryonic stages, resulted in a striking 'no heart' phenotype. Knockdown of Son and Pdss2, among others, caused structural and functional defects, including reduced or abolished contractility, respectively. In summary, using a combination of human genetics and cardiac model systems, this study identified new genes as candidates for causing human congenital heart disease, with particular emphasis on ribosomal genes, such as RPL13. This powerful, novel approach of combining cardiac phenotyping in human MCPs and in the in vivo Drosophila heart at high throughput will allow for testing large numbers of CHD candidates, based on patient genomic data, and for building upon existing genetic networks involved in heart development and disease.
Viswanathan, G. K., Shwartz, D., Losev, Y., Arad, E., Shemesh, C., Pichinuk, E., Engel, H., Raveh, A., Jelinek, R., Cooper, I., Gosselet, F., Gazit, E. and Segal, D. (2019). Purpurin modulates Tau-derived VQIVYK fibrillization and ameliorates Alzheimer's disease-like symptoms in animal model. Cell Mol Life Sci. PubMed ID: 31562564
Summary:
Neurofibrillary tangles of the Tau protein and plaques of the amyloid beta peptide are hallmarks of Alzheimer's disease (AD), which is characterized by the conversion of monomeric proteins/peptides into misfolded beta-sheet rich fibrils. Halting the fibrillation process and disrupting the existing aggregates are key challenges for AD drug development. In a previous study in vitro high-throughput screening was performed for the identification of potent inhibitors of Tau aggregation using a proxy model, a highly aggregation-prone hexapeptide fragment (306)VQIVYK(311) (termed PHF6) derived from Tau. This study has characterized a hit molecule from that screen as a modulator of Tau aggregation using in vitro, in silico, and in vivo techniques. This molecule, an anthraquinone derivative named Purpurin, inhibited ~ 50% of PHF6 fibrillization in vitro at equimolar concentration and disassembled pre-formed PHF6 fibrils. In silico studies showed that Purpurin interacted with key residues of PHF6, which are responsible for maintaining its beta-sheets conformation. Isothermal titration calorimetry and surface plasmon resonance experiments with PHF6 and full-length Tau (FL-Tau), respectively, indicated that Purpurin interacted with PHF6 predominantly via hydrophobic contacts and displayed a dose-dependent complexation with FL-Tau. Purpurin was non-toxic when fed to Drosophila and it significantly ameliorated the AD-related neurotoxic symptoms of transgenic flies expressing WT-FL human Tau (hTau) plausibly by inhibiting Tau accumulation and reducing Tau phosphorylation. Purpurin also reduced hTau accumulation in cell culture overexpressing hTau. Importantly, Purpurin efficiently crossed an in vitro human blood-brain barrier model. These findings suggest that Purpurin could be a potential lead molecule for AD therapeutics.
Xiao, C., Hull, D., Qiu, S., Yeung, J., Zheng, J., Barwell, T., Robertson, R. M. and Seroude, L. (2019). Expression of Heat shock protein 70 is insufficient to extend Drosophila melanogaster longevity. G3 (Bethesda). PubMed ID: 31624139
Summary:
It has been known for over 20 years that Drosophila melanogaster flies with twelve additional copies of the hsp70 gene encoding the 70 kDa heat shock protein lives longer after a non-lethal heat treatment. Since the heat treatment also induces the expression of additional heat shock proteins, the biological effect can be due either to HSP70 acting alone or in combination. This study used the UAS/GAL4 system to determine whether hsp70 is sufficient to affect the longevity and the resistance to thermal, oxidative or desiccation stresses of the whole organism. It was observed that HSP70 expression in the nervous system or muscles has no effect on longevity or stress resistance but ubiquitous expression reduces the life span of males. It was also observed that the down-regulation of Hsp70 using RNAi did not affect longevity.
Xie, L., Gu, X., Okamoto, K., Westermark, G. T. and Leifer, K. (2019). 3D analysis of human islet amyloid polypeptide crystalline structures in Drosophila melanogaster. PLoS One 14(10): e0223456. PubMed ID: 31600260
Summary:
Expression of the Alzheimer's disease associated polypeptide Abeta42 and the human polypeptide hormone islet amyloid polypeptide (hIAPP) and the prohormone precursor (hproIAPP) in neurons of Drosophila melanogaster leads to the formation of protein aggregates in the fat body tissue surrounding the brain. This study determined the structure of these membrane-encircled protein aggregates using transmission electron microscopy (TEM) and observed the dissolution of protein aggregates after starvation. Electron tomography (ET) as an extension of transmission electron microscopy revealed that these aggregates were comprised of granular subunits having a diameter of 20 nm aligned into highly ordered structures in all three dimensions. This 3D structural analysis provides novel insight into the aggregation process of hIAPP in the fat body tissue of Drosophila melanogaster.

Tuesday, December 19th - Chromatin and chromosomes

Koval, L., Proshkina, E., Shaposhnikov, M. and Moskalev, A. (2019). The role of DNA repair genes in radiation-induced adaptive response in Drosophila melanogaster is differential and conditional. Biogerontology. PubMed ID: 31624983
Summary:
Studies in human and mammalian cell cultures have shown that induction of DNA repair mechanisms is required for the formation of stimulation effects of low doses of ionizing radiation, named "hormesis". Nevertheless, the role of cellular defense mechanisms in the formation of radiation-induced hormesis at the level of whole organism remains poorly studied. The aim of this work was to investigate the role of genes involved in different mechanisms and stages of DNA repair in radioadaptive response and radiation hormesis by lifespan parameters in Drosophila melanogaster. Genes were studied that control DNA damage sensing (D-Gadd45, Hus1, mnk), nucleotide excision repair (mei-9, mus210, Mus209), base excision repair (Rrp1), DNA double-stranded break repair by homologous recombination (Brca2, spn-B, okr) and non-homologous end joining (Ku80, WRNexo), and the Mus309 gene that participates in several mechanisms of DNA repair. The obtained results demonstrate that in flies with mutations in studied genes radioadaptive response and radiation hormesis are absent or appear to a lesser extent than in wild-type Canton-S flies. Chronic exposure of gamma-radiation in a low dose during pre-imaginal stages of development leads to an increase in expression of the studied DNA repair genes, which is maintained throughout the lifespan of flies. However, the activation of conditional ubiquitous overexpression of DNA repair genes does not induce resistance to an acute exposure to gamma-radiation and reinforces its negative impact.
Suda, K., Muraoka, Y., Ortega-Yanez, A., Yoshida, H., Kizu, F., Hochin, T., Kimura, H. and Yamaguchi, M. (2019). Reduction of Rpd3 suppresses defects in locomotive ability and neuronal morphology induced by the knockdown of Drosophila SLC25A46 via an epigenetic pathway. Exp Cell Res: 111673. PubMed ID: 31614134
Summary:
Mitochondrial dysfunction causes various diseases. Mutations in the SLC25A46 gene have been identified in mitochondrial diseases that are sometimes classified as Charcot-Marie-Tooth disease type 2, optic atrophy, and Leigh syndrome. A homolog of SLC25A46 was identified in Drosophila and designated as dSLC25A46 (CG5755). Previous work has established mitochondrial disease model targeting of dSLC25A46, which causes locomotive dysfunction and morphological defects at neuromuscular junctions, such as reduced synaptic branch lengths and decreased numbers of boutons. To investigate the involvement of epigenetic regulators in mitochondrial diseases, candidate epigenetic regulators that interact with human SLC25A46 were identifed by searching Gene Expression Omnibus (GEO). It was discovered that HDAC1 binds to several SLC25A46 genomic regions in human cultured CD4 (+) cells, and attempts were made to prove this in an in vivo Drosophila model. By demonstrating that Rpd3, Drosophila HDAC1, regulates the histone H4K8 acetylation state in dSLC25A46 genomic regions, this study confirmed that Rpd3 is a novel epigenetic regulator modifying the phenotypes observed with the mitochondrial disease model targeting of dSLC25A46. The functional reduction of Rpd3 rescued the deficient locomotive ability and aberrant morphology of motoneurons at presynaptic terminals induced by the dSLC25A46 knockdown. The present results suggest that the inhibition of HDAC1 suppresses the pathogenic processes that lead to the degeneration of motoneurons in mitochondrial diseases.
Renschler, G., Richard, G., Valsecchi, C. I. K., Toscano, S., Arrigoni, L., Ramirez, F. and Akhtar, A. (2019). Hi-C guided assemblies reveal conserved regulatory topologies on X and autosomes despite extensive genome shuffling. Genes Dev. PubMed ID: 31601616
Summary:
Genome rearrangements that occur during evolution impose major challenges on regulatory mechanisms that rely on three-dimensional genome architecture. This study developed a scaffolding algorithm and generated chromosome-length assemblies from Hi-C data for studying genome topology in three distantly related Drosophila species. Extensive genome shuffling between these species was observed with one synteny breakpoint after approximately every six genes. A/B compartments, a set of large gene-dense topologically associating domains (TADs), and spatial contacts between high-affinity sites (HAS) located on the X chromosome are maintained over 40 million years, indicating architectural conservation at various hierarchies. Evolutionary conserved genes cluster in the vicinity of HAS, while HAS locations appear evolutionarily flexible, thus uncoupling functional requirement of dosage compensation from individual positions on the linear X chromosome. Therefore, 3D architecture is preserved even in scenarios of thousands of rearrangements highlighting its relevance for essential processes such as dosage compensation of the X chromosome.
Kocak, E., Dykstra, S., Nemeth, A., Coughlin, C. G., Rodgers, K. and McVey, M. (2019). The Drosophila melanogaster PIF1 helicase promotes survival during replication stress and processive DNA synthesis during double-strand gap repair. Genetics. PubMed ID: 31537623
Summary:
PIF1 is a 5' to 3' DNA helicase that can unwind double-stranded DNA and disrupt nucleic acid-protein complexes. In Saccharomyces, Pif1 plays important roles in mitochondrial and nuclear genome maintenance, telomere length regulation, unwinding of G-quadruplex structures, and DNA synthesis during break-induced replication. Some, but not all, of these functions are shared with other eukaryotes. To gain insight into the evolutionarily conserved functions of PIF1, pif1 null mutants were created in Drosophila. pif1 mutant larvae exposed to high concentrations of hydroxyurea, but not other DNA damaging agents, were found to experience reduced survival to adulthood. Embryos lacking PIF1 fail to segregate their chromosomes efficiently during early nuclear divisions, consistent with a defect in DNA replication. Furthermore, loss of the BRCA2 protein, which is required for stabilization of stalled replication forks in metazoans, causes synthetic lethality in third instar larvae lacking either PIF1 or the polymerase delta subunit POL32. Interestingly, pif1 mutants have a reduced ability to synthesize DNA during repair of a double-stranded gap, but only in the absence of POL32. Together, these results support a model in which Drosophila PIF1 functions with POL32 during times of replication stress but acts independently of POL32 to promote synthesis during double-strand gap repair.
Kohzaki, H., Asano, M., Murakami, Y. and Mazo, A. (2020). Epigenetic regulation affects gene amplification in Drosophila development. Front Biosci (Landmark Ed) 25: 632-645. PubMed ID: 31585908
Summary:
In Drosophila melanogaster, in response to developmental transcription factors, and by repeated initiation of DNA replication of four chorion genes, ovarian follicle cells, form an onion skin-type structure at the replication origins. The DNA replication machinery is conserved from yeast to humans. Subunits of the origin recognition complex (ORC) is comprised of Orc1, Orc2, and Cdc6 genes. While mutations of Orc1 and Orc2 and not Cdc6 can be lethal, overexpression of these genes lead to female sterility. Ecdysone, is a steroidal prohormone of the major insect molting hormone 20-hydroxyecdysone that in Drosophila, triggers molting, metamorphosis, and oogenesis. To this end, several ecdysone receptor (EcR) binding sites were identified around gene amplification loci. It was also found that H3K4 was trimethylated at chorion gene amplification origins, but not at the act1 locus. Female mutants overexpressing Lsd1 (a dimethyl histone H3K4 demethylase) or Lid (a trimethyl histone H3K4 demethylase), but not a Lid mutant, were sterile. The data suggest that ecdysone signaling determines which origin initiates DNA replication and contributes to the development. Screening strategies using Drosophila offer the opportunity for development of drugs that reduce gene amplification and alter histone modification associated with epigenetic effects.
Fernandez, J., Bloomer, H., Kellam, N. and LaRocque, J. R. (2019). Chromosome preference during homologous recombination repair of DNA double-strand breaks in Drosophila melanogaster. G3 (Bethesda). PubMed ID: 31519746
Summary:
DNA double-strand breaks (DSBs) are especially toxic DNA lesions that, if left unrepaired, can lead to wide-ranging genomic instability. Of the pathways available to repair DSBs, the most accurate is homologous recombination (HR), where a homologous sequence is used as a donor template to restore genetic information at the break site. While much of the biochemical aspects of HR repair have been characterized, how the repair machinery locates and discriminates between potential homologous donor templates throughout the genome remains elusive. This study used Drosophila melanogaster to investigate whether there is a preference between intrachromosomal and interhomolog donor sequences in mitotically dividing cells. The results demonstrate that, although interhomolog HR is possible and frequent if another donor template is not available, intrachromosomal donor templates are highly preferred. This is true even if the interhomolog donor template is less diverged than the intrachromosomal donor template. Thus, despite the stringent requirements for homology, the chromosomal location of the donor template plays a more significant role in donor template choice.

Monday, November 18th - Evolution

Rodriguez-Exposito, E., Garcia-Gonzalez, F. and Polak, M. (2019). Individual and synergistic effects of male external genital traits in sexual selection. J Evol Biol. PubMed ID: 31554023
Summary:
Male genital traits exhibit extraordinary interspecific phenotypic variation. This remarkable and general evolutionary trend is widely considered to be the result of sexual selection. However, a good understanding of whether or how individual genital traits function in different competitive arenas (episodes of sexual selection), or how different genital traits may interact to influence competitive outcomes, is still not available. This study used an experimental approach based on high-precision laser phenotypic engineering to address these outstanding questions, focusing on three distinct sets of micron-scale external (nonintromittent) genital spines in male Drosophila kikkawai Burla (Diptera: Drosophilidae). Elimination of the large pair of spines on the male secondary claspers sharply reduced male ability to copulate, yet elimination of the other sets of spines on the primary and secondary claspers had no significant effects on copulation probability. Intriguingly, both the large spines on the secondary claspers and the cluster of spines on the primary claspers were found to independently promote male competitive fertilization success. Moreover, when large and small secondary clasper spines were simultaneously shortened in individual males, these males suffered greater reductions in fertilization success relative to males whose traits were altered individually, providing evidence for synergistic effects of external genital traits on fertilization success. Overall, the results are significant in demonstrating that a given genital trait (the large spines on the secondary claspers) can function in different episodes of sexual selection, and distinct genital traits may interact in sexual selection. The results offer an important contribution to evolutionary biology by demonstrating an understudied selective mechanism, operating via subtle trait interactions in a post-insemination context, by which genital traits may be co-evolving.
Stewart, N. B. and Rogers, R. L. (2019). Chromosomal rearrangements as a source of new gene formation in Drosophila yakuba. PLoS Genet 15(9): e1008314. PubMed ID: 31545792
Summary:
The origins of new genes are among the most fundamental questions in evolutionary biology. Understanding of the ways that new genetic material appears and how that genetic material shapes population variation remains incomplete. De novo genes and duplicate genes are a key source of new genetic material on which selection acts. To better understand the origins of these new gene sequences, this study explored the ways that structural variation might alter expression patterns and form novel transcripts. Evidence is provided that chromosomal rearrangements are a source of novel genetic variation that facilitates the formation of de novo exons in Drosophila. 51 cases were found of de novo exon formation created by chromosomal rearrangements in 14 strains of D. yakuba. These new genes inherit transcription start signals and open reading frames when the 5' end of existing genes are combined with previously untranscribed regions. Such new genes would appear with novel peptide sequences, without the necessity for secondary transitions from non-coding RNA to protein. This mechanism of new peptide formations contrasts with canonical theory of de novo gene progression requiring non-coding intermediaries that must acquire new mutations prior to loss via pseudogenization. Hence, these mutations offer a means to de novo gene creation and protein sequence formation in a single mutational step, answering a long standing open question concerning new gene formation. Gene expression changes were identified for 134 existing genes, indicating that these mutations can alter gene regulation. Population variability for chromosomal rearrangements is considerable, with 2368 rearrangements observed across 14 inbred lines. More rearrangements were identified on the X chromosome than any of the autosomes, suggesting the X is more susceptible to chromosome alterations. Together, these results suggest that chromosomal rearrangements are a source of variation in populations that is likely to be important to explain genetic and therefore phenotypic diversity.
Luecke, D. M. and Kopp, A. (2019). Sex-specific evolution of relative leg size in Drosophila prolongata results from changes in the intersegmental coordination of tissue growth. Evolution. PubMed ID: 31595502
Summary:
Evolution of relative organ size is the most prolific source of morphological diversity, yet the underlying molecular mechanisms that modify growth control are largely unknown. Models where organ proportions have undergone recent evolutionary changes hold the greatest promise for understanding this process. Uniquely among Drosophila species, D. prolongata displays a dramatic, male-specific increase in the size of its forelegs relative to other legs. By comparing leg development between males and females of D. prolongata and its closest relative D. carrolli, this study shows that the exaggerated male forelegs are produced by a sex- and segment-specific increase in mitosis during the final larval instar. Intersegmental compensatory control, where smaller leg primordia grow at a faster rate, is observed in both species and sexes. However, the equlibrium growth rates that determine the final relative proportion between the first and second legs have shifted in male D. prolongata compared both to conspecific females and to D. carrolli. It is suggested that the observed developmental changes that produce new adult proportions reflect an interplay between conserved growth coordination mechanisms and evolving organ-specific growth targets.
Mai, D., Nalley, M. J. and Bachtrog, D. (2019). Patterns of genomic differentiation in the Drosophila nasuta species complex. Mol Biol Evol. PubMed ID: 31556453
Summary:
The Drosophila nasuta species complex contains over a dozen recently diverged species that are distributed widely across South-East Asia, and which show varying degrees of pre- and post-zygotic isolation. This study assembled a high-quality genome for D. albomicans using single-molecule sequencing and chromatin conformation capture and draft genomes for 11 additional species and 67 individuals across the clade, to infer the species phylogeny and patterns of genetic diversity in this group. The assembly recovers entire chromosomes, and the origin of this radiation was dated to about 2 million years ago. Despite low levels of overall differentiation, most species or subspecies show clear clustering into their designated taxonomic groups using population genetics and phylogenetic methods. Local evolutionary history is heterogeneous across the genome, and differs between the autosomes and the X chromosome for species in the sulfurigaster subgroup, likely due to autosomal introgression. This study establishes the nasuta species complex as a promising model system to further characterize the evolution of pre- and post-zygotic isolation in this clade.
Sharp, N. P. and Whitlock, M. C. (2019). No evidence of positive assortative mating for genetic quality in fruit flies. Proc Biol Sci 286(1912): 20191474. PubMed ID: 31575372
Summary:
In sexual populations, the effectiveness of selection will depend on how gametes combine with respect to genetic quality. If gametes with deleterious alleles are likely to combine with one another, deleterious genetic variation can be more easily purged by selection. Assortative mating, where there is a positive correlation between parents in a phenotype of interest such as body size, is often observed in nature, but does not necessarily reveal how gametes ultimately combine with respect to genetic quality itself. This study manipulated genetic quality in fruit fly populations using an inbreeding scheme designed to provide an unbiased measure of mating patterns. While inbred flies had substantially reduced reproductive success, their gametes did not combine with those of other inbred flies more often than expected by chance, indicating a lack of positive assortative mating. Instead, a negative correlation was detected in genetic quality between parents, i.e. disassortative mating, which diminished with age. This pattern is expected to reduce the genetic variance for fitness, diminishing the effectiveness of selection. How mechanisms of sexual selection could produce a pattern of disassortative mating is discussed. This study highlights that sexual selection has the potential to either increase or decrease genetic load.
Siddiq, M. A. and Thornton, J. W. (2019). Fitness effects but no temperature-mediated balancing selection at the polymorphic Adh gene of Drosophila melanogaster. Proc Natl Acad Sci U S A. PubMed ID: 31594844
Summary:
Polymorphism in the alcohol dehydrogenase (ADH) protein of Drosophila melanogaster, like genetic variation in many other enzymes, has long been hypothesized to be maintained by a selective trade-off between thermostability and enzyme activity. Two major Adh variants, named Fast and Slow, are distributed along latitudinal clines on several continents. The balancing selection trade-off hypothesis posits that Fast is favored at high latitudes because it metabolizes alcohol faster, whereas Slow is favored at low latitudes because it is more stable at high temperatures. This study use biochemical and physiological assays of precisely engineered genetic variants to directly test this hypothesis. As predicted, the Fast protein has higher catalytic activity than Slow, and both the Fast protein and regulatory variants linked to it confer greater ethanol tolerance on transgenic animals. No evidence was found of a temperature-mediated trade-off: The Fast protein is not less stable or active at high temperatures, and Fast alleles increase ethanol tolerance and survivorship at all temperatures tested. Further, analysis of a population genomic dataset reveals no signature of balancing selection in the Adh gene. These results provide strong evidence against balancing selection driven by a stability/activity trade-off in Adh, and they justify caution about this hypothesis for other enzymes except those for which it has been directly tested. These findings tentatively suggest that environment-specific selection for the Fast allele, coupled with demographic history, may have produced the observed pattern of Adh variation.

Friday, November 15th - Adult Neural Development and Function

Sheng, C., Javed, U., Rosenthal, J., Yin, J., Qin, B. and Yuan, Q. (2019). Time-lapse live imaging and quantification of fast dendritic branch dynamics in developing Drosophila neurons. J Vis Exp(151). PubMed ID: 31609313
Summary:
Highly motile dendritic filopodia are widely present in neurons at early developmental stages. These exploratory dynamic branches sample the surrounding environment and initiate contacts with potential synaptic partners. Although the connection between dendritic branch dynamics and synaptogenesis is well established, how developmental and activity-dependent processes regulate dendritic branch dynamics is not well understood. This is partly due to the technical difficulties associated with the live imaging and quantitative analyses of these fine structures using an in vivo system. A method was establised to study dendrite dynamics using Drosophila larval ventral lateral neurons (LNvs), which can be individually labeled using genetic approaches and are accessible for live imaging. Taking advantage of this system, protocols were developed to capture branch dynamics of the whole dendritic arbor of a single labeled LNv through time-lapse live imaging. Post-processing was performed to improve image quality through drift correction and deconvolution, followed by analyzing branch dynamics at the single-branch level by annotating spatial positions of all branch terminals. Lastly, R scripts and specific parameters were developed to quantify branch dynamics using the coordinate information generated by the terminal tracing. Collectively, this protocol allows achieving of a detailed quantitative description of branch dynamics of the neuronal dendritic arbor with high temporal and spatial resolution. The methods developed in this study are generally applicable to sparsely labeled neurons in both in vitro and in vivo conditions.
Mohapatra, P. and Menuz, K. (2019). Molecular profiling of the Drosophila antenna reveals conserved genes underlying olfaction in insects. G3 (Bethesda). PubMed ID: 31527046
Summary:
Repellent odors are widely used to prevent insect-borne diseases, making it imperative to identify the conserved molecular underpinnings of their olfactory systems. Currently, little is known about the molecules supporting odor signaling beyond the odor receptors themselves. Most known molecules function in one of two classes of olfactory sensilla, single-walled or double-walled, which have differing morphology and odor response profiles. This study took two approaches to discover novel genes that contribute to insect olfaction in the periphery. Drosophila melanogaster amos mutants were transcriptionally profiled that lack trichoid and basiconic sensilla, the single-walled sensilla in this species. This revealed 187 genes whose expression is enriched in these sensilla, including pickpocket ion channels and neuromodulator GPCRs that could mediate signaling pathways unique to single-walled sensilla. For a second approach, 141 antennal-enriched (AE) genes were computationally identified that are more than ten times as abundant in D. melanogaster antennae as in other tissues or whole-body extracts and are thus likely to play a role in olfaction. Unambiguous orthologs of AE genes were identified in the genomes of four distantly related insect species, and most identified orthologs were expressed in the antenna of these species. Further analysis revealed that nearly half of the 141 AE genes are localized specifically to either single or double-walled sensilla. Functional annotation suggests the AE genes include signaling molecules and enzymes that could be involved in odorant degradation. Together, these two resources provide a foundation for future studies investigating conserved mechanisms of odor signaling.
Schlichting, M., Weidner, P., Diaz, M., Menegazzi, P., Dalla Benetta, E., Helfrich-Forster, C. and Rosbash, M. (2019). Light-mediated circuit switching in the Drosophila neuronal clock network. Curr Biol 29(19): 3266-3276. PubMed ID: 31564496
Summary:
The circadian clock is a timekeeper but also helps adapt physiology to the outside world. This is because an essential feature of clocks is their ability to adjust (entrain) to the environment, with light being the most important signal. Whereas cryptochrome-mediated entrainment is well understood in Drosophila, integration of light information via the visual system lacks a neuronal or molecular mechanism. This study shows that a single photoreceptor subtype is essential for long-day adaptation. These cells activate key circadian neurons, namely the large ventral-lateral neurons (lLNvs), which release the neuropeptide pigment-dispersing factor (PDF). RNAi and rescue experiments show that PDF from these cells is necessary and sufficient for delaying the timing of the evening (E) activity in long-day conditions. This contrasts to PDF that derives from the small ventral-lateral neurons (sLNvs), which are essential for constant darkness (DD) rhythmicity. Using a cell-specific CRISPR/Cas9 assay, this study shows that lLNv-derived PDF directly interacts with neurons important for E activity timing. Interestingly, this pathway is specific for long-day adaptation and appears to be dispensable in equinox or DD conditions. The results therefore indicate that external cues cause a rearrangement of neuronal hierarchy, which contributes to behavioral plasticity.
Roussou, I. G., Papanikolopoulou, K., Savakis, C. and Skoulakis, E. M. C. (2019). Drosophila Bruton's Tyrosine Kinase regulates habituation latency and facilitation in distinct mushroom body neurons. J Neurosci. PubMed ID: 31530645
Summary:
Habituation is the adaptive behavioral outcome of processes engaged in timely devaluation of non-reinforced repetitive stimuli, but the neuronal circuits and molecular mechanisms that underlie them are not well-understood. To gain insights into these processes this study developed and characterized a habituation assay to repetitive footshocks in mixed sex Drosophila groups, and has demonstrated that acute neurotransmission from adult alpha/beta mushroom body (MB) neurons prevents premature stimulus devaluation. This study demonstrates that activity of the non-receptor Tyrosine kinase Btk protein is required within these neurons to prevent premature habituation. Significantly, it was also demonstrated that the complementary process of timely habituation to the repetitive stimulation is facilitated by alpha /beta MB neurons and also requires Btk activity. Hence the results provide initial insights into molecular mechanisms engaged in footshock habituation within distinct MB neurons. Importantly, Btk attenuation specifically within alpha /beta neurons leads to defective habituation, which is readily reversible by administration of the antipsychotics Clozapine and Risperidone suggesting that the loss of the kinase may dysregulate monoamine receptors within these neurons, whose activity underlies the failure to habituate.
Molina-Obando, S., Vargas-Fique, J. F., Henning, M., Gur, B., Schladt, T. M., Akhtar, J., Berger, T. K. and Silies, M. (2019). ON selectivity in Drosophila vision is a multisynaptic process involving both glutamatergic and GABAergic inhibition. Elife 8. PubMed ID: 31535971
Summary:
Sensory systems sequentially extract increasingly complex features. ON and OFF pathways, for example, encode increases or decreases of a stimulus from a common input. This ON/OFF pathway split is thought to occur at individual synaptic connections through a sign-inverting synapse in one of the pathways. This study showed that ON selectivity is a multisynaptic process in the Drosophila visual system. A pharmacogenetics approach demonstrates that both glutamatergic inhibition through GluClalpha and GABAergic inhibition through Rdl mediate ON responses. Although neurons postsynaptic to the glutamatergic ON pathway input L1 lose all responses in GluClalpha mutants, they are resistant to a cell-type-specific loss of GluClalpha. This shows that ON selectivity is distributed across multiple synapses, and raises the possibility that cell-type-specific manipulations might reveal similar strategies in other sensory systems. Thus, sensory coding is more distributed than predicted by simple circuit motifs, allowing for robust neural processing.
Schlichting, M., Diaz, M. M., Xin, J. and Rosbash, M. (2019). Neuron-specific knockouts indicate the importance of network communication to Drosophila rhythmicity. Elife 8. PubMed ID: 31613223
Summary:
Animal circadian rhythms persist in constant darkness and are driven by intracellular transcription-translation feedback loops. Although these cellular oscillators communicate, isolated mammalian cellular clocks continue to tick away in darkness without intercellular communication. To investigate these issues in Drosophila, behavior as well as molecular rhythms were assayed within individual brain clock neurons while blocking communication within the ca. 150 neuron clock network. CRISPR-mediated neuron-specific circadian clock knockouts were also generated. The results point to two key clock neuron groups: loss of the clock within both regions but neither one alone has a strong behavioral phenotype in darkness; communication between these regions also contributes to circadian period determination. Under these dark conditions, the clock within one region persists without network communication. The clock within the famous PDF-expressing s-LNv neurons however was strongly dependent on network communication, likely because clock gene expression within these vulnerable sLNvs depends on neuronal firing or light.

Thursday, November 11th - Model Systems

Kezos, J. N., Phillips, M. A., Thomas, M. D., Ewunkem, A. J., Rutledge, G. A., Barter, T. T., Santos, M. A., Wong, B. D., Arnold, K. R., Humphrey, L. A., Yan, A., Nouzille, C., Sanchez, I., Cabral, L. G., Bradley, T. J., Mueller, L. D., Graves, J. L., Jr. and Rose, M. R. (2019). Genomics of early cardiac dysfunction and mortality in obese Drosophila melanogaster. Physiol Biochem Zool 92(6): 591-611. PubMed ID: 31603376
Summary:
In experimental evolution, functional demands are imposed on laboratory populations of model organisms using selection. After enough generations of such selection, the resulting populations constitute excellent material for physiological research. An intense selection regime for increased starvation resistance was imposed on 10 large outbred Drosophila populations. The selection responses were observed of starvation and desiccation resistance, metabolic reserves, and heart robustness via electrical pacing. Furthermore, the pooled genomes of these populations were sequenced. As expected, significant increases in starvation resistance and lipid content were found in 10 intensely selected SCO populations. The selection regime also improved desiccation resistance, water content, and glycogen content among these populations. Additionally, the average rate of cardiac arrests in 10 obese SCO populations was double the rate of the 10 ancestral CO populations. Age-specific mortality rates were increased at early adult ages by selection. Genomic analysis revealed a large number of single nucleotide polymorphisms across the genome that changed in frequency as a result of selection. These genomic results were similar to those obtained in the laboratory from less direct selection procedures. The combination of extensive genomic and phenotypic differentiation between these 10 populations and their ancestors makes them a powerful system for the analysis of the physiological underpinnings of starvation resistance.
Kiss, A. A., Somlyai-Popovics, N., Kiss, M., Boldogkoi, Z., Csiszar, K. and Mink, M. (2019). Type IV collagen is essential for proper function of integrin-mediated adhesion in Drosophila muscle fibers. Int J Mol Sci 20(20). PubMed ID: 31623094
Summary:
Congenital muscular dystrophy (CMD), a subgroup of myopathies is a genetically and clinically heterogeneous group of inherited muscle disorders and is characterized by progressive muscle weakness, fiber size variability, fibrosis, clustered necrotic fibers, and central myonuclei present in regenerating muscle. Type IV collagen (COL4A1) mutations have recently been identified in patients with intracerebral, vascular, renal, ophthalmologic pathologies and congenital muscular dystrophy, consistent with diagnoses of Walker-Warburg Syndrome or Muscle-Eye-Brain disease. Morphological characteristics of muscular dystrophy have also been demonstrated Col4a1 mutant mice. Yet, several aspects of the pathomechanism of COL4A1-associated muscle defects remained largely uncharacterized. Based on the results of genetic, histological, molecular, and biochemical analyses in an allelic series of Drosophila col4a1 mutants, evidence is provided that col4a1 mutations arise by transitions in glycine triplets, associate with severely compromised muscle fibers within the single-layer striated muscle of the common oviduct, characterized by loss of sarcomere structure, disintegration and streaming of Z-discs, indicating an essential role for the COL4A1 protein. Features of altered cytoskeletal phenotype include actin bundles traversing over sarcomere units, amorphous actin aggregates, atrophy, and aberrant fiber size. The mutant COL4A1-associated defects appear to recapitulate integrin-mediated adhesion phenotypes observed in RNA-inhibitory Drosophila. These results provide insight into the mechanistic details of COL4A1-associated muscle disorders and suggest a role for integrin-collagen interaction in the maintenance of sarcomeres.
Mascolo, E., Barile, A., Mecarelli, L. S., Amoroso, N., Merigliano, C., Massimi, A., Saggio, I., Hansen, T., Tramonti, A., Di Salvo, M. L., Barbetti, F., Contestabile, R. and Verni, F. (2019). The expression of four pyridoxal kinase (PDXK) human variants in Drosophila impacts on genome integrity. Sci Rep 9(1): 14188. PubMed ID: 31578392
Summary:
In eukaryotes, pyridoxal kinase (PDXK) acts in vitamin B6 salvage pathway to produce pyridoxal 5'-phosphate (PLP), the active form of the vitamin, which is implicated in numerous crucial metabolic reactions. In Drosophila, mutations in the dPdxk gene cause chromosome aberrations (CABs) and increase glucose content in larval hemolymph. Both phenotypes are rescued by the expression of the wild type human PDXK counterpart. This study expressed, in dPdxk1 mutant flies, four PDXK human variants: three (D87H, V128I and H246Q) listed in databases, and one (A243G) found in a genetic screening in patients with diabetes. Differently from human wild type PDXK, none of the variants was able to completely rescue CABs and glucose content elicited by dPdxk1 mutation. Biochemical analysis of D87H, V128I, H246Q and A243G proteins revealed reduced catalytic activity and/or reduced affinity for PLP precursors which justify this behavior. Although these variants are rare in population and carried in heterozygous condition, these findings suggest that in certain metabolic contexts and diseases in which PLP levels are reduced, the presence of these PDXK variants could threaten genome integrity and increase cancer risk.
Mori, A., Hatano, T., Inoshita, T., Shiba-Fukushima, K., Koinuma, T., Meng, H., Kubo, S. I., Spratt, S., Cui, C., Yamashita, C., Miki, Y., Yamamoto, K., Hirabayashi, T., Murakami, M., Takahashi, Y., Shindou, H., Nonaka, T., Hasegawa, M., Okuzumi, A., Imai, Y. and Hattori, N. (2019). Parkinson's disease-associated iPLA2-VIA/PLA2G6 regulates neuronal functions and alpha-synuclein stability through membrane remodeling. Proc Natl Acad Sci U S A 116(41): 20689-20699. PubMed ID: 31548400
Summary:
Mutations in the iPLA2-VIA/PLA2G6 gene are responsible for PARK14-linked Parkinson's disease (PD) with alpha-synucleinopathy. However, it is unclear how iPLA2-VIA mutations lead to alpha-synuclein (alpha-Syn) aggregation and dopaminergic (DA) neurodegeneration. This study reports that iPLA2-VIA-deficient Drosophila exhibits defects in neurotransmission during early developmental stages and progressive cell loss throughout the brain, including degeneration of the DA neurons. Lipid analysis of brain tissues reveals that the acyl-chain length of phospholipids is shortened by iPLA2-VIA loss, which causes endoplasmic reticulum (ER) stress through membrane lipid disequilibrium. The introduction of wild-type human iPLA2-VIA or the mitochondria-ER contact site-resident protein C19orf12 in iPLA2-VIA-deficient flies rescues the phenotypes associated with altered lipid composition, ER stress, and DA neurodegeneration, whereas the introduction of a disease-associated missense mutant, iPLA2-VIA A80T, fails to suppress these phenotypes. The acceleration of alpha-Syn aggregation by iPLA2-VIA loss is suppressed by the administration of linoleic acid, correcting the brain lipid composition. These findings suggest that membrane remodeling by iPLA2-VIA is required for the survival of DA neurons and alpha-Syn stability.
Nicholson, H. E., Tariq, Z., Housden, B. E., Jennings, R. B., Stransky, L. A., Perrimon, N., Signoretti, S. and Kaelin, W. G., Jr. (2019). HIF-independent synthetic lethality between CDK4/6 inhibition and VHL loss across species. Sci Signal 12(601). PubMed ID: 31575731
Summary:
Inactivation of the VHL tumor suppressor gene is the signature initiating event in clear cell renal cell carcinoma (ccRCC), the most common form of kidney cancer, and causes the accumulation of hypoxia-inducible factor 2alpha (HIF-2alpha; see Drosophila Similar). HIF-2alpha inhibitors are effective in some ccRCC cases, but both de novo and acquired resistance have been observed in the laboratory and in the clinic. This study identified synthetic lethality between decreased activity of cyclin-dependent kinases 4 and 6 (CDK4/6) and VHL inactivation in two species (human and Drosophila) and across diverse human ccRCC cell lines in culture and xenografts. Although HIF-2alpha transcriptionally induced the CDK4/6 partner cyclin D1, HIF-2alpha was not required for the increased CDK4/6 requirement of VHL(-/-) ccRCC cells. Accordingly, the antiproliferative effects of CDK4/6 inhibition were synergistic with HIF-2alpha inhibition in HIF-2alpha-dependent VHL(-/-) ccRCC cells and not antagonistic with HIF-2alpha inhibition in HIF-2alpha-independent cells. These findings support testing CDK4/6 inhibitors as treatments for ccRCC, alone and in combination with HIF-2alpha inhibitors.
Qiu, W., Chen, X., Tian, Y., Wu, D., Du, M. and Wang, S. (2019). Protection against oxidative stress and anti-aging effect in Drosophila of royal jelly-collagen peptide. Food Chem Toxicol: 110881. PubMed ID: 31622731
Summary:
Dietary peptide has been of great interest because of its perspective in nutrition and health of human body. The aim of this study was to develop a dietary nutritional supplement exerting both antioxidant and anti-aging effects. Peptide, named as ERJ-CP, was prepared by mixing enzyme-treated royal jelly (ERJ) with collagen peptide (CP), showing stronger antioxidant activity in vitro. Drosophila was used as model animal to investigate anti-aging effect of ERJ-CP in vivo. ERJ-CP significantly prolonged the average life span of Drosophila treated with H2O2 and paraquat, reducing malondialdehyde (MDA) and protein carbonyl (PCO) levels in Drosophila. In addition, 3mg/mL of ERJ-CP could prolong the lifespan of natural aging Drosophila by 11.16%. ERJ-CP could up-regulate the levels of total superoxide dismutase (T-SOD), glutathione peroxidase (GSH-Px), catalase (CAT) and down-regulate the contents of MDA and PCO. Moreover, the intake of ERJ-CP increased the food consumption, weight gain and exercise capacity of Drosophila. The results showed that ERJ-CP played a protective role in both antioxidant and anti-aging effects on Drosophila, and the anti-aging effect may be achieved by alleviating oxidative damage. It suggests that ERJ-CP could be developed as a health-promoting ingredient with antioxidant and anti-aging effects for human body.

Wednesday, November 13th - Cell Cycle

Lie-Jensen, A., Ivanauskiene, K., Malerod, L., Jain, A., Tan, K. W., Laerdahl, J. K., Liestol, K., Stenmark, H. and Haglund, K. (2019). Centralspindlin Recruits ALIX to the Midbody during Cytokinetic Abscission in Drosophila via a Mechanism Analogous to Virus Budding. Curr Biol. PubMed ID: 31607533
Summary:
Abscission, the final step of cytokinesis, cleaves the thin intercellular bridge connecting the two daughter cells. The scaffold protein ALIX is a core component of the abscission machinery with an evolutionarily conserved role in midbody recruitment of ESCRT-III, which mediates the final cut. In mammalian cells, the centralspindlin complex recruits the major midbody organizer CEP55 that directly binds and recruits ALIX and ESCRT-I, which in turn cooperatively recruit ESCRT-III. However, CEP55 is missing in Drosophila melanogaster and other invertebrates, and it is unknown how the abscission machinery is recruited to the midbody in the absence of CEP55. This study addresses how Drosophila ALIX is recruited to the midbody. Surprisingly, ALIX localizes to the midbody via its V-domain, independently of the GPPX3Y motif in the proline-rich region that recruits human ALIX. It was elucidated that the centralspindlin component Pavarotti (H.s.MKLP1) interacts with the V-domain of ALIX to recruit it to the midbody. Specifically, the results indicate that an LxxLF motif in Pavarotti directly interacts with a conserved hydrophobic pocket in the ALIX V-domain, which in human ALIX binds (L)YPXnL/LxxLF motifs of virus proteins. Thus, this study identifies that ALIX is recruited by an analogous mechanism during abscission in Drosophila as during virus budding in mammalian cells and an ancestral role for centralspindlin in recruiting the abscission machinery to the midbody.
Pavlova, G. A. et al. (2019). RNAi-mediated depletion of the NSL complex subunits leads to abnormal chromosome segregation and defective centrosome duplication in Drosophila mitosis. PLoS Genet 15(9): e1008371. PubMed ID: 31527906
Summary:
The Drosophila Nonspecific Lethal (NSL) complex is a major transcriptional regulator of housekeeping genes. It contains at least seven subunits that are conserved in the human KANSL complex: Nsl1/Wah (KANSL1), Dgt1/Nsl2 (KANSL2), Rcd1/Nsl3 (KANSL3), Rcd5 (MCRS1), MBD-R2 (PHF20), Wds (WDR5) and Mof (MOF/KAT8). Previous studies have shown that Dgt1, Rcd1 and Rcd5 are implicated in centrosome maintenance. This study analyzed the mitotic phenotypes caused by RNAi-mediated depletion of Rcd1, Rcd5, MBD-R2 or Wds in greater detail. Depletion of any of these proteins in Drosophila S2 cells led to defects in chromosome segregation. Consistent with these findings, Rcd1, Rcd5 and MBD-R2 RNAi cells showed reduced levels of both Cid/CENP-A and the kinetochore component Ndc80. In addition, RNAi against any of the four genes negatively affected centriole duplication. In Wds-depleted cells, the mitotic phenotypes were similar but milder than those observed in Rcd1-, Rcd5- or MBD-R2-deficient cells. RT-qPCR experiments and interrogation of published datasets revealed that transcription of many genes encoding centromere/kinetochore proteins (e.g., cid, Mis12 and Nnf1b), or involved in centriole duplication (e.g., Sas-6, Sas-4 and asl) is substantially reduced in Rcd1, Rcd5 and MBD-R2 RNAi cells, and to a lesser extent in wds RNAi cells. During mitosis, both Rcd1-GFP and Rcd5-GFP accumulate at the centrosomes and the telophase midbody, MBD-R2-GFP is enriched only at the chromosomes, while Wds-GFP accumulates at the centrosomes, the kinetochores, the midbody, and on a specific chromosome region. Collectively, these results suggest that the mitotic phenotypes caused by Rcd1, Rcd5, MBD-R2 or Wds depletion are primarily due to reduced transcription of genes involved in kinetochore assembly and centriole duplication.
Huang, A., Kremser, L., Schuler, F., Wilflingseder, D., Lindner, H., Geley, S. and Lusser, A. (2019). Phosphorylation of Drosophila CENP-A on serine 20 regulates protein turn-over and centromere-specific loading. Nucleic Acids Res. PubMed ID: 31535131
Summary:
Centromeres are specialized chromosomal regions epigenetically defined by the presence of the histone H3 variant CENP-A. CENP-A is required for kinetochore formation which is essential for chromosome segregation during mitosis. Spatial restriction of CENP-A to the centromere is tightly controlled. Its overexpression results in ectopic incorporation and the formation of potentially deleterious neocentromeres in yeast, flies and in various human cancers. This study showed that Drosophila CENP-A is phosphorylated at serine 20 (S20) by casein kinase II and that in mitotic cells, the phosphorylated form is enriched on chromatin. Importantly, the results reveal that S20 phosphorylation regulates the turn-over of prenucleosomal CENP-A by the SCFPpa-proteasome pathway and that phosphorylation promotes removal of CENP-A from ectopic but not from centromeric sites in chromatin. Multiple lines of evidence are provided for a crucial role of S20 phosphorylation in controlling restricted incorporation of CENP-A into centromeric chromatin in flies. Modulation of the phosphorylation state of S20 may provide the cells with a means to fine-tune CENP-A levels in order to prevent deleterious loading to extra-centromeric sites.
Pauleau, A. L., Bergner, A., Kajtez, J. and Erhardt, S. (2019). The checkpoint protein Zw10 connects CAL1-dependent CENP-A centromeric loading and mitosis duration in Drosophila cells. PLoS Genet 15(9): e1008380. PubMed ID: 31553715
Summary:
A defining feature of centromeres is the presence of the histone H3 variant CENP-A that replaces H3 in a subset of centromeric nucleosomes. In Drosophila cultured cells CENP-A deposition at centromeres takes place during the metaphase stage of the cell cycle and strictly depends on the presence of its specific chaperone CAL1. How CENP-A loading is restricted to mitosis is unknown. Overexpression of CAL1 is associated with increased CENP-A levels at centromeres and uncouples CENP-A loading from mitosis. Moreover, CENP-A levels inversely correlate with mitosis duration suggesting crosstalk of CENP-A loading with the regulatory machinery of mitosis. Mitosis length is influenced by the spindle assembly checkpoint (SAC), and this study found that CAL1 interacts with the SAC protein and RZZ complex component Zw10 and thus constitutes the anchor for the recruitment of RZZ. Therefore, CAL1 controls CENP-A incorporation at centromeres both quantitatively and temporally, connecting it to the SAC to ensure mitotic fidelity.
Hatkevich, T., Boudreau, V., Rubin, T., Maddox, P. S., Huynh, J. R. and Sekelsky, J. (2019). Centromeric SMC1 promotes centromer clustering and stabilizes meiotic homolog pairing. PLoS Genet 15(10): e1008412. PubMed ID: 31609962
Summary:
During meiosis, each chromosome must selectively pair and synapse with its own unique homolog to enable crossover formation and subsequent segregation. How homolog pairing is maintained in early meiosis to ensure synapsis occurs exclusively between homologs is unknown. This study aimed to further understand this process by examining the meiotic defects of a unique Drosophila mutant, Mcm5A7. Mcm5A7 mutants are proficient in homolog pairing at meiotic onset yet fail to maintain pairing as meiotic synapsis ensues, causing seemingly normal synapsis between non-homologous loci. This pairing defect corresponds with a reduction of SMC1-dependent centromere clustering at meiotic onset. Overexpressing SMC1 in this mutant significantly restores centromere clustering, homolog pairing, and crossover formation. These data indicate that the initial meiotic pairing of homologs is not sufficient to yield synapsis exclusively between homologs and provide a model in which meiotic homolog pairing must be stabilized by centromeric SMC1 to ensure proper synapsis.
Deger, N., Yang, Y., Lindsey-Boltz, L. A., Sancar, A. and Selby, C. P. (2019). Drosophila, which lacks canonical transcription-coupled repair proteins, performs transcription-coupled repair. J Biol Chem. PubMed ID: 31624146
Summary:
Previous work with the classic T4 endonuclease V digestion of DNA from irradiated Drosophila cells followed by Southern hybridization led to the conclusion that Drosophila lacks transcription-coupled dnarep (TCR). This conclusion was reinforced by the Drosophila Genome Project which revealed that Drosophila lacks Cockayne syndrome WD repeat protein (CSA), CSB, or UV-stimulated scaffold protein A (UVSSA) homologs, whose orthologs are present in eukaryotes ranging from Arabidopsis to humans that carry out TCR. A recently developed in vivo excision assay and the excision repair-sequencing (XR-Seq) method have enabled genome-wide analysis of nucleotide excision repair in various organisms at single-nucleotide resolution and in a strand-specific manner. Using these methods, it was discovered that Drosophila S2 cells carry out robust TCR comparable to that observed in mammalian cells. These findings provide critical new insights into the mechanisms of TCR among various different species.

Tuesday November 12th - Signaling

Garcia De Las Bayonas, A., Philippe, J. M., Lellouch, A. C. and Lecuit, T. (2019). Distinct RhoGEFs activate apical and junctional contractility under control of G proteins during epithelial morphogenesis. Curr Biol. PubMed ID: 31522942
Summary:
Small RhoGTPases direct cell shape changes and movements during tissue morphogenesis. Their activities are tightly regulated in space and time to specify the desired pattern of actomyosin contractility that supports tissue morphogenesis. This is expected to stem from polarized surface stimuli and from polarized signaling processing inside cells. This study examined this general problem in the context of cell intercalation that drives extension of the Drosophila ectoderm. In the ectoderm, G protein-coupled receptors (GPCRs) and their downstream heterotrimeric G proteins (Galpha and Gbetagamma) activate Rho1 both medial-apically, where it exhibits pulsed dynamics, and at junctions, where its activity is planar polarized. However, the mechanisms responsible for polarizing Rho1 activity are unclear. This study reports that distinct guanine exchange factors (GEFs) activate Rho1 in these two cellular compartments. RhoGEF2 acts uniquely to activate medial-apical Rho1 but is recruited both medial-apically and at junctions by Galpha12/13-GTP, also called Concertina (Cta) in Drosophila. On the other hand, Dp114RhoGEF (Dp114), a newly characterized RhoGEF, is required for cell intercalation in the extending ectoderm, where it activates Rho1 specifically at junctions. Its localization is restricted to adherens junctions and is under Gbeta13F/Ggamma1 control. Furthermore, Gbeta13F/Ggamma1 activates junctional Rho1 and exerts quantitative control over planar polarization of Rho1. Finally, this study found that Dp114RhoGEF is absent in the mesoderm, arguing for a tissue-specific control over junctional Rho1 activity. These results clarify the mechanisms of polarization of Rho1 activity in different cellular compartments and reveal that distinct GEFs are sensitive tuning parameters of cell contractility in remodeling epithelia.
Moss-Taylor, L., Upadhyay, A., Pan, X., Kim, M. J. and O'Connor, M. B. (2019). Body size and tissue-scaling is regulated by motoneuron-derived activinbeta in Drosophila melanogaster. Genetics. PubMed ID: 31585954
Summary:
Correct scaling of body and organ size is crucial for proper development and survival of all organisms. Perturbations in circulating hormones, including insulins and steroids, are largely responsible for changing body size in response to both genetic and environmental factors. Such perturbations typically produce adults whose organs and appendages scale proportionately with final size. The identity of additional factors that might contribute to scaling of organs and appendages with body size is unknown. This study reports that loss-of-function mutations in Drosophila Activinbeta (Actbeta), a member of the TGF-beta superfamily, lead to production of small larvae/pupae and undersized rare adult escapers. Morphometric measurements of escaper adult appendage size (wings, legs), as well as heads, thoraxes, and abdomens, reveal a disproportional reduction in abdominal size compared to other tissues. Similar size measurements of selected Actbeta mutant larval tissues demonstrate that somatic muscle size is disproportionately smaller when compared to fat body, salivary glands, prothoracic glands, imaginal discs and brain. This study also showed that Actbeta control of body size is dependent on canonical signaling through the transcription-factor dSmad2 and that it modulates the growth rate, but not feeding behavior, during the third instar period. Tissue and cell-specific knockdown and overexpression studies reveal that motoneuron derived Actbeta is essential for regulating proper body size and tissue scaling. These studies suggest that, unlike in vertebrates where Myostatin, and certain other Activin-like factors act as systemic negative regulators of muscle mass, in Drosophila Actbeta is a positive regulator of muscle mass that is directly delivered to muscles by motoneurons. The importance of these findings in coordinating proportional scaling of insect muscle mass to appendage size is discussed.
Palu, R. A. S., Ong, E., Stevens, K., Chung, S., Owings, K. G., Goodman, A. G. and Chow, C. Y. (2019). Natural genetic variation screen in Drosophila identifies Wnt signaling, mitochondrial metabolism, and redox homeostasis genes as modifiers of apoptosis. G3 (Bethesda). PubMed ID: 31570502
Summary:
Apoptosis is the primary cause of degeneration in a number of neuronal, muscular, and metabolic disorders. These diseases are subject to a great deal of phenotypic heterogeneity in patient populations, primarily due to differences in genetic variation between individuals. This creates a barrier to effective diagnosis and treatment. Understanding how genetic variation influences apoptosis could lead to the development of new therapeutics and better personalized treatment approaches. This study examined the impact of the natural genetic variation in the Drosophila Genetic Reference Panel (DGRP) on two models of apoptosis-induced retinal degeneration: overexpression of p53 or reaper (rpr). A number of known apoptotic, neural, and developmental genes were identified as candidate modifiers of degeneration. Gene Set Enrichment Analysis (GSEA) was used to identify pathways that harbor genetic variation that impact these apoptosis models, including Wnt signaling, mitochondrial metabolism, and redox homeostasis. Finally, many of these candidates were demonstrated to have a functional effect on apoptosis and degeneration. These studies provide a number of avenues for modifying genes and pathways of apoptosis-related disease.
Gahr, B. M., Brandle, F., Zimmermann, M. and Nagel, A. C. (2019). An RBPJ-Drosophila model reveals dependence of RBPJ protein stability on the formation of transcription-regulator complexes. Cells 8(10). PubMed ID: 31615108
Summary:
Notch signaling activity is mediated by the transcriptional regulator RBPJ in a complex with activated Notch (NICD). Analysis of Notch pathway regulation in humans is hampered by a partial redundancy of the four Notch receptor copies, yet RBPJ is solitary, allowing its study in model systems. In Drosophila melanogaster, the RBPJ orthologue is encoded by Suppressor of Hairless [Su(H)]. Using genome engineering, this study replaced Su(H) by murine RBPJ in order to study its function in the fly. In fact, RBPJ largely substitutes for Su(H)'s function, yet subtle phenotypes reflect increased Notch signaling activity. Accordingly, the binding of RBPJ to Hairless (H) protein, the general Notch antagonist in Drosophila, was considerably reduced compared to that of Su(H). An H-binding defective RBPJ(LLL) mutant matched the respective Su(H)(LLL) allele: homozygotes were lethal due to extensive Notch hyperactivity. Moreover, RBPJ(LLL) protein accumulated at lower levels than wild type RBPJ, except in the presence of NICD. Apparently, RBPJ protein stability depends on protein complex formation with either H or NICD, similar to Su(H), demonstrating that the murine homologue underlies the same regulatory mechanisms as Su(H) in Drosophila. These results underscore the importance of regulating the availability of RBPJ protein to correctly mediate Notch signaling activity in the fly.
Miller, S. W., Movsesyan, A., Zhang, S., Fernandez, R. and Posakony, J. W. (2019). Evolutionary emergence of Hairless as a novel component of the Notch signaling pathway. Elife 8. PubMed ID: 31545167
Summary:
Suppressor of Hairless [Su(H)], the transcription factor at the end of the Notch pathway in Drosophila, utilizes the Hairless protein to recruit two co-repressors, Groucho (Gro) and C-terminal Binding Protein (CtBP), indirectly. Hairless is present only in the Pancrustacea, raising the question of how Su(H) in other protostomes gains repressive function. This study shows that Su(H) from a wide array of arthropods, molluscs, and annelids includes motifs that directly bind Gro and CtBP; thus, direct co-repressor recruitment is ancestral in the protostomes. How did Hairless come to replace this ancestral paradigm? The discovery of a protein (S-CAP) in Myriapods and Chelicerates that contains a motif similar to the Su(H)-binding domain in Hairless has revealed a likely evolutionary connection between Hairless and Metastasis-associated (MTA) protein, a component of the NuRD complex. Sequence comparison and widely conserved microsynteny suggest that S-CAP and Hairless arose from a tandem duplication of an ancestral MTA gene.
Fulford, A. D., Holder, M. V., Frith, D., Snijders, A. P., Tapon, N. and Ribeiro, P. S. (2019). Casein kinase 1 family proteins promote Slimb-dependent Expanded degradation. Elife 8. PubMed ID: 31567070
Summary:
Hippo signalling integrates diverse stimuli related to epithelial architecture to regulate tissue growth and cell fate decisions. The Hippo kinase cascade represses the growth-promoting transcription co-activator Yorkie. The FERM protein Expanded is one of the main upstream Hippo signalling regulators in Drosophila as it promotes Hippo kinase signalling and directly inhibits Yorkie. To fulfil its function, Expanded is recruited to the plasma membrane by the polarity protein Crumbs. However, Crumbs-mediated recruitment also promotes Expanded turnover via a phosphodegron-mediated interaction with a Slimb/beta-TrCP SCF E3 ligase complex. This study shows that the Casein Kinase 1 (CKI) family is required for Expanded phosphorylation. CKI expression promotes Expanded phosphorylation and interaction with Slimb/beta-TrCP. Conversely, CKI depletion in S2 cells impairs Expanded degradation downstream of Crumbs. In wing imaginal discs, CKI loss leads to elevated Expanded and Crumbs levels. Thus, phospho-dependent Expanded turnover ensures a tight coupling of Hippo pathway activity to epithelial architecture.

Friday, November 8th - Evolution

Allan, C. W. and Matzkin, L. M. (2019). Genomic analysis of the four ecologically distinct cactus host populations of Drosophila mojavensis. BMC Genomics 20(1): 732. PubMed ID: 31606030
Summary:
Relationships between an organism and its environment can be fundamental in the understanding how populations change over time and species arise. Local ecological conditions can shape variation at multiple levels, among these are the evolutionary history and trajectories of coding genes. This study examines the rate of molecular evolution at protein-coding genes throughout the genome in response to host adaptation in the cactophilic Drosophila mojavensis. These insects are intimately associated with cactus necroses, developing as larvae and feeding as adults in these necrotic tissues. Drosophila mojavensis is composed of four isolated populations across the deserts of western North America and each population has adapted to utilize different cacti that are chemically, nutritionally, and structurally distinct. High coverage Illumina sequencing was performed on three previously unsequenced populations of D. mojavensis. Genomes were assembled using the previously sequenced genome of D. mojavensis from Santa Catalina Island (USA) as a template. Protein coding genes were aligned across all four populations and rates of protein evolution were determined for all loci using a several approaches. Loci that exhibited elevated rates of molecular evolution tend to be shorter, have fewer exons, low expression, be transcriptionally responsive to cactus host use and have fixed expression differences across the four cactus host populations. Fast evolving genes were involved with metabolism, detoxification, chemosensory reception, reproduction and behavior. Results of this study give insight into the process and the genomic consequences of local ecological adaptation.
Himmel, N. J., Letcher, J. M., Sakurai, A., Gray, T. R., Benson, M. N. and Cox, D. N. (2019). Drosophila menthol sensitivity and the precambrian origins of transient receptor potential-dependent chemosensation. Philos Trans R Soc Lond B Biol Sci 374(1785): 20190369. PubMed ID: 31544603
Summary:
Transient receptor potential (TRP) cation channels are highly conserved, polymodal sensors which respond to a wide variety of stimuli. Perhaps most notably, TRP channels serve critical functions in nociception and pain. A growing body of evidence suggests that transient receptor potential melastatin (TRPM) and transient receptor potential ankyrin (TRPA) thermal and electrophile sensitivities predate the protostome-deuterostome split (greater than 550 Ma). However, TRPM and TRPA channels are also thought to detect modified terpenes (e.g. menthol). Although terpenoids like menthol are thought to be aversive and/or harmful to insects, mechanistic sensitivity studies have been largely restricted to chordates. Furthermore, it is unknown if TRP-menthol sensing is as ancient as thermal and/or electrophile sensitivity. Combining genetic, optical, electrophysiological, behavioural and phylogenetic approaches, this study tested the hypothesis that insect TRP channels play a conserved role in menthol sensing. Topical application of menthol to Drosophila melanogaster larvae was found to elicit a Trpm- and TrpA1-dependent nocifensive rolling behaviour, which requires activation of Class IV nociceptor neurons. Further, in characterizing the evolution of TRP channels, the hypotheses were put forward that three previously undescribed TRPM channel clades (basal, alphaTRPM and betaTRPM), as well as TRPs with residues critical for menthol sensing, were present in ancestral bilaterians.
Bracewell, R., Chatla, K., Nalley, M. J. and Bachtrog, D. (2019). Dynamic turnover of centromeres drives karyotype evolution in Drosophila. Elife 8. PubMed ID: 31524597
Summary:
Centromeres are the basic unit for chromosome inheritance, but their evolutionary dynamics is poorly understood. This study generate high-quality reference genomes for multiple Drosophila obscura group species to reconstruct karyotype evolution. All chromosomes in this lineage were ancestrally telocentric and the creation of metacentric chromosomes in some species was driven by de novo seeding of new centromeres at ancestrally gene-rich regions, independently of chromosomal rearrangements. The emergence of centromeres resulted in a drastic size increase due to repeat accumulation, and dozens of genes previously located in euchromatin are now embedded in pericentromeric heterochromatin. Metacentric chromosomes secondarily became telocentric in the pseudoobscura subgroup through centromere repositioning and a pericentric inversion. The former (peri)centric sequences left behind shrunk dramatically in size after their inactivation, yet contain remnants of their evolutionary past, including increased repeat-content and heterochromatic environment. Centromere movements are accompanied by rapid turnover of the major satellite DNA detected in (peri)centromeric regions.
Lewis, M. T. and Hamby, K. A. (2019). Differential impacts of yeasts on feeding behavior and development in larval Drosophila suzukii (Diptera:Drosophilidae). Sci Rep 9(1): 13370. PubMed ID: 31527678
Summary:
Larval Drosophila encounter and feed on a diverse microbial community within fruit. In particular, free-living yeast microbes provide a source of dietary protein critical for development. However, successional changes to the fruit microbial community may alter host quality through impacts on relative protein content or yeast community composition. For many species of Drosophila, fitness benefits from yeast feeding vary between individual yeast species, indicating differences in yeast nutritional quality. To better understand these associations, this study evaluated how five species of yeast impacted feeding preference and development in larval Drosophila suzukii. Larvae exhibited a strong attraction to the yeast Hanseniaspora uvarum in pairwise yeast feeding assays. However, larvae also performed most poorly on diets containing H. uvarum, a mismatch in preference and performance that suggests differences in yeast nutritional quality are not the primary factor driving larval feeding behavior. Together, these results demonstrate that yeast plays a critical role in D. suzukii's ecology and that larvae may have developed specific yeast associations. Further inquiry, including systematic comparisons of Drosophila larval yeast associations more broadly, will be necessary to understand patterns of microbial resource use in larvae of D. suzukii and other frugivorous species.
Hironaka, K. I., Fujimoto, K. and Nishimura, T. (2019). Optimal scaling of critical size for metamorphosis in the genus Drosophila. iScience 20: 348-358. PubMed ID: 31610371
Summary:
Juveniles must reach a critical body size to become a mature adult. Molecular determinants of critical size have been studied, but the evolutionary importance of critical size is still unclear. Using nine fly species, this study showed that interspecific variation in organism size can be explained solely by species-specific critical size. The observed variation in critical size quantitatively agrees with the interspecific scaling relationship predicted by the life history model, which hypothesizes that critical size mediates an energy allocation switch between juvenile and adult tissues. The mechanism underlying critical size scaling is explained by an inverse relationship between growth duration and growth rate, which cancels out their contributions to the final size. Finally, evolutionary changes in growth duration can be traced back to the scaling of ecdysteroid hormone dynamics. It is concluded that critical size adaptively optimizes energy allocation, and has a central role in organism size determination.
Leitao, A. B., Bian, X., Day, J. P., Pitton, S., Demir, E. and Jiggins, F. M. (2019). Independent effects on cellular and humoral immune responses underlie genotype-by-genotype interactions between Drosophila and parasitoids. PLoS Pathog 15(10): e1008084. PubMed ID: 31589659
Summary:
It is common to find abundant genetic variation in host resistance and parasite infectivity within populations, with the outcome of infection frequently depending on genotype-specific interactions. Underlying these effects are complex immune defenses that are under the control of both host and parasite genes. Extensive variation in Drosophila melanogaster's immune response was found against the parasitoid wasp Leptopilina boulardi. Some aspects of the immune response, such as phenoloxidase activity, are predominantly affected by the host genotype. Some, such as upregulation of the complement-like protein Tep1, are controlled by the parasite genotype. Others, like the differentiation of immune cells called lamellocytes, depend on the specific combination of host and parasite genotypes. These observations illustrate how the outcome of infection depends on independent genetic effects on different aspects of host immunity. These observations provide a physiological mechanism to generate phenomena like epistasis and genotype-interactions that underlie models of coevolution.

Thursday, November 7th - RNA and Transposons

Cordeiro, J., Carvalho, T. L., Valente, V. and Robe, L. J. (2019). Evolutionary history and classification of Micropia retroelements in Drosophilidae species. PLoS One 14(10): e0220539. PubMed ID: 31622354
Summary:
Transposable elements (TEs) have the main role in shaping the evolution of genomes and host species, contributing to the creation of new genes and promoting rearrangements frequently associated with new regulatory networks. Micropia belongs to the Ty3/Gypsy group of long terminal repeats (LTR) retroelements and comprises one of the least studied Drosophila transposable elements. This study assessed the evolutionary history of Micropia within Drosophilidae, while trying to assist in the classification of this TE. At first, searches were performed of Micropia presence in the genome of natural populations from several species. Then, based on searches within online genomic databases, Micropia-like sequences were retrieved from the genomes of distinct Drosophilidae species. The knowledge of Micropia distribution within Drosophila species. The Micropia retroelements consist of an array of divergent sequences, which was subdivided into 20 subfamilies. Even so, a patchy distribution of Micropia sequences within the Drosophilidae phylogeny could be identified, with incongruences between the species phylogeny and the Micropia phylogeny. Comparing the pairwise synonymous distance (dS) values between Micropia and three host nuclear sequences, several cases were found of unexpectedly high levels of similarity between Micropia sequences in divergent species. All these findings provide a hypothesis to the evolution of Micropia within Drosophilidae, which include several events of vertical and horizontal transposon transmission, associated with ancestral polymorphisms and recurrent Micropia sequences diversification.
Guan, L. and Grigoriev, A. (2019). Age-related Argonaute loading of ribosomal RNA fragments. Microrna. PubMed ID: 31538909
Summary:
Accumulating evidence points to the functional roles of rRNA derived fragments (rRFs), often considered degradation byproducts. Small RNAs, including miRNAs and tRNA-derived fragments (tRFs), have been implicated in the aging process and this study considered rRFs in this context. A computational analysis was performed of Argonaute-loaded rRFs in Drosophila melanogaster to study rRF changes with age. rRF abundance in Ago1 and Ago2 was considered at 3 and 30 days to identify Ago1-guided and Ago2-guided fragments. Putative seed sequences were sought in rRFs based on frequent matches of sliding k-mer windows to the conserved regions of 12 Drosophila genomes. Putative targets (containing matches to seeds identified in four rRFs) were predicted and their functional enrichments were studied using Gene Ontology. Precise cleavage sites of distinct rRF isoforms were identified from both nuclear and mitochondrial rRNAs. The most prominent rRF isoforms were enriched in Ago2 at 3 days and that loading strongly decreased with age. For less abundant rRFs, loading of Ago2-guided rRFs generally increased in Ago2, whereas Ago1-guided rRFs revealed diverse patterns. The distribution of seed matches in targets suggested that rRFs may bind to various conserved regions of many genes, possibly via miRNA-like seed-based mechanisms. These observations suggest that rRFs may be functional molecules, with age-dependent Argonaute loading, comparable to that of miRNAs and tRFs. The putative rRF targets showed significant enrichment in developmental processes and biological regulation, similar to tRFs and consistent with a possible involvement of these newly identified small RNAs in the Drosophila aging.
Barr, J., Gilmutdinov, R., Wang, L., Shidlovskii, Y. and Schedl, P. (2019). The Drosophila CPEB protein Orb specifies oocyte fate by a 3'UTR dependent autoregulatory loop. Genetics. PubMed ID: 31594794
Summary:
orb encodes one of the two fly CPEB proteins. These widely conserved proteins bind to the 3'UTRs of target mRNAs and activate or repress their translation. This study shows that a positive autoregulatory loop driven by the orb gene propels the specification of oocyte identity in Drosophila egg chambers. Oocyte fate specification is mediated by a 3'UTR dependent mechanism that concentrates orb mRNAs and proteins in one of the two pro-oocytes in the 16 cell germline cyst. When the orb 3'UTR is deleted, orb mRNA and protein fail to localize and all sixteen cells become nurse cells. In WT, the oocyte is specified when orb and other gene products concentrate in a single cell in region 2b of the germarium. A partially functional orb 3'UTR replacement delays oocyte specification until the egg chambers reach stage 2 of oogenesis. Prior to this point, orb mRNA and protein are unlocalized, as are other markers of oocyte identity, and the oocyte is not specified. After stage 2, approximately 50% of the chambers successfully localize orb in a single cell, and this cell assumes oocyte identity. In the remaining chambers, the orb autoregulatory loop is not activated and no oocyte is formed. Finally, maintenance of oocyte identity requires continuous orb activity.
Kong, J., Han, H., Bergalet, J., Bouvrette, L. P. B., Hernandez, G., Moon, N. S., Vali, H., Lecuyer, E. and Lasko, P. (2019). A ribosomal protein S5 isoform is essential for oogenesis and interacts with distinct RNAs in Drosophila melanogaster. Sci Rep 9(1): 13779. PubMed ID: 31551467
Summary:
In Drosophila melanogaster there are two genes encoding ribosomal protein S5, RpS5a and RpS5b. This study demonstrates that RpS5b is required for oogenesis. Females lacking RpS5b produce ovaries with numerous developmental defects that undergo widespread apoptosis in mid-oogenesis. Females lacking germline RpS5a are fully fertile, but germline expression of interfering RNA targeting germline RpS5a in an RpS5b mutant background worsened the RpS5b phenotype and blocked oogenesis before egg chambers form. A broad spectrum of mRNAs co-purified in immunoprecipitations with RpS5a, while RpS5b-associated mRNAs were specifically enriched for GO terms related to mitochondrial electron transport and cellular metabolic processes. Consistent with this, RpS5b mitochondrial fractions are depleted for proteins linked to oxidative phosphorylation and mitochondrial respiration, and RpS5b mitochondria tended to form large clusters and had more heterogeneous morphology than those from controls. It is concluded that RpS5b-containing ribosomes preferentially associate with particular mRNAs and serve an essential function in oogenesis.
Lo Piccolo, L., Mochizuki, H. and Nagai, Y. (2019). The lncRNA hsromega regulates arginine dimethylation of human FUS to cause its proteasomal degradation in Drosophila. J Cell Sci. PubMed ID: 31519807
Summary:
The lncRNAs play structural and regulatory roles on RNA-binding proteins (RBPs). However, the mechanisms by which lncRNAs regulate the neurodegenerative-causative RBP like FUS remain poorly understood. This study shows that knockdown of the lncRNA hsromega causes a shift in the methylation status of human FUS from mono- (MMA) to di-methylated (DMA) arginine via upregulation of the argininemethyl transferases 5 (PRMT5). This novel regulatory role is critical to FUS toxicity since the PRMT5-dependent dimethylation of FUS is required for its proteasomal degradation and causes a reduction of high levels of FUS. Moreover, this study shows that an increase of FUS determines a decline of both PRMT 1 and 5 transcripts leading to an accumulation of neurotoxic MMA-FUS. Therefore, overexpression of either PRMT1 or PRMT5 is able to rescue the FUS toxicity. These results highlight a novel role of lncRNAs in post-translation modification (PTM) of FUS and suggest a causal relationship between lncRNAs and dysfunctional PRMTs in the pathogenesis of FUSopathies.
Osumi, K., Sato, K., Murano, K., Siomi, H. and Siomi, M. C. (2019). Essential roles of Windei and nuclear monoubiquitination of Eggless/SETDB1 in transposon silencing. EMBO Rep: e48296. PubMed ID: 31576653
Summary:
Eggless/SETDB1 (Egg), the only essential histone methyltransferase (HMT) in Drosophila, plays a role in gene repression, including piRNA-mediated transposon silencing in the ovaries. Previous studies suggested that Egg is post-translationally modified and showed that Windei (Wde) regulates Egg nuclear localization through protein-protein interaction. Monoubiquitination of mammalian SETDB1 is necessary for the HMT activity. Using cultured ovarian somatic cells, this study shows that Egg is monoubiquitinated and phosphorylated but that only monoubiquitination is required for piRNA-mediated transposon repression. Egg monoubiquitination occurs in the nucleus. Egg has its own nuclear localization signal, and the nuclear import of Egg is Wde-independent. Wde recruits Egg to the chromatin at target gene silencing loci, but their interaction is monoubiquitin-independent. The abundance of nuclear Egg is governed by that of nuclear Wde. These results illuminate essential roles of nuclear monoubiquitination of Egg and the role of Wde in piRNA-mediated transposon repression.

Wednesday, November 6th - Behavior

Khodaei, L. and Long, T. A. F. (2019). Kin recognition and co-operative foraging in Drosophila melanogaster larvae. J Evol Biol. PubMed ID: 31454451
Summary:
A long-standing goal for biologists and social scientists is to understand the factors that lead to the evolution and maintenance of co-operative behaviour between conspecifics. To that end, the fruit fly, Drosophila melanogaster, is becoming an increasingly popular model species to study sociality, however, most of the research to date has focused on adult behaviours. This study set out to examine group feeding behaviour by larvae and to determine whether the degree of relatedness between individuals mediates the expression co-operation. In a series of assays, the average degree of relatedness was manipulated in groups of third instar larvae that were faced with resource scarcity, and measured the size, frequency and composition of feeding clusters, as well as the fitness benefits associated with co-operation. The results suggest that larval D. melanogaster are capable of kin recognition (something that has not been previously described in this species), as clusters were more numerous, larger and involved more larvae, when more closely related kin were present in the social environment. These findings are discussed in the context of the correlated fitness-associated benefits of co-operation, the potential mechanisms by which individuals may recognize kin, and how that kinship may play an important role in facilitating the manifestation of this co-operative behaviour.
Varma, V., Krishna, S., Srivastava, M., Sharma, V. K. and Sheeba, V. (2019). Accuracy of fruit-fly eclosion rhythms evolves by strengthening circadian gating rather than developmental fine-tuning. Biol Open 8(8). PubMed ID: 31455663
Summary:
Fruit flies (Drosophila melanogaster) eclose from their pupae mainly around dawn. The timing of eclosion is thought to confer adaptive benefits to the organisms and thus shows remarkable accuracy. This study examined the relative contributions of gating of eclosion by the circadian clock versus clock-independent developmental rates and light-induced responses in the eclosion phenotype exhibited by fly populations that have evolved greater accuracy in eclosion rhythms compared to controls. The results showed that stocks that have evolved greater accuracy of eclosion rhythms due to artificial selection do not show reduced individual variation in pupariation and pigmentation time compared to controls, though they do exhibit lower variation in eclosion time. Selected stocks also did not show lower variation in eclosion time under constant light conditions in contrast to the greater accuracy seen under light-dark cycles. Moreover, manipulations of developmental rate by varying larval density and inducing eclosion by changing onset of light phase did not alter the eclosion profile of selected stocks as much as it did controls, since selected stocks largely restricted eclosion to the daytime. These results suggest that fly populations selected for greater accuracy have evolved accurate eclosion rhythms primarily by strengthening circadian gating of eclosion rather than due to fine-tuning of clock-independent developmental processes.
Abhilash, L., Ghosh, A. and Sheeba, V. (2019). Selection for timing of eclosion results in co-evolution of temperature responsiveness in Drosophila melanogaster. J Biol Rhythms: 748730419877315. PubMed ID: 31608742
Summary:
Circadian rhythms in adult eclosion of Drosophila are postulated to be regulated by a pair of coupled oscillators: one is the master clock that is light sensitive and temperature compensated and the other that is a slave oscillator whose period is temperature sensitive and whose phase is reflected in the overt behavior. Within this framework, it was reasoned that in populations of Drosophila melanogaster that have been artificially selected for highly divergent phases of eclosion rhythm, there may be changes in this network of the master-slave oscillator system, via changes in the temperature-sensitive oscillator and/or the coupling of the light- and temperature-sensitive oscillators. Light/dark cycles were used in conjunction with different constant ambient temperatures and two different amplitudes of temperature cycles in an overall cool or warm temperature and analyzed phases, gate width, and normalized amplitude of the rhythms in each of these conditions. The populations selected for eclosion in the morning (early flies) do not vary their phases with change in temperature regimes, whereas the populations selected for eclosion in the evening (late flies) show phase lability of up to ~5 h. These results imply a genetic correlation between timing of behavior and temperature sensitivity of the circadian clock.
Gunel, S., Rhodin, H., Morales, D., Campagnolo, J. H., Ramdya, P. and Fua, P. (2019). DeepFly3D, a deep learning-based approach for 3D limb and appendage tracking in tethered, adult Drosophila. Elife 8. PubMed ID: 31584428
Summary:
Studying how neural circuits orchestrate limbed behaviors requires the precise measurement of the positions of each appendage in 3-dimensional (3D) space. Deep neural networks can estimate 2-dimensional (2D) pose in freely behaving and tethered animals. However, the unique challenges associated with transforming these 2D measurements into reliable and precise 3D poses have not been addressed for small animals including the fly, Drosophila melanogaster. This study presents DeepFly3D, a software that infers the 3D pose of tethered, adult Drosophila using multiple camera images. DeepFly3D does not require manual calibration, uses pictorial structures to automatically detect and correct pose estimation errors, and uses active learning to iteratively improve performance.More accurate unsupervised behavioral embedding was demonstrated using 3D joint angles rather than commonly used 2D pose data. Thus, DeepFly3D enables the automated acquisition of Drosophila behavioral measurements at an unprecedented level of detail for a variety of biological applications.
Massey, J. H., Chung, D., Siwanowicz, I., Stern, D. L. and Wittkopp, P. J. (2019). The yellow gene influences Drosophila male mating success through sex comb melanization. Elife 8. PubMed ID: 31612860
Summary:
Drosophila melanogaster males perform a series of courtship behaviors that, when successful, result in copulation with a female. For over a century, mutations in the yellow gene, named for its effects on pigmentation, have been known to reduce male mating success. Prior work has suggested that yellow influences mating behavior through effects on wing extension, song, and/or courtship vigor. This study ruled out these explanations, as well as effects on the nervous system more generally, and found instead that the effects of yellow on male mating success are mediated by its effects on pigmentation of male-specific leg structures called sex combs. Loss of yellow expression in these modified bristles reduces their melanization, which changes their structure and causes difficulty grasping females prior to copulation. These data illustrate why the mechanical properties of anatomy, not just neural circuitry, must be considered to fully understand the development and evolution of behavior.
Gonzalez, D., Rihani, K., Neiers, F., Poirier, N., Fraichard, S., Gotthard, G., Chertemps, T., Maibeche, M., Ferveur, J. F. and Briand, L. (2019). The Drosophila odorant-binding protein 28a is involved in the detection of the floral odour ss-ionone. Cell Mol Life Sci. PubMed ID: 31564000
Summary:
Odorant-binding proteins (OBPs) are small soluble proteins that are thought to transport hydrophobic odorants across the aqueous sensillar lymph to olfactory receptors. A recent study revealed that OBP28a, one of the most abundant Drosophila OBPs, is not required for odorant transport, but acts in buffering rapid odour variation in the odorant environment. To further unravel and decipher its functional role, recombinant OBP28a was expressed, and its binding specificity was characterized. Using a fluorescent binding assay, it was found that OBP28a binds a restricted number of floral-like chemicals, including ss-ionone, with an affinity in the micromolar range. The X-ray crystal structure was solved of OBP28a, which showed extensive conformation changes upon ligand binding. Mutant flies genetically deleted for the OBP28a gene showed altered responses to ss-ionone at a given concentration range, supporting its essential role in the detection of specific compounds present in the natural environment of the fly.

Tuesday, November 5th - Adult Neural Development and Function

Mancini, N., Hranova, S., Weber, J., Weiglein, A., Schleyer, M., Weber, D., Thum, A. S. and Gerber, B. (2019). Reversal learning in Drosophila larvae. Learn Mem 26(11): 424-435. PubMed ID: 31615854
Summary:
Adjusting behavior to changed environmental contingencies is critical for survival, and reversal learning provides an experimental handle on such cognitive flexibility. This study investigated reversal learning in larval Drosophila. Using odor-taste associations, olfactory reversal learning was established in the appetitive and the aversive domain, using either fructose as a reward or high-concentration sodium chloride as a punishment, respectively. Reversal learning is demonstrated both in differential and in absolute conditioning, in either valence domain. In differential conditioning, the animals are first trained such that an odor A is paired, for example, with the reward whereas odor B is not (A+/B); this is followed by a second training phase with reversed contingencies (A/B+). In absolute conditioning, odor B is omitted, such that the animals are first trained with paired presentations of A and reward, followed by unpaired training in the second training phase. The results reveal "true" reversal learning in that the opposite associative effects of both the first and the second training phase are detectable after reversed-contingency training. In what is a surprisingly quick, one-trial contingency adjustment in the Drosophila larva, the present study establishes a simple and genetically easy accessible study case of cognitive flexibility.
Dickerson, B. H., de Souza, A. M., Huda, A. and Dickinson, M. H. (2019). Flies regulate wing motion via active control of a dual-Function gyroscope. Curr Biol. PubMed ID: 31607538
Summary:
Flies execute their remarkable aerial maneuvers using a set of wing steering muscles, which are activated at specific phases of the stroke cycle. The activation phase of these muscles-which determines their biomechanical output - arises via feedback from mechanoreceptors at the base of the wings and structures unique to flies called halteres. Evolved from the hindwings, the tiny halteres oscillate at the same frequency as the wings, although they serve no aerodynamic function and are thought to act as gyroscopes. Like the wings, halteres possess minute control muscles whose activity is modified by descending visual input, raising the possibility that flies control wing motion by adjusting the motor output of their halteres, although this hypothesis has never been directly tested. Using genetic techniques possible in Drosophila melanogaster, this study tested the hypothesis that visual input during flight modulates haltere muscle activity and that this, in turn, alters the mechanosensory feedback that regulates the wing steering muscles. The results suggest that rather than acting solely as a gyroscope to detect body rotation, halteres also function as an adjustable clock to set the spike timing of wing motor neurons, a specialized capability that evolved from the generic flight circuitry of their four-winged ancestors. In addition to demonstrating how the efferent control loop of a sensory structure regulates wing motion, these results provide insight into the selective scenario that gave rise to the evolution of halteres.
Marmor-Kollet, N., Gutman, I., Issman-Zecharya, N. and Schuldiner, O. (2019). Glial derived TGF-beta instructs axon midline stopping. Front Mol Neurosci 12: 232. PubMed ID: 31611773
Summary:
A fundamental question that underlies the proper wiring and function of the nervous system is how axon extension stops during development. However, mechanistic understanding of axon stopping is currently poor. The stereotypic development of the Drosophila mushroom body (MB) provides a unique system in which three types of anatomically distinct neurons (gamma, alpha'/beta', and alpha/beta) develop and interact to form a complex neuronal structure. All three neuronal types innervate the ipsi-lateral side and do not cross the midline. Plum, an immunoglobulin (Ig) superfamily protein that was previously shown to function as a TGF-beta accessory receptor, is required within MB alpha/beta neurons for their midline stopping. Overexpression of Plum within MB neurons is sufficient to induce retraction of alpha/beta axons. As expected, rescue experiments revealed that Plum likely functions in alpha/beta neurons and mediates midline stopping via the downstream effector RhoGEF2. Finally, glial-derived Myoglianin (Myo) was identified as the major TGF-beta ligand that instructs midline stopping of MB neurons. Taken together, this study strongly suggests that TGF-beta signals originating from the midline facilitate midline stopping of alpha/beta neuron in a Plum dependent manner.
Liu, L. Y., Long, X., Yang, C. P., Miyares, R. L., Sugino, K., Singer, R. H. and Lee, T. (2019). Mamo decodes hierarchical temporal gradients into terminal neuronal fate. Elife 8. PubMed ID: 31545163
Summary:
Temporal patterning is a seminal method of expanding neuronal diversity. This study has unravel a mechanism decoding neural stem cell temporal gene expression and transforming it into discrete neuronal fates. This mechanism is characterized by hierarchical gene expression. First, Drosophila neuroblasts express opposing temporal gradients of RNA-binding proteins, Imp and Syp. These proteins promote or inhibit chinmo translation, yielding a descending neuronal gradient. Together, first and second-layer temporal factors define a temporal expression window of BTB-zinc finger nuclear protein, Mamo. The precise temporal induction of Mamo is achieved via both transcriptional and post-transcriptional regulation. Finally, Mamo is essential for the temporally defined, terminal identity of alpha'/beta' mushroom body neurons and identity maintenance. This study describes a straightforward paradigm of temporal fate specification where diverse neuronal fates are defined via integrating multiple layers of gene regulation. The neurodevelopmental roles of orthologous/related mammalian genes suggest a fundamental conservation of this mechanism in brain development.
Deutsch, D., Clemens, J., Thiberge, S. Y., Guan, G. and Murthy, M. (2019). Shared song detector neurons in Drosophila male and female brains drive sex-specific behaviors. Curr Biol 29(19): 3200-3215.e3205. PubMed ID: 31564492
Summary:
Males and females often produce distinct responses to the same sensory stimuli. How such differences arise-at the level of sensory processing or in the circuits that generate behavior-remains largely unresolved across sensory modalities. This issue was addressed in the acoustic communication system of Drosophila. During courtship, males generate time-varying songs, and each sex responds with specific behaviors. Male and female behavioral tuning was characterized for all aspects of song, and feature tuning was shown to be similar between sexes, suggesting sex-shared song detectors drive divergent behaviors. Higher-order neurons in the Drosophila brain, called pC2, were identified that are tuned for multiple temporal aspects of one mode of the male's song and drive sex-specific behaviors. Neurons were also uncovered that are specifically tuned to an acoustic communication signal and reside at the sensory-motor interface, flexibly linking auditory perception with sex-specific behavioral responses.
Delventhal, R., O'Connor, R. M., Pantalia, M. M., Ulgherait, M., Kim, H. X., Basturk, M. K., Canman, J. C. and Shirasu-Hiza, M. (2019). Dissection of central clock function in Drosophila through cell-specific CRISPR-mediated clock gene disruption. Elife 8. PubMed ID: 31613218
Summary:
In Drosophila, ~150 neurons expressing molecular clock proteins regulate circadian behavior. Sixteen of these neurons secrete the neuropeptide Pdf and have been called 'master pacemakers' because they are essential for circadian rhythms. A subset of Pdf(+) neurons (the morning oscillator) regulates morning activity and communicates with other non-Pdf(+) neurons, including a subset called the evening oscillator. It has been assumed that the molecular clock in Pdf(+) neurons is required for these functions. To test this, Gal4-UAS based CRISPR tools were developed and validated for cell-specific disruption of key molecular clock components, period and timeless. While loss of the molecular clock in both the morning and evening oscillators eliminates circadian locomotor activity, the molecular clock in either oscillator alone is sufficient to rescue circadian locomotor activity in the absence of the other. This suggests that clock neurons do not act in a hierarchy but as a distributed network to regulate circadian activity.

Monday, November 4th - Disease models

Kasza, K. E., Supriyatno, S. and Zallen, J. A. (2019). Cellular defects resulting from disease-related myosin II mutations in Drosophila. Proc Natl Acad Sci U S A. PubMed ID: 31615886
Summary:
The nonmuscle myosin II motor protein produces forces that are essential to driving the cell movements and cell shape changes that generate tissue structure. Mutations in myosin II that are associated with human diseases are predicted to disrupt critical aspects of myosin function, but the mechanisms that translate altered myosin activity into specific changes in tissue organization and physiology are not well understood. This study used the Drosophila embryo to model human disease mutations that affect myosin motor activity. Using in vivo imaging and biophysical analysis, it was shown that engineering human MYH9-related disease mutations into Drosophila myosin II produces motors with altered organization and dynamics that fail to drive rapid cell movements, resulting in defects in epithelial morphogenesis. In embryos that express the Drosophila myosin motor variants R707C or N98K and have reduced levels of wild-type myosin, myosin motors are correctly planar polarized and generate anisotropic contractile tension in the tissue. However, expression of these motor variants is associated with a cellular-scale reduction in the speed of cell intercalation, resulting in a failure to promote full elongation of the body axis. In addition, these myosin motor variants display slowed turnover and aberrant aggregation at the cell cortex, indicating that mutations in the motor domain influence mesoscale properties of myosin organization and dynamics. These results demonstrate that disease-associated mutations in the myosin II motor domain disrupt specific aspects of myosin localization and activity during cell intercalation, linking molecular changes in myosin activity to defects in tissue morphogenesis.
Grandon, B., Rincheval-Arnold, A., Jah, N., Corsi, J. M., Araujo, L. M., Glatigny, S., Prevost, E., Roche, D., Chiocchia, G., Guenal, I., Gaumer, S. and Breban, M. (2019). HLA-B27 alters BMP/TGFbeta signalling in Drosophila, revealing putative pathogenic mechanism for spondyloarthritis. Ann Rheum Dis. PubMed ID: 31563893
Summary:
The human leucocyte antigen (HLA)-B27 confers an increased risk of spondyloarthritis (SpA) by unknown mechanism. The objective of this work was to uncover HLA-B27 non-canonical properties that could explain its pathogenicity, using a new Drosophila model. Transgenic Drosophila expressing the SpA-associated HLA-B*27:04 or HLA-B*27:05 subtypes, or the non-associated HLA-B*07:02 allele, were generated alone or in combination with human beta2-microglobulin (hbeta2m), under tissue-specific drivers. Loss of crossveins in the wings and a reduced eye phenotype were observed after expression of HLA-B*27:04 or HLA-B*27:05 in Drosophila but not in fruit flies expressing the non-associated HLA-B*07:02 allele. These HLA-B27-induced phenotypes required the presence of hbeta2m that allowed expression of well-folded HLA-B conformers at the cell surface. Loss of crossveins resulted from a dominant negative effect of HLA-B27 on the type I bone morphogenetic protein (BMP) receptor saxophone (Sax) with which it interacted, resulting in elevated Mothers against decapentaplegic (Mad, a Drosophila receptor-mediated Smad) phosphorylation. Likewise, in immune cells from patients with SpA, HLA-B27 specifically interacted with activin receptor-like kinase-2 (ALK2), the mammalian Sax ortholog, at the cell surface and elevated Smad phosphorylation was observed in response to activin A and transforming growth factor beta (TGFbeta). It is concluded that antagonistic interaction of HLA-B27 with ALK2, which exerts inhibitory functions on the TGFbeta/BMP signalling pathway at the cross-road between inflammation and ossification, could adequately explain SpA development.
Hsieh, Y. C., Guo, C., Yalamanchili, H. K., Abreha, M., Al-Ouran, R., Li, Y., Dammer, E. B., Lah, J. J., Levey, A. I., Bennett, D. A., De Jager, P. L., Seyfried, N. T., Liu, Z. and Shulman, J. M. (2019). Tau-mediated disruption of the spliceosome triggers cryptic RNA splicing and neurodegeneration in Alzheimer's disease. Cell Rep 29(2): 301-316.e310. PubMed ID: 31597093
Summary:
In Alzheimer's disease (AD), spliceosomal proteins with critical roles in RNA processing aberrantly aggregate and mislocalize to Tau neurofibrillary tangles. This study tested the hypothesis that Tau-spliceosome interactions disrupt pre-mRNA splicing in AD. In human postmortem brain with AD pathology, Tau coimmunoprecipitates with spliceosomal components. In Drosophila, pan-neuronal Tau expression triggers reductions in multiple core and U1-specific spliceosomal proteins, and genetic disruption of these factors, including SmB, U1-70K, and U1A, enhances Tau-mediated neurodegeneration. It was further shown that loss of function in SmB, encoding a core spliceosomal protein, causes decreased survival, progressive locomotor impairment, and neuronal loss, independent of Tau toxicity. Lastly, RNA sequencing reveals a similar profile of mRNA splicing errors in SmB mutant and Tau transgenic flies, including intron retention and non-annotated cryptic splice junctions. In human brains, cryptic splicing errors were confirmed in association with neurofibrillary tangle burden. These results implicate spliceosome disruption and the resulting transcriptome perturbation in Tau-mediated neurodegeneration in AD.
Higham, J. P., Hidalgo, S., Buhl, E. and Hodge, J. J. L. (2019). Restoration of olfactory memory in Drosophila overexpressing human Alzheimer's disease associated Tau by manipulation of L-type Ca(2+) channels. Front Cell Neurosci 13: 409. PubMed ID: 31551716
Summary:
The cellular underpinnings of memory deficits in Alzheimer's disease (AD) are poorly understood. This study utilized the tractable neural circuits sub-serving memory in Drosophila to investigate the role of impaired Ca(2+) handling in memory deficits caused by expression of human 0N4R isoform of tau which is associated with AD. Expression of tau in mushroom body neuropils, or a subset of mushroom body output neurons, led to impaired memory. By using the Ca(2+) reporter GCaMP6f, changes were pbserved in Ca(2+) signaling when tau was expressed in these neurons, an effect that could be blocked by the L-type Ca(2+) channel antagonist nimodipine or reversed by RNAi knock-down of the L-type channel gene. The L-type Ca(2+) channel itself is required for memory formation, however, RNAi knock-down of the L-type Ca(2+) channel in neurons overexpressing human tau resulted in flies whose memory is restored to levels equivalent to wild-type. Expression data suggest that Drosophila L-type Ca(2+) channel mRNA levels are increased upon tau expression in neurons, thus contributing to the effects observed on memory and intracellular Ca(2+) homeostasis. Together, these Ca(2+) imaging and memory experiments suggest that expression of the 0N4R isoform of human tau increases the number of L-type Ca(2+) channels in the membrane resulting in changes in neuronal excitability that can be ameliorated by RNAi knockdown or pharmacological blockade of L-type Ca(2+) channels. This highlights a role for L-type Ca(2+) channels in tauopathy and their potential as a therapeutic target for AD.
Cassidy, D., Epiney, D., Salameh, C., Zhou, L. T., Salomon, R. N., Schirmer, A. E., McVey, M. and Bolterstein, E. (2019). Evidence for premature aging in a Drosophila model of Werner syndrome. Exp Gerontol: 110733. PubMed ID: 31518666
Summary:
Werner syndrome (WS) is an autosomal recessive progeroid disease characterized by patients' early onset of aging, increased risk of cancer and other age-related pathologies. WS is caused by mutations in WRN, a RecQ helicase that has essential roles responding to DNA damage and preventing genomic instability. While human WRN has both an exonuclease and helicase domain, Drosophila WRNexo has high genetic and functional homology to only the exonuclease domain of WRN. Like WRN-deficient human cells, Drosophila WRNexo null mutants (WRNexo(Delta)) are sensitive to replication stress, demonstrating mechanistic similarities between these two models. Compared to age-matched wild-type controls, WRNexo(Delta) flies exhibit increased physiological signs of aging, such as shorter lifespans, higher tumor incidence, muscle degeneration, reduced climbing ability, altered behavior, and reduced locomotor activity. Interestingly, these effects are more pronounced in females suggesting sex-specific differences in the role of WRNexo in aging. This and future mechanistic studies will contribute to linking faulty DNA repair mechanisms with the process of aging.
Castillo-Quan, J. I., Tain, L. S., Kinghorn, K. J., Li, L., Gronke, S., Hinze, Y., Blackwell, T. K., Bjedov, I. and Partridge, L. (2019). A triple drug combination targeting components of the nutrient-sensing network maximizes longevity. Proc Natl Acad Sci U S A 116(42): 20817-20819. PubMed ID: 31570569
Summary:
Increasing life expectancy is causing the prevalence of age-related diseases to rise, and there is an urgent need for new strategies to improve health at older ages. Reduced activity of insulin/insulin-like growth factor signaling (IIS) and mechanistic target of rapamycin (mTOR) nutrient-sensing signaling network can extend lifespan and improve health during aging in diverse organisms. However, the extensive feedback in this network and adverse side effects of inhibition imply that simultaneous targeting of specific effectors in the network may most effectively combat the effects of aging. This study shows that the mitogen-activated protein kinase kinase (MEK) inhibitor trametinib, the mTOR complex 1 (mTORC1) inhibitor rapamycin, and the glycogen synthase kinase-3 (GSK-3) inhibitor lithium act additively to increase longevity in Drosophila. Remarkably, the triple drug combination increased lifespan by 48%. Furthermore, the combination of lithium with rapamycin cancelled the latter's effects on lipid metabolism. In conclusion, a polypharmacology approach of combining established, prolongevity drug inhibitors of specific nodes may be the most effective way to target the nutrient-sensing network to improve late-life health.

Friday, November 1st - Embryonic Development

Reding, K., Chen, M., Lu, Y., Cheatle Jarvela, A. M. and Pick, L. (2019). Shifting roles of Drosophila pair-rule gene orthologs: segmental expression and function in the milkweed bug Oncopeltus fasciatus. Development 146(17). PubMed ID: 31444220
Summary:
The discovery of pair-rule genes (PRGs) in Drosophila revealed the existence of an underlying two-segment-wide prepattern directing embryogenesis. The milkweed bug Oncopeltus fasciatus, a hemimetabolous insect, is a more representative arthropod: most of its segments form sequentially after gastrulation. This study reports the expression and function of orthologs of the complete set of nine Drosophila PRGs in Oncopeltus Seven Of-PRG-orthologs are expressed in stripes in the primordia of every segment, rather than every other segment; Of-runt is PR-like and several orthologs are also expressed in the segment addition zone. RNAi-mediated knockdown of Of-odd-skipped, paired and sloppy-paired impacted all segments, with no indication of PR-like register. Of-E75A is expressed in PR-like stripes, although it is not expressed in this way in Drosophila, demonstrating the existence of an underlying PR-like prepattern in Oncopeltus These findings reveal that a switch occurred in regulatory circuits, leading to segment formation: while several holometabolous insects are 'Drosophila-like', using PRG orthologs for PR patterning, most Of-PRGs are expressed segmentally in Oncopeltus, a more basally branching insect. Thus, an evolutionarily stable phenotype - segment formation - is directed by alternate regulatory pathways in diverse species.
Fehr, D., Handzlik, J. E., Manu and Loh, Y. (2019). Classification-Based Inference of Dynamical Models of Gene Regulatory Networks. G3 (Bethesda). PubMed ID: 31624138
Summary:
Cell-fate decisions during development are controlled by densely interconnected gene regulatory networks (GRNs) consisting of many genes. The switch-like nature of gene regulation can be exploited to break the gene circuit inference problem into two simpler optimization problems that are amenable to computationally efficient supervised learning techniques. This study presents FIGR (Fast Inference of Gene Regulation), a novel classification-based inference approach to determining gene circuit parameters. FIGR's effectiveness was demonstrated on synthetic data generated from random gene circuits of up to 50 genes as well as experimental data from the gap gene system of Drosophila melanogaster, a benchmark for inferring dynamical GRN models. FIGR is faster than global non-linear optimization by a factor of 600 and its computational complexity scales much better with GRN size. On a practical level, FIGR can accurately infer the biologically realistic gap gene network in under a minute on desktop-class hardware instead of requiring hours of parallel computing. It is anticipated that FIGR would enable the inference of much larger biologically realistic GRNs than was possible before.
Silver, J. T., Wirtz-Peitz, F., Simoes, S., Pellikka, M., Yan, D., Binari, R., Nishimura, T., Li, Y., Harris, T. J. C., Perrimon, N. and Tepass, U. (2019). Apical polarity proteins recruit the RhoGEF Cysts to promote junctional myosin assembly. J Cell Biol. PubMed ID: 31409654
Summary:
The spatio-temporal regulation of small Rho GTPases is crucial for the dynamic stability of epithelial tissues. However, how RhoGTPase activity is controlled during development remains largely unknown. To explore the regulation of Rho GTPases in vivo, this study analyzed the Rho GTPase guanine nucleotide exchange factor (RhoGEF) Cysts, the Drosophila orthologue of mammalian p114RhoGEF, GEF-H1, p190RhoGEF, and AKAP-13. Loss of Cysts causes a phenotype that closely resembles the mutant phenotype of the apical polarity regulator Crumbs. This phenotype can be suppressed by the loss of basolateral polarity proteins, suggesting that Cysts is an integral component of the apical polarity protein network. Cysts is recruited to the apico-lateral membrane through interactions with the Crumbs complex and Bazooka/Par3. Cysts activates Rho1 at adherens junctions and stabilizes junctional myosin. Junctional myosin depletion is similar in Cysts- and Crumbs-compromised embryos. Together, these findings indicate that Cysts is a downstream effector of the Crumbs complex and links apical polarity proteins to Rho1 and myosin activation at adherens junctions, supporting junctional integrity and epithelial polarity.
Jiang, T. and Harris, T. J. C. (2019). Par-1 controls the composition and growth of cortical actin caps during Drosophila embryo cleavage. J Cell Biol. PubMed ID: 31641019
Summary:
Cell structure depends on the cortex, a thin network of actin polymers and additional proteins underlying the plasma membrane. The cell polarity kinase Par-1 is required for cells to form following syncytial Drosophila embryo development. This requirement stems from Par-1 promoting cortical actin caps that grow into dome-like metaphase compartments for dividing syncytial nuclei. The actin caps are a composite material of Diaphanous (Dia)-based actin bundles interspersed with independently formed, Arp2/3-based actin puncta. Par-1 and Dia colocalize along extended regions of the bundles, and both are required for the bundles and for each other's bundle-like localization, consistent with an actin-dependent self-reinforcement mechanism. Par-1 helps establish or maintain these bundles in a cortical domain with relatively low levels of the canonical formin activator Rho1-GTP. Arp2/3 is required for displacing the bundles away from each other and toward the cap circumference, suggesting interactions between these cytoskeletal components could contribute to the growth of the cap into a metaphase compartment.
Liu, B., Sung, H. W. and Grosshans, J. (2019). Multiple functions of the essential gene PpV in Drosophila early development. G3 (Bethesda). PubMed ID: 31484673
Summary:
Protein phosphatase V (PpV) encodes the Drosophila homologue of the evolutionarily conserved Protein Phosphatase 6 (PP6). The physiological and developmental functions of PpV/PP6 have not been well characterized due to lack of a genetically defined mutant. This study identified a PpV non-sense mutation and describes multiple mutant phenotypes in oogenesis and early embryogenesis. Specifically, it was found that the defects in chromosome segregation during nuclear cycles are related to AuroraA function, which is consistent with the interaction of PP6 and AuroraA in mammalian cells. Surprisingly, a PpV function was identified specifically in blastoderm cell cycle but not in cell proliferation in the follicle epithelium or larval wing imaginal discs. Embryos from PpV germline clones frequently undergo an extra nuclear division cycle. By epistasis analysis, it was found that PpV functions in parallel with tribbles, but independently of auroraA for the remodeling of the nuclear cycles. Taken together, this study reports novel developmental functions of PpV and provides a framework for further genetic analysis under physiological conditions.
Pare, A. C., Naik, P., Shi, J., Mirman, Z., Palmquist, K. H. and Zallen, J. A. (2019). An LRR receptor-Teneurin system directs planar polarity at compartment boundaries. Dev Cell 51(2):208-221
Summary:
Epithelial cells dynamically self-organize in response to extracellular spatial cues relayed by cell-surface receptors. During convergent extension in Drosophila, Toll-related receptors direct planar polarized cell rearrangements that elongate the head-to-tail axis. However, many cells establish polarity in the absence of Toll receptor activity, indicating the presence of additional spatial cues. This study demonstrates that the leucine-rich-repeat receptor Tartan and the teneurin Ten-m provide critical polarity signals at epithelial compartment boundaries. The Tartan and Ten-m extracellular domains interact in vitro, and Tartan promotes Ten-m localization to compartment boundaries in vivo. Tartan and Ten-m are shown to be necessary for the planar polarity and organization of compartment boundary cells. Moreover, ectopic stripes of Tartan and Ten-m are sufficient to induce myosin accumulation at stripe boundaries. These results demonstrate that the Tartan/Ten-m and Toll receptor systems together create a high-resolution network of spatial cues that guides cell behavior during convergent extension.
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