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Current papers in developmental biology and gene function


Monday November 30th, 2020 - Disease Models

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Otte, C. G., Fortuna, T. R., Mann, J. R., Gleixner, A. M., Ramesh, N., Pyles, N. J., Pandey, U. B. and Donnelly, C. J. (2020). Optogenetic TDP-43 nucleation induces persistent insoluble species and progressive motor dysfunction in vivo. Neurobiol Dis: 105078. PubMed ID: 32927062
TDP-43 (see Drosophila Tdp-43) is a predominantly nuclear DNA/RNA binding protein that is often mislocalized into insoluble cytoplasmic inclusions in post-mortem patient tissue in a variety of neurodegenerative disorders, most notably, Amyotrophic Lateral Sclerosis (ALS), a fatal and progressive neuromuscular disorder. The underlying causes of TDP-43 proteinopathies remain unclear, but recent studies indicate the formation of these protein assemblies is driven by aberrant phase transitions of RNA deficient TDP-43. Technical limitations have prevented an understanding of how TDP-43 proteinopathy relates to disease pathogenesis. Current animal models of TDP-43 proteinopathy often rely on overexpression of wild-type TDP-43 to non-physiological levels that may initiate neurotoxicity through nuclear gain of function mechanisms, or by the expression of disease-causing mutations found in only a fraction of ALS patients. New technologies allowing for light-responsive control of subcellular protein crowding provide a promising approach to drive intracellular protein aggregation, as has been previously demonstrated in vitro. This study presents a model for the optogenetic induction of TDP-43 aggregation in Drosophila that recapitulates key biochemical features seen in patient pathology, most notably light-inducible persistent insoluble species and progressive motor dysfunction. These data describe a photokinetic in vivo model that could be as a future platform to identify novel genetic and pharmacological modifiers of diseases associated with TDP-43 neuropathology.
Andrew, D. R., Moe, M. E., Chen, D., Tello, J. A., Doser, R. L., Conner, W. E., Ghuman, J. K. and Restifo, L. L. (2020). Spontaneous motor-behavior abnormalities in two Drosophila models of neurodevelopmental disorders. J Neurogenet: 1-22. PubMed ID: 33164597
Boys with fragile X syndrome (FXS), a leading monogenic cause of intellectual disability, often display repetitive behaviors, a core feature of autism. This study characterized spontaneous-motor-behavior phenotypes of Drosophila dfmr1 mutants, an established model for FXS. Individual 1-day-old adult flies, with mature nervous systems, were recorded in small arenas. Young dfmr1 mutants spent excessive time grooming, with increased bout number and duration; both were rescued by transgenic wild-type dfmr1(+). By two grooming-pattern measures, dfmr1-mutant flies showed elevated repetitions consistent with perseveration, which is common in FXS. In addition, the mutant flies display a preference for grooming posterior body structures, and an increased rate of grooming transitions from one site to another. The possibility was raised that courtship and circadian rhythm defects, previously reported for dfmr1 mutants, are complicated by excessive grooming. Significantly increased grooming was also observed in CASK mutants, despite their dramatically decreased walking phenotype. The mutant flies, a model for human CASK-related neurodevelopmental disorders, displayed consistently elevated grooming indices throughout the assay, but transient locomotory activation immediately after placement in the arena. Based on published data identifying FMRP-target transcripts and functional analyses of mutations causing human genetic neurodevelopmental disorders, the following proteins are proposed as candidate mediators of excessive repetitive behaviors in FXS: CaMKIIα, NMDA receptor subunits 2A and 2B, NLGN3, and SHANK3. Together, these fly-mutant phenotypes and mechanistic insights provide starting points for drug discovery to identify compounds that reduce dysfunctional repetitive behaviors.
Aqsa, Sarkar, S. (2020). Age dependent trans-cellular propagation of human tau aggregates in Drosophila disease models. Brain Res: 147207. PubMed ID: 33212022
Tauopathies is a class of neurodegenerative disorders which involves the transformation of physiological tau (see Drosophila Tau) into pathogenic tau. One of the prime causes reported to drive this conversion is tau hyperphosphorylation and the subsequent propagation of pathogenic protein aggregates across the nervous system. Although past attempts have been made to deduce the details of tau propagation, yet not much is known about its mechanism. A better understanding of this aspect of disease pathology can prove to be beneficial for the development of diagnostic and therapeutic approaches. For the first time, this study demonstrates that the human tau possesses an intrinsic property to spread trans-cellularly in the fly nervous system irrespective of the tau allele or the neuronal tissue type. Aggregate migration restricted by targeted down-regulation of a specific kinase, elucidates the role of hyper-phosphorylation in its movement. Contrary to the previous models, this study delivers an easy and rapid in-vivo model for comprehensive examination of tau migration pathology. Henceforth, the developed model would not only be immensely helpful in uncovering the mechanistic in-depths of tau propagation pathology but also aid in modifier and/or drug screening for amelioration of tauopathies.
Park, J. H., Chung, C. G., Seo, J., Lee, B. H., Lee, Y. S., Kweon, J. H. and Lee, S. B. (2020). C9orf72-Associated Arginine-Rich Dipeptide Repeat Proteins Reduce the Number of Golgi Outposts and Dendritic Branches in Drosophila Neurons. Mol Cells 43(9): 821-830. PubMed ID: 32975212
Altered dendritic morphology is frequently observed in various neurological disorders including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD)This study investigated dendritic morphological defects caused by dipeptide repeat protein (DPR) toxicity associated with G4C2 expansion mutation of C9orf72 (the leading genetic cause of ALS and FTD) in Drosophila neurons. Among the five DPRs produced by repeat-associated non-ATG translation of G4C2 repeats, this study found that arginine-rich DPRs (PR and GR) led to the most significant reduction in dendritic branches and plasma membrane (PM) supply in Class IV dendritic arborization (C4 da) neurons. Furthermore, expression of PR and GR reduced the number of Golgi outposts (GOPs) in dendrites. In Drosophila brains, expression of PR, but not GR, led to a significant reduction in the mRNA level of CrebA, a transcription factor regulating the formation of GOPs. Overexpressing CrebA in PR-expressing C4 da neurons mitigated PM supply defects and restored the number of GOPs, but the number of dendritic branches remained unchanged, suggesting that other molecules besides CrebA may be involved in dendritic branching. Taken together, these results provide valuable insight into the understanding of dendritic pathology associated with C9-ALS/FTD.
Newton, H., Wang, Y. F., Camplese, L., Mokochinski, J. B., Kramer, H. B., Brown, A. E. X., Fets, L. and Hirabayashi, S. (2020). Systemic muscle wasting and coordinated tumour response drive tumourigenesis. Nat Commun 11(1): 4653. PubMed ID: 32938923
Cancer cells demand excess nutrients to support their proliferation, but how tumours exploit extracellular amino acids during systemic metabolic perturbations remain incompletely understood. This study used a Drosophila model of high-sugar diet (HSD)-enhanced tumourigenesis to uncover a systemic host-tumour metabolic circuit that supports tumour growth. Coordinate induction of systemic muscle wasting is demonstrated with tumour-autonomous Yorkie-mediated SLC36-family amino acid transporter expression as a proline-scavenging programme to drive tumourigenesis. Indole-3-propionic acid was identified as an optimal amino acid derivative to rationally target the proline-dependency of tumour growth. Insights from this whole-animal Drosophila model provide a powerful approach towards the identification and therapeutic exploitation of the amino acid vulnerabilities of tumourigenesis in the context of a perturbed systemic metabolic network.
Tazelaar, G. H. P., Boeynaems, S., De Decker, M., ...., Veldink, J. H. and van Es, M. A. (2020). ATXN1 repeat expansions confer risk for amyotrophic lateral sclerosis and contribute to TDP-43 mislocalization. Brain Commun 2(2): fcaa064. PubMed ID: 32954321
Increasingly, repeat expansions are being identified as part of the complex genetic architecture of amyotrophic lateral sclerosis. To date, several repeat expansions have been genetically associated with the disease: intronic repeat expansions in C9orf72, polyglutamine expansions in ATXN2 and polyalanine expansions in NIPA1. Together with previously published data, the identification of an amyotrophic lateral sclerosis patient with a family history of spinocerebellar ataxia type 1, caused by polyglutamine expansions in ATXN1, suggested a similar disease association for the repeat expansion in ATXN1. A large-scale international study was therefore performed in 11,700 individuals, in which a significant association was shown between intermediate ATXN1 repeat expansions and amyotrophic lateral sclerosis. Subsequent functional experiments have shown that ATXN1 reduces the nucleocytoplasmic ratio of TDP-43 and enhances amyotrophic lateral sclerosis phenotypes in Drosophila, further emphasizing the role of polyglutamine repeat expansions in the pathophysiology of amyotrophic lateral sclerosis.

Friday, November 27th - Adult Physiology

Ceder, M. M., Aggarwal, T., Hosseini, K., Maturi, V., Patil, S., Perland, E., Williams, M. J. and Fredriksson, R. (2020). CG4928 Is Vital for Renal Function in Fruit Flies and Membrane Potential in Cells: A First In-Depth Characterization of the Putative Solute Carrier UNC93A. Front Cell Dev Biol 8: 580291. PubMed ID: 33163493
The number of transporter proteins that are not fully characterized is immense. This study used Drosophila melanogaster and human cell lines to perform a first in-depth characterization of CG4928, an ortholog to the human UNC93A, of which little is known. Solute carriers regulate and maintain biochemical pathways important for the body, and malfunctioning transport is associated with multiple diseases. Based on phylogenetic analysis, CG4928 is closely related to human UNC93A and has a secondary and a tertiary protein structure and folding similar to major facilitator superfamily transporters. Ubiquitous knockdown of CG4928 causes flies to have a reduced secretion rate from the Malpighian tubules; altering potassium content in the body and in the Malpighian tubules, homologous to the renal system; and results in the development of edema. The edema could be rescued by using amiloride, a common diuretic, and by maintaining the flies on ion-free diets. CG4928-overexpressing cells did not facilitate the transport of sugars and amino acids; however, proximity ligation assay revealed that CG4928 co-localized with TASK(1) channels. Overexpression of CG4928 resulted in induced apoptosis and cytotoxicity, which could be restored when cells were kept in high-sodium media. Furthermore, the basal membrane potential was observed to be disrupted. Taken together, the results indicate that CG4928 is of importance for generating the cellular membrane potential by an unknown manner. However, it is speculated that it most likely acts as a regulator or transporter of potassium flows over the membrane.
Martelli, F., Zhongyuan, Z., Wang, J., Wong, C. O., Karagas, N. E., Roessner, U., Rupasinghe, T., Venkatachalam, K., Perry, T., Bellen, H. J. and Batterham, P. (2020). Low doses of the neonicotinoid insecticide imidacloprid induce ROS triggering neurological and metabolic impairments in Drosophila. Proc Natl Acad Sci U S A. PubMed ID: 32989137
Declining insect population sizes are provoking grave concern around the world as insects play essential roles in food production and ecosystems. Environmental contamination by intense insecticide usage is consistently proposed as a significant contributor, among other threats. Many studies have demonstrated impacts of low doses of insecticides on insect behavior, but have not elucidated links to insecticidal activity at the molecular and cellular levels. In this study, the histological, physiological, and behavioral impacts of imidacloprid was investigated in Drosophila melanogaster, an experimental organism exposed to insecticides in the field. Oxidative stress is a key factor in the mode of action of this insecticide at low doses. Imidacloprid produces an enduring flux of Ca(2+) into neurons and a rapid increase in levels of reactive oxygen species (ROS) in the larval brain. It affects mitochondrial function, energy levels, the lipid environment, and transcriptomic profiles. Use of RNAi to induce ROS production in the brain recapitulates insecticide-induced phenotypes in the metabolic tissues, indicating that a signal from neurons is responsible. Chronic low level exposures in adults lead to mitochondrial dysfunction, severe damage to glial cells, and impaired vision. The potent antioxidant, N-acetylcysteine amide (NACA), reduces the severity of a number of the imidacloprid-induced phenotypes, indicating a causal role for oxidative stress. Given that other insecticides are known to generate oxidative stress, this research has wider implications. The systemic impairment of several key biological functions, including vision, reported here would reduce the resilience of insects facing other environmental challenges.
Thompson, J. B., Su, O. O., Yang, N. and Bauer, J. H. (2020). Sleep-length differences are associated with altered longevity in the fruit fly Drosophila melanogaster. Biol Open 9(9). PubMed ID: 32938639
Sleep deprivation has been shown to negatively impact health outcomes, leading to decreased immune responses, memory loss, increased activity of stress and inflammatory pathways, weight gain, and even behavioral changes. These observations suggest that sleep deprivation substantially interferes with important physiological functions, including metabolic pathways of energy utilization. Many of those phenotypes are correlated with age, suggesting that disrupted sleep may interfere with the aging process. However, little is known about how sleep disruption affects aging and longevity. This study investigated this relationship using eight representative fruit fly lines from the Sleep Inbred Panel (SIP). The SIP consists of 39 inbred lines that display extreme short- and long-sleep patterns, and constitutes a crucial Drosophila community resource for investigating the mechanisms of sleep regulation. The data show that flies with short-sleep periods have ∼16% longer life span, as well as reduced aging rate, compared to flies with long-sleep. In contrast, disrupting normal circadian rhythm reduces fly longevity. Short-sleep SIP flies moreover show slight metabolic differences to long-sleep lines, and to flies with disrupted circadian rhythm. These data suggest that the inbred SIP lines engage sleep mechanisms that are distinct from the circadian clock system.
Malkeyeva, D., Kiseleva, E. and Fedorova, S. (2020). Small heat shock protein Hsp67Bc plays a significant role in Drosophila melanogaster cold-stress tolerance. J Exp Biol. PubMed ID: 32943578
Hsp67Bc in Drosophila melanogaster is a member of the small heat shock protein family, the main function of which is to prevent the aggregation of misfolded or damaged proteins. Hsp67Bc interacts with Starvin and Hsp23, which are known to be a part of the cold-stress response in the fly during the recovery phase. This study investigated the role of the Hsp67Bc gene in the cold-stress response. In adult Drosophila, Hsp67Bc expression was shown to increase after cold stress and decrease after 1.5 h of recovery, indicating the involvement of Hsp67Bc in short-term stress recovery. A deletion in the D. melanogaster Hsp67Bc gene was implemented using imprecise excision of a P-element, and the cold tolerance of Hsp67Bc-null mutants was analyzed at different developmental stages. Hsp67Bc-null homozygous flies were found to be viable and fertile but display varying cold-stress tolerance throughout the stages of ontogenesis: the survival after cold stress is slightly impaired in late 3(rd) instar larvae, unaffected in pupae, and notably affected in adult females. Moreover, the recovery from chill coma is delayed in Hsp67Bc-null adults of both sexes. In addition, the deletion in the Hsp67Bc gene caused more prominent up-regulation of Hsp70 following cold stress, suggesting the involvement of Hsp70 in compensation of the lack of the Hsp67Bc protein. Taken together, these results suggest that Hsp67Bc is involved in the recovery of flies from a comatose state and contributes to the protection of the fruit fly from cold stress.
Kim, H., Kim, M. and Kim, M. S. (2020). Facilitating fructose-driven metabolism exerts a protective effect on anoxic stress in Drosophila. Insect Mol Biol. PubMed ID: 32920918
Hypoxic stress is linked to various cardiovascular disorders (e.g., stroke, myocardial infarction), mediated, at least in part, by a reduction in ATP synthesis. Fructose-driven glycolysis is proposed as an alternative pathway capable of sustaining ATP production even under anoxic conditions. This study tested the hypothesis that facilitating fructose-driven metabolism exerts a protective effect against anoxic stress in Drosophila. Genetically modified flies with the human fructose transporter (GluT5) and ketohexokinase (KHK) genes downstream of upstream activating sequence (UAS) were constructed. The GAL4-UAS system was confirmed to: i) increase the expression of GluT5 and KHK in a tissue-specific and a time-dependent manner (i.e., whole flies [with Act5c-gene switch GAL4 driver], neurons [with elav-gene switch GAL4 driver]) and ii) reduce mortality of flies when placed under anoxic stress. Taken together, these data suggest that increasing fructose metabolism may be a clinically relevant approach to minimize hypoxia-induced cellular damage.
Pereira, M. T., Brock, K. and Musselman, L. P. (2020). Meep, a Novel Regulator of Insulin Signaling, Supports Development and Insulin Sensitivity via Maintenance of Protein Homeostasis in Drosophila melanogaster. G3 (Bethesda). PubMed ID: 32998936
Insulin signaling is critical for developmental growth and adult homeostasis, yet the downstream regulators of this signaling pathway are not completely understood. Using the model organism Drosophila melanogaster, a genomic approach was undertaken to identify novel mediators of insulin signaling. These studies led to the identification of Meep, encoded by the gene CG32335. Expression of this gene is both insulin receptor- and diet-dependent. Meep was specifically required in the developing fat body to tolerate a high-sugar diet (HSD). Meep is not essential on a control diet, but when reared on an HSD, knockdown of meep causes hyperglycemia, reduced growth, developmental delay, pupal lethality, and reduced longevity. These phenotypes stem in part from Meep's role in promoting insulin sensitivity and protein stability. This work suggests a critical role for protein homeostasis in development during overnutrition. Because Meep is conserved and obesity-associated in mammals, future studies on Meep may help to understand the role of proteostasis in insulin-resistant type 2 diabetes.

Wednesday, November 25th - Behavior

Abe, M. S. (2020). Functional advantages of Levy walks emerging near a critical point. Proc Natl Acad Sci U S A 117(39): 24336-24344. PubMed ID: 32929032
A special class of random walks, so-called Lévy walks, has been observed in a variety of organisms ranging from cells, insects, fishes, and birds to mammals, including humans. Although their prevalence is considered to be a consequence of natural selection for higher search efficiency, some findings suggest that Lévy walks might also be epiphenomena that arise from interactions with the environment. Therefore, why they are common in biological movements remains an open question. Based on some evidence that Lévy walks are spontaneously generated in the brain and the fact that power-law distributions in Lévy walks can emerge at a critical point, it was hypothesized that the advantages of Lévy walks might be enhanced by criticality. However, the functional advantages of Lévy walks are poorly understood. This study modeled nonlinear systems for the generation of locomotion and showed that Lévy walks emerging near a critical point had optimal dynamic ranges for coding information. This discovery suggested that Lévy walks could change movement trajectories based on the magnitude of environmental stimuli. This study then showed that the high flexibility of Lévy walks enabled switching exploitation/exploration based on the nature of external cues. Finally, this study analyzed the movement trajectories of freely moving Drosophila larvae and showed empirically that the Lévy walks may emerge near a critical point and have large dynamic range and high flexibility. The results suggest that the commonly observed Lévy walks emerge near a critical point and could be explained on the basis of these functional advantages.
Bentzur, A., Ben-Shaanan, S., Benichou, J. I. C., Costi, E., Levi, M., Ilany, A. and Shohat-Ophir, G. (2020). Early Life Experience Shapes Male Behavior and Social Networks in Drosophila. Curr Biol. PubMed ID: 33186552
Living in a group creates a complex and dynamic environment in which behavior of individuals is influenced by and affects the behavior of others. Although social interaction and group living are fundamental adaptations exhibited by many organisms, little is known about how prior social experience, internal states, and group composition shape behavior in groups. This study presents an analytical framework for studying the interplay between social experience and group interaction in Drosophila melanogaster. The complexity of interactions in a group was simplified using a series of experiments in which the social experience and motivational states of individuals were controlled to compare behavioral patterns and social networks of groups under different conditions. Social enrichment promotes the formation of distinct group structure that is characterized by high network modularity, high inter-individual and inter-group variance, high inter-individual coordination, and stable social clusters. Using environmental and genetic manipulations, this study showed that visual cues and cVA-sensing neurons are necessary for the expression of social interaction and network structure in groups. Finally, the formation of group behavior and structure was exploited in heterogenous groups composed of flies with distinct internal states, and emergent structures were documented that are beyond the sum of the individuals that constitute it. These results demonstrate that fruit flies exhibit complex and dynamic social structures that are modulated by the experience and composition of different individuals within the group. This paves the path for using simple model organisms to dissect the neurobiology of behavior in complex social environments.
Xiao, C. and Qiu, S. (2020). Frequency-specific modification of locomotor components by the white gene in Drosophila melanogaster adult flies. Genes Brain Behav: e12703. PubMed ID: 32964643
The classic eye-color gene white (w) in Drosophila melanogaster (fruitfly) has unexpected behavioral consequences. How w affects locomotion of adult flies is largely unknown. This study shows that a mutant allele (w(1118)) selectively increases locomotor components at relatively high frequencies (> 0.1 Hz). The (w(1118)) flies had reduced transcripts of w(+) from the 5' end of the gene. Male flies of (w(1118)) walked continuously in circular arenas while the wildtype Canton-S walked intermittently. Through careful control of genetic and cytoplasmic backgrounds, this study found that the (w(1118)) locus was associated with continuous walking. w(1118) -carrying male flies showed increased median values of path length per second (PPS) and 5-min path length compared with w(+) -carrying males. Additionally, flies carrying 2-4 genomic copies of mini-white(+) (mw(+)) in the w(1118) background showed suppressed median PPSs and decreased 5-min path length compared with controls, and the suppression was dependent on the copy number of mw(+). Analysis of the time-series (i.e. PPSs over time) by Fourier transform indicated that w(1118) was associated with increased locomotor components at relatively high frequencies (> 0.1 Hz). The addition of multiple genomic copies of mw(+) (2-4 copies) suppressed the high-frequency components. Lastly, the downregulation of w(+) in neurons but not glial cells resulted in increased high-frequency components. It is concluded that mutation of w modified the locomotion in adult flies by selectively increasing high-frequency locomotor components.
Shahandeh, M. P., Brock, C. and Turner, T. L. (2020). Light dependent courtship behavior in Drosophila simulans and D. melanogaster. PeerJ 8: e9499. PubMed ID: 32742789
Differences in courtship signals and perception are well-known among Drosophila species. One such described difference is the dependency on light, and thus presumably vision, for copulation success. Many studies have described a difference in light-dependent copulation success between D. melanogaster and D. simulans, identifying D. simulans as a light-dependent species, and D. melanogaster as a light-independent one. However, many of these studies use assays of varying design and few strains to represent the entire species. This study attempted to better characterize this purported difference using 11 strains of each species, paired by collection location, in behavioral assays conducted at two different exposure times. While there is a species-wide difference in magnitude of light-dependent copulation success, D. melanogaster copulation success is, on average, still impaired in the dark at both exposure times measured. Additionally, there is significant variation in strain-specific ability to copulate in the dark in both species across two different exposure times. This variation correlates strongly with longitude in D. melanogaster, but not in D. simulans. It is hypothesized that differences in species history and demography may explain behavioral variation. Finally, courtship assays were used to show that light-dependent copulation success in one D. simulans strain is driven in part by both males and females. Potential differences in courtship signals and/or signal importance between these species are discussed and potential for further comparative studies for functional characterization.
Rouse, J., McDowall, L., Mitchell, Z., Duncan, E. J. and Bretman, A. (2020). Social competition stimulates cognitive performance in a sex-specific manner. Proc Biol Sci 287(1935): 20201424. PubMed ID: 32933446
Social interactions are thought to be a critical driver in the evolution of cognitive ability. Cooperative interactions, such as pair bonding, rather than competitive interactions have been largely implicated in the evolution of increased cognition. This is despite competition traditionally being a very strong driver of trait evolution. Males of many species track changes in their social environment and alter their reproductive strategies in response to anticipated levels of competition. This study predicts this to be cognitively challenging. Using a Drosophila melanogaster model, it was possible to distinguish between the effects of a competitive environment versus generic social contact by exposing flies to same-sex same-species competition versus different species partners, shown to present non-competitive contacts. Males increase olfactory learning/memory and visual memory after exposure to conspecific males only, a pattern echoed by increased expression of synaptic genes and an increased need for sleep. For females, largely not affected by mating competition, the opposite pattern was seen. The results indicate that specific social contacts dependent on sex, not simply generic social stimulation, may be an important evolutionary driver for cognitive ability in fruit flies.
Wang, F., Wang, K., Forknall, N., Parekh, R. and Dickson, B. J. (2020). Circuit and Behavioral Mechanisms of Sexual Rejection by Drosophila Females. Curr Biol. PubMed ID: 32795445
The mating decisions of Drosophila melanogaster females are primarily revealed through either of two discrete actions: opening of the vaginal plates to allow copulation, or extrusion of the ovipositor to reject the male. Both actions are triggered by the male courtship song, and both are dependent upon the female's mating status. Virgin females are more likely to open their vaginal plates in response to song; mated females are more likely to extrude their ovipositor. This study examine the neural cause and behavioral consequence of ovipositor extrusion. This study shows that the DNp13 descending neurons act as command-type neurons for ovipositor extrusion and that ovipositor extrusion is an effective deterrent only when performed by females that have previously mated. The DNp13 neurons respond to male song via direct synaptic input from the pC2l auditory neurons. Mating status does not modulate the song responses of DNp13 neurons, but rather how effectively they can engage the motor circuits for ovipositor extrusion. Evidence is presented that mating status information is mediated by ppk(+) sensory neurons in the uterus, which are activated upon ovulation. Vaginal plate opening and ovipositor extrusion are thus controlled by anatomically and functionally distinct circuits, highlighting the diversity of neural decision-making circuits even in the context of closely related behaviors with shared exteroceptive and interoceptive inputs.

Tuesday November 24th - Adult neural development and function

Fernandez-Chiappe, F., Hermann-Luibl, C., Peteranderl, A., Reinhard, N., Senthilan, P. R., Hieke, M., Selcho, M., Yoshii, T., Shafer, O. T., Muraro, N. I. and Helfrich-Forster, C. (2020). Dopamine signaling in wake promoting clock neurons is not required for the normal regulation of sleep in Drosophila. J Neurosci. PubMed ID: 33172977
Dopamine is a wake-promoting neuromodulator in mammals and fruit flies. In Drosophila melanogaster, the network of clock neurons that drives sleep/activity cycles comprises both wake- and sleep-promoting cell types. The large and small ventrolateral neurons (l-LN(v)s and s-LN(v)s) have been identified as wake-promoting neurons within the clock neuron network. The l-LN(v)s are innervated by dopaminergic neurons, and earlier work proposed that dopamine signaling raises cAMP levels in the l-LN(v)s and thus induces excitatory electrical activity (action potential firing), which results in wakefulness and inhibits sleep. This study tested this hypothesis by combining cAMP imaging and patch-clamp recordings in isolated brains. Dopamine application indeed increases cAMP levels and depolarizes the l-LN(v)s, but surprisingly, it does not result in increased firing rates. Down-regulation of the excitatory dopamine receptor, Dop1R1 in the l- and s-LN(v)s, but not of Dop1R2, abolished the depolarization of l-LN(v)s in response to dopamine. This indicates that dopamine signals via Dop1R1 to the l-LN(v)s. Down-regulation of Dop1R1 or Dop1R2 receptors in the l- and s-LN(v)s does not affect sleep in males. Unexpectedly, a moderate decrease of daytime sleep was found with down-regulation of Dop1R1 and of nighttime sleep with down-regulation of Dop1R2. Since the l-LN(v)s do not utilize Dop1R2 receptors and the s-LN(v)s also respond to dopamine, it is concluded that the s-LN(v)s are responsible for the observed decrease in nighttime sleep. In summary, dopamine signaling in the wake-promoting LN(v)s is not required for daytime arousal, but likely promotes nighttime sleep via the s-LN(v)s.
Tare, M., Chimata, A. V., Gogia, N., Narwal, S., Deshpande, P. and Singh, A. (2020). An E3 ubiquitin ligase, cullin-4 regulates retinal differentiation in Drosophila eye. Genesis: e23395. PubMed ID: 32990387
During organogenesis, cell proliferation is followed by the differentiation of specific cell types to form an organ. Any aberration in differentiation can result in developmental defects, which can result in a partial to a near-complete loss of an organ. This study employed the Drosophila eye model to understand the genetic and molecular mechanisms involved in the process of differentiation. In a forward genetic screen, cullin-4 (cul-4), which encodes an E3 ubiquitin ligase, was found to play an important role in retinal differentiation. During development, cul-4 is known to be involved in protein degradation, regulation of genomic stability, and regulation of cell cycle. Previously, it was reported that cul-4 regulates cell death during eye development by downregulating Wingless (Wg)/Wnt signaling pathway. Loss-of-function of cul-4 results in a reduced eye phenotype, which can be due to onset of cell death. However, loss-of-function of cul-4 also affects retinal development by downregulating retinal determination (RD) gene expression. Early markers of retinal differentiation are dysregulated in cul-4 loss of function conditions, indicating that cul-4 is necessary for differentiation. Furthermore, loss-of-function of cul-4 ectopically induces expression of negative regulators of eye development like Wg and Homothorax (Hth). During eye development, Wg is known to block the progression of a synchronous wave of differentiation referred to as Morphogenetic furrow (MF). In cul-4 loss-of-function background, expression of dpp-lacZ, a MF marker, is significantly downregulated. These data suggest a new role of cul-4 in retinal differentiation. These studies may have significant bearings on understanding of early eye development.
Blum, I. D., Keleş, M. F., Baz, E. S., Han, E., Park, K., Luu, S., Issa, H., Brown, M., Ho, M. C. W., Tabuchi, M., Liu, S. and Wu, M. N. (2020). Astroglial Calcium Signaling Encodes Sleep Need in Drosophila. Curr Biol. PubMed ID: 33186550
Sleep is under homeostatic control, whereby increasing wakefulness generates sleep need and triggers sleep drive. However, the molecular and cellular pathways by which sleep need is encoded are poorly understood. In addition, the mechanisms underlying both how and when sleep need is transformed to sleep drive are unknown. Using ex vivo and in vivo imaging, this study shows in Drosophila that astroglial Ca(2+) signaling increases with sleep need. This signaling is dependent on a specific L-type Ca(2+) channel and is necessary for homeostatic sleep rebound. Thermogenetically increasing Ca(2+) in astrocytes induces persistent sleep behavior, and this phenotype was exploited to conduct a genetic screen for genes required for the homeostatic regulation of sleep. From this large-scale screen, TyrRII, a monoaminergic receptor required in astrocytes for sleep homeostasis, was identifed. TyrRII levels rise following sleep deprivation in a Ca(2+)-dependent manner, promoting further increases in astrocytic Ca(2+) and resulting in a positive-feedback loop. Moreover, these findings suggest that astrocytes then transmit this sleep need to a sleep drive circuit by upregulating and releasing the interleukin-1 analog Spätzle, which then acts on Toll receptors on R5 neurons. These findings define astroglial Ca(2+) signaling mechanisms encoding sleep need and reveal dynamic properties of the sleep homeostatic control system.
Durrieu, M., Wystrach, A., Arrufat, P., Giurfa, M. and Isabel, G. (2020). Fruit flies can learn non-elemental olfactory discriminations. Proc Biol Sci 287(1938): 20201234. PubMed ID: 33171086
Associative learning allows animals to establish links between stimuli based on their concomitance. In the case of Pavlovian conditioning, a single stimulus A (the conditional stimulus, CS) is reinforced unambiguously with an unconditional stimulus (US) eliciting an innate response. This conditioning constitutes an 'elemental' association to elicit a learnt response from A(+) without US presentation after learning. However, associative learning may involve a 'complex' CS composed of several components. In that case, the compound may predict a different outcome than the components taken separately, leading to ambiguity and requiring the animal to perform so-called non-elemental discrimination. This study focussed on such a non-elemental task, the negative patterning (NP) problem, and provides the first evidence of NP solving in Drosophila. Drosophila were shown to learn to discriminate a simple component (A or B) associated with electric shocks (+) from an odour mixture composed either partly (called 'feature-negative discrimination' A(+) versus AB(-)) or entirely (called 'NP' A(+)B(+) versus AB(-)) of the shock-associated components. Furthermore, this study shows that conditioning repetition results in a transition from an elemental to a configural representation of the mixture required to solve the NP task, highlighting the cognitive flexibility of Drosophila.
Grabowska, M. J., Jeans, R., Steeves, J. and van Swinderen, B. (2020). Oscillations in the central brain of Drosophila are phase locked to attended visual features. Proc Natl Acad Sci U S A. PubMed ID: 33177231
Object-based attention describes the brain's capacity to prioritize one set of stimuli while ignoring others. Human research suggests that the binding of diverse stimuli into one attended percept requires phase-locked oscillatory activity in the brain. Even insects display oscillatory brain activity during visual attention tasks, but it is unclear if neural oscillations in insects are selectively correlated to different features of attended objects. This question was addressed by recording local field potentials in the Drosophila central complex, a brain structure involved in visual navigation and decision making. Attention was found to selectively increase the neural gain of visual features associated with attended objects; attention could be redirected to unattended objects by activation of a reward circuit. Attention was associated with increased beta (20- to 30-Hz) oscillations that selectively locked onto temporal features of the attended visual objects. These results suggest a conserved function for the beta frequency range in regulating selective attention to salient visual features.
Yao, P. J., Eren, E., Petralia, R. S., Gu, J. W., Wang, Y. X. and Kapogiannis, D. (2020). Mitochondrial Protrusions in Neuronal Cells. iScience 23(9): 101514. PubMed ID: 32942173
Mitochondrial function relies on multiple quality control mechanisms, including the release of mitochondrial vesicles. To investigate the ultrastructure and prevalence of mitochondrial membranous protrusions (and, by extension, vesicles) in neurons, mitochondria were surveyed in rat and planarian brains using transmission electron microscopy (EM). Mitochondrial protrusions mostly extend from the outer membrane. Leveraging available 3D EM datasets of the brain, mitochondrial protrusions were further analyzed in neurons of mouse and Drosophila brains, identifying high-resolution spatial views of these protrusions. To assess whether the abundance of mitochondrial protrusions and mitochondria-derived vesicles respond to cellular stress, neurons expressing fluorescently tagged mitochondrial markers were examined using confocal microscopy with Airyscan; increased numbers of mitochondrial protrusions and vesicles were found with mild stress. Future studies using improved spatial resolution with added temporal information may further define the functional implications of mitochondrial protrusions and vesicles in neurons.

Monday, November 23rd - Signaling

Hood, S. E., Kofler, X. V., Chen, Q., Scott, J., Ortega, J. and Lehmann, M. (2020). Nuclear translocation ability of Lipin differentially affects gene expression and survival in fed and fasting Drosophila. J Lipid Res. PubMed ID: 32989002
Lipins are eukaryotic proteins with functions in lipid synthesis and the homeostatic control of energy balance. They execute these functions by acting as phosphatidate phosphatase enzymes in the cytoplasm and by changing gene expression after translocation into the cell nucleus, in particular under fasting conditions. This study asked whether nuclear translocation and enzymatic activity of Drosophila Lipin serve essential functions and how gene expression changes, under both fed and fasting conditions, when nuclear translocation is impaired. To address these questions, a Lipin null mutant, a mutant expressing Lipin lacking a nuclear localization signal (Lipin(ΔNLS)), and a mutant expressing enzymatically dead Lipin were created. The data support the conclusion that the enzymatic, but not the nuclear gene regulatory activity of Lipin, is essential for survival. Notably, adult Lipin(ΔNLS) flies were not only viable but also exhibited improved life expectancy. In contrast, they were highly susceptible to starvation. Both the improved life expectancy in the fed state and the decreased survival in the fasting state correlated with changes in metabolic gene expression. Moreover, increased life expectancy of fed flies was associated with a decreased metabolic rate. Interestingly, in addition to metabolic genes, genes involved in feeding behavior and the immune response were mis-regulated in Lipin(ΔNLS) flies. Combined, these data suggest that nuclear activity of Lipin influences the genomic response to nutrient availability with effects on life expectancy and starvation resistance. Thus, nutritional or therapeutic approaches that aim at lowering nuclear translocation of lipins in humans may be worth exploring.
Woodling, N. S., Rajasingam, A., Minkley, L. J., Rizzo, A. and Partridge, L. (2020). Independent glial subtypes delay development and extend healthy lifespan upon reduced insulin-PI3K signalling. BMC Biol 18(1): 124. PubMed ID: 32928209
The increasing age of global populations highlights the urgent need to understand the biological underpinnings of ageing. To this end, inhibition of the insulin/insulin-like signalling (IIS) pathway can extend healthy lifespan in diverse animal species, but with trade-offs including delayed development. It is possible that distinct cell types underlie effects on development and ageing; cell-type-specific strategies could therefore potentially avoid negative trade-offs when targeting diseases of ageing, including prevalent neurodegenerative diseases. The highly conserved diversity of neuronal and non-neuronal (glial) cell types in the Drosophila nervous system makes it an attractive system to address this possibility. This study has thus investigated whether IIS in distinct glial cell populations differentially modulates development and lifespan in Drosophila. Glia-specific IIS inhibition, using several genetic means, delays development while extending healthy lifespan. The effects on lifespan can be recapitulated by adult-onset IIS inhibition, whereas developmental IIS inhibition is dispensable for modulation of lifespan. Notably, the effects observed on both lifespan and development act through the PI3K branch of the IIS pathway and are dependent on the transcription factor FOXO. Finally, IIS inhibition in several glial subtypes can delay development without extending lifespan, whereas the same manipulations in astrocyte-like glia alone are sufficient to extend lifespan without altering developmental timing. These findings reveal a role for distinct glial subpopulations in the organism-wide modulation of development and lifespan, with IIS in astrocyte-like glia contributing to lifespan modulation but not to developmental timing. These results enable a more complete picture of the cell-type-specific effects of the IIS network, a pathway whose evolutionary conservation in humans make it tractable for therapeutic interventions. These findings therefore underscore the necessity for cell-type-specific strategies to optimise interventions for the diseases of ageing.
Wolfstetter, G., Pfeifer, K., Backman, M., Masudi, T. A., Mendoza-Garcia, P., Chen, S., Sonnenberg, H., Sukumar, S. K., Uckun, E., Varshney, G. K., Uv, A. and Palmer, R. H. (2020). Identification of the Wallenda JNKKK as an Alk suppressor reveals increased competitiveness of Alk-expressing cells. Sci Rep 10(1): 14954. PubMed ID: 32917927
Anaplastic lymphoma kinase (Alk) is a receptor tyrosine kinase of the insulin receptor super-family that functions as oncogenic driver in a range of human cancers such as neuroblastoma. In order to investigate mechanisms underlying Alk oncogenic signaling, a genetic suppressor screen was conducted in Drosophila melanogaster. This screen identified multiple loci important for Alk signaling, including members of Ras/Raf/ERK-, Pi3K-, and STAT-pathways as well as tailless (tll) and foxo whose orthologues NR2E1/TLX and FOXO3 are transcription factors implicated in human neuroblastoma. Many of the identified suppressors were also able to modulate signaling output from activated oncogenic variants of human ALK, suggesting that this screen identified targets likely relevant in a wide range of contexts. Interestingly, two misexpression alleles of wallenda (wnd, encoding a leucine zipper bearing kinase similar to human DLK and LZK) were among the strongest suppressors. Alk expression leads to a growth advantage and induces cell death in surrounding cells. These results suggest that Alk activity conveys a competitive advantage to cells, which can be reversed by over-expression of the JNK kinase kinase Wnd.
Harsh, S. and Eleftherianos, I. (2020). Tumor induction in Drosophila imaginal epithelia triggers modulation of fat body lipid droplets. Biochimie 179: 65-68. PubMed ID: 32946989
Understanding of cancer-specific metabolic changes is currently unclear. In recent years, the fruit fly Drosophila melanogaster with its powerful genetic tools has become an attractive model for studying both tumor autonomous and the systemic processes resulting from the tumor growth. This study investigated the effect of tumorigenesis on the modulation of lipid droplets (LDs) in the larval fat bodies (mammalian equivalent of adipose tissue). Notch signaling was overexpressed alone or in combination with the developmental regulator Myocyte enhancer factor 2 (Mef2) using wing-specific and eye-specific drivers, the size of LDs in the fat body of the different tumor bearing larvae was quantified, and the expression of genes associated with lipolysis and lipogenesis was estimated. Hyperplastic and neoplastic tumor induced by overexpression of Notch and co-expression of Notch and Mef2 respectively were found to trigger impaired lipid metabolism marked by increased size of fat body LDs. The impaired lipid metabolism in tumor carrying larvae is linked to the altered expression of genes that participate in lipolysis and lipogenesis. These findings reveal modulation of LDs as one of the host's specific response upon tumor initiation. This information could potentially uncover mechanisms for designing innovative approaches to modulate cancer growth.
Ugbode, C., Garnham, N., Fort-Aznar, L., Evans, G. J. O., Chawla, S. and Sweeney, S. T. (2020). JNK signalling regulates antioxidant responses in neurons. Redox Biol 37: 101712. PubMed ID: 32949970
Reactive oxygen species (ROS) are generated during physiological bouts of synaptic activity and as a consequence of pathological conditions in the central nervous system. How neurons respond to and distinguish between ROS in these different contexts is currently unknown. In Drosophila mutants with enhanced JNK activity, lower levels of ROS are observed and these animals are resistant to both changes in ROS and changes in synapse morphology induced by oxidative stress. In wild type flies, disrupting JNK-AP-1 signalling perturbs redox homeostasis suggesting JNK activity positively regulates neuronal antioxidant defense. This hypothesis was validated in mammalian neurons, finding that JNK activity regulates the expression of the antioxidant gene Srxn-1, in a c-Jun dependent manner. This study describes a conserved 'adaptive' role for neuronal JNK in the maintenance of redox homeostasis that is relevant to several neurodegenerative diseases.
Moore, R., Vogt, K., Acosta Martin, A. E., Shire, P., Zeidler, M. and Smythe, E. (2020). Integration of JAK/STAT receptor-ligand trafficking, signalling and gene expression in Drosophila melanogaster cells. J Cell Sci. PubMed ID: 32917740
The JAK/STAT pathway is an essential signalling cascade required for multiple processes during development and for adult homeostasis. A key question in understanding this pathway is how it is regulated in different cell contexts. This study examined how endocytic processing contributes to signalling by the single cytokine receptor, Domeless, in Drosophila melanogaster cells. An evolutionarily conserved di-Leu motif was identified that is required for Domeless internalisation; endocytosis is required for activation of a subset of Domeless targets. These data indicate that endocytosis both qualitatively and quantitatively regulates Domeless signalling. STAT92E, the single STAT transcription factor in Drosophila, appears to be the target of endocytic regulation and and these studies show that phosphorylation of STAT92E on Tyr704, while necessary, is not always sufficient for target transcription. Finally, a conserved residue, Thr702, was identified that is essential for Tyr704 phosphorylation. Taken together, these findings identify previously unknown aspects of JAK/STAT pathway regulation likely to play key roles in the spatial and temporal regulation of signalling in vivo.

Friday, November 20th - Gonad Development

Hagen, J. F. D., Mendes, C. C., Booth, S. R., Jimenez, J. F., Tanaka, K. M., Franke, F. A., Baudouin-Gonzalez, L., Ridgway, A. M., Arif, S., Nunes, M. D. S. and McGregor, A. P. (2020). Unravelling the genetic basis for the rapid diversification of male genitalia between Drosophila species. Mol Biol Evol. PubMed ID: 32931587
In the last 240,000 years, males of the Drosophila simulans species clade have evolved striking differences in the morphology of their epandrial posterior lobes and claspers (surstyli). These appendages are used for grasping the female during mating and so their divergence is most likely driven by sexual selection. Mapping studies indicate a highly polygenic and generally additive genetic basis for these morphological differences. However, there is only a limited understanding of the gene regulatory networks that control the development of genital structures and how they evolved to result in this rapid phenotypic diversification. This study used new D. simulans/D. mauritiana introgression lines on chromosome 3L to generate higher resolution maps of posterior lobe and clasper differences between these species. RNA-seq was carried out on the developing genitalia of both species to identify the expressed genes and those that are differentially expressed between the two species. This allowed testing of the function of expressed positional candidates during genital development in D. melanogaster. Several new genes were found to be involved in the development and possibly the evolution of these genital structures, including the transcription factors Hairy and Grunge. Furthermore, it was discovered that during clasper development Hairy negatively regulates tartan (trn), a gene known to contribute to divergence in clasper morphology. Taken together, these results provide new insights into the regulation of genital development and how this has evolved between species.
Weber, J., Kabakci, Z., Chaurasia, S., Brunner, E. and Lehner, C. F. (2020). Chromosome separation during Drosophila male meiosis I requires separase-mediated cleavage of the homolog conjunction protein UNO. PLoS Genet 16(10): e1008928. PubMed ID: 33001976
Regular chromosome segregation during the first meiotic division requires prior pairing of homologous chromosomes into bivalents. During canonical meiosis, linkage between homologous chromosomes is maintained until late metaphase I by chiasmata resulting from meiotic recombination in combination with distal sister chromatid cohesion. Separase-mediated elimination of cohesin from chromosome arms at the end of metaphase I permits terminalization of chiasmata and homolog segregation to opposite spindle poles during anaphase I. Interestingly, separase is also required for bivalent splitting during meiosis I in Drosophila males, where homologs are conjoined by an alternative mechanism independent of meiotic recombination and cohesin. This study reports the identification of a novel alternative homolog conjunction protein encoded by the previously uncharacterized gene univalents only (uno). The univalents that are present in uno null mutants at the start of meiosis I, instead of normal bivalents, are segregated randomly. In wild type, UNO protein is detected in dots associated with bivalent chromosomes and most abundantly at the localized pairing site of the sex chromosomes. UNO is cleaved by separase. Expression of a mutant UNO version with a non-functional separase cleavage site restores homolog conjunction in a uno null background. However, separation of bivalents during meiosis I is completely abrogated by this non-cleavable UNO version. Therefore, it is proposed that homolog separation during Drosophila male meiosis I is triggered by separase-mediated cleavage of UNO.
Smolko, A. E., Shapiro-Kulnane, L. and Salz, H. K. (2020). An autoregulatory switch in sex-specific phf7 transcription causes loss of sexual identity and tumors in the Drosophila female germline. Development 147(17). PubMed ID: 32816970
Maintenance of germ cell sexual identity is essential for reproduction. Entry into the spermatogenesis or oogenesis pathway requires that the appropriate gene network is activated and the antagonist network is silenced. For example, in Drosophila female germ cells, forced expression of the testis-specific PHD finger protein 7 (PHF7) disrupts oogenesis, leading to either an agametic or germ cell tumor phenotype. This study shows that PHF7-expressing ovarian germ cells inappropriately express hundreds of genes, many of which are male germline genes. The majority of genes under PHF7 control in female germ cells are not under PHF7 control in male germ cells, suggesting that PHF7 is acting in a tissue-specific manner. Remarkably, transcriptional reprogramming includes a positive autoregulatory feedback mechanism in which ectopic PHF7 overcomes its own transcriptional repression through promoter switching. Furthermore, it was found that tumorigenic capacity is dependent on the dosage of phf7 This study reveals that ectopic PHF7 in female germ cells leads to a loss of sexual identity and the promotion of a regulatory circuit that is beneficial for tumor initiation and progression.
Wang, M., Chen, X., Wu, Y., Zheng, Q., Chen, W., Yan, Y., Luan, X., Shen, C., Fang, J., Zheng, B. and Yu, J. (2020). RpS13 controls the homeostasis of germline stem cell niche through Rho1-mediated signals in the Drosophila testis. Cell Prolif: e12899. PubMed ID: 32896929
The stem cell niche regulates the renewal and differentiation of germline stem cells (GSCs) in Drosophila. Previous work has identified a series of genes encoding ribosomal proteins that may contribute to the self-renewal and differentiation of GSCs. However, the mechanisms that maintain and differentiate GSCs in their niches were not well understood. Flies were used to generate tissue-specific gene knockdown. Small interfering RNAs were used to knockdown genes in S2 cells. qRT-PCR was used to examine the relative mRNA expression level. TUNEL staining or flow cytometry assays were used to detect cell survival. Immunofluorescence was used to determine protein localization and expression pattern. Using a genetic manipulation approach, this study investigated the role of ribosomal protein S13 (RpS13) in testes and S2 cells. RpS13 was shown to be required for the self-renewal and differentiation of GSCs. RpS13 regulates cell proliferation and apoptosis. Mechanistically, RpS13 regulates the expression of ribosome subunits and could moderate the expression of the Rho1, DE-cad and Arm proteins. Notably, Rho1 imitated the phenotype of RpS13 in both Drosophila testes and S2 cells, and recruited cell adhesions, which was mediated by the DE-cad and Arm proteins. These findings uncover a novel mechanism of RpS13 that mediates Rho1 signals in the stem cell niche of the Drosophila testis.
Dutta, P., Nath, S., Li, J. and Li, W. X. (2020). Drosophila SERTAD domain protein Taranis is required in somatic cells for maintenance of male germline stem cells. Dev Dyn. PubMed ID: 32969117
Polycomb proteins are essential for maintaining stem cell identity across different stem cell niches. However, how they function to maintain stem cell niches is not fully understood. This study shows that the SERTAD protein Taranis (Tara), which is a Polycomb-trithorax group protein, is expressed in the adult testis niche and plays a role in its maintenance in Drosophila. tara is expressed in early cyst cells, likely including somatic cyst stem cells (CySCs) of Drosophila male testis tip region, which houses both germline and somatic cyst stem cells along with the hub cells, forming the stem cell niche. Consistent with its expression, it was found that, while loss of tara in germline cells only had minimal effects, tara knockdown in all cells or only in somatic cells of the niche reduced the number of not only somatic cells, but also germline stem cells (GSCs). It was further found that Tara might antagonize Notch signaling in CySCs to maintain the stem cell niche. These studies suggest that Tara might function in somatic CySCs for GSC maintenance in the Drosophila testis.
Faber, A. I. E., van der Zwaag, M., Schepers, H., Eggens-Meijer, E., Kanon, B., C, I. J., Kuipers, J., Giepmans, B. N. G., Freire, R., Grzeschik, N. A., Rabouille, C. and Sibon, O. C. M. (2020). Vps13 is required for timely removal of nurse cell corpses. Development. PubMed ID: 32994170
Programmed cell death and consecutive removal of cellular remnants is essential for development. During late stages of Drosophila melanogaster oogenesis, the small somatic follicle cells that surround the large nurse cells, promote non-apoptotic nurse cell death, subsequently engulf them, and contribute to the timely removal of nurse cell corpses. This study identify a role for Vps13 in the timely removal of nurse cell corpses downstream of developmental programmed cell death. Vps13 is an evolutionary conserved peripheral membrane protein associated with membrane contact sites and lipid transfer. Vps13 is expressed in late nurse cells and persistent nurse cell remnants are observed when Vps13 is depleted from nurse cells but not from follicle cells. Microscopic analysis revealed enrichment of Vps13 in close proximity to the plasma membrane and the endoplasmic reticulum in nurse cells undergoing degradation. Ultrastructural analysis uncovered the presence of an underlying Vps13-dependent membranous structure in close association with the plasma membrane. The newly identified structure and function suggests the presence of a Vps13-dependent process required for complete degradation of bulky remnants of dying cells.

Thursday, November 19th - Methods

Wang, Y., Jeong, H., Yoon, B. J. and Qian, X (2020). ClusterM: a scalable algorithm for computational prediction of conserved protein complexes across multiple protein interaction networks. BMC Genomics 21(Suppl 10): 615. PubMed ID: 33208103
The current computational methods on identifying conserved protein complexes across multiple Protein-Protein Interaction (PPI) networks suffer from the lack of explicit modeling of the desired topological properties within conserved protein complexes as well as their scalability. To overcome those issues, this paper proposes a scalable algorithm—ClusterM—for identifying conserved protein complexes across multiple PPI networks through the integration of network topology and protein sequence similarity information. ClusterM overcomes the computational barrier that existed in previous methods, where the complexity escalates exponentially when handling an increasing number of PPI networks; and it is able to detect conserved protein complexes with both topological separability and cohesive protein sequence conservation. On two independent compendiums of PPI networks from Saccharomyces cerevisiae (Sce, yeast), Drosophila melanogaster (Dme, fruit fly), Caenorhabditis elegans (Cel, worm), and Homo sapiens (Hsa, human), this study demonstrates that ClusterM outperforms other state-of-the-art algorithms by a significant margin and is able to identify de novo conserved protein complexes across four species that are missed by existing algorithms. ClusterM can better capture the desired topological property of a typical conserved protein complex, which is densely connected within the complex while being well-separated from the rest of the networks. Furthermore, these experiments have shown that ClusterM is highly scalable and efficient when analyzing multiple PPI networks.
Port, F., Starostecka, M. and Boutros, M. (2020). Multiplexed conditional genome editing with Cas12a in Drosophila. Proc Natl Acad Sci U S A 117(37): 22890-22899. PubMed ID: 32843348
CRISPR-Cas genome engineering has revolutionized biomedical research by enabling targeted genome modification with unprecedented ease. In the popular model organism Drosophila melanogaster, gene editing has so far relied exclusively on the prototypical CRISPR nuclease Cas9. Additional CRISPR systems could expand the genomic target space, offer additional modes of regulation, and enable the independent manipulation of genes in different cells of the same animal. This study describes a platform for efficient Cas12a gene editing in Drosophila. This study shows that Cas12a from Lachnospiraceae bacterium, but not Acidaminococcus spec., can mediate robust gene editing in vivo. In combination with most CRISPR RNAs (crRNAs), LbCas12a activity is high at 29 °C, but low at 18 °C, enabling modulation of gene editing by temperature. LbCas12a can directly utilize compact crRNA arrays that are substantially easier to construct than Cas9 single-guide RNA arrays, facilitating multiplex genome engineering. Furthermore, conditional expression of LbCas12a is sufficient to mediate tightly controlled gene editing in a variety of tissues, allowing detailed analysis of gene function in a multicellular organism. A variant of LbCas12a with a D156R point mutation was tested; it has substantially higher activity and outperforms a state-of-the-art Cas9 system in identifying essential genes. Cas12a gene editing expands the genome-engineering toolbox in Drosophila and will be a powerful method for the functional annotation of the genome. This work also presents a fully genetically encoded Cas12a system in an animal, laying out principles for the development of similar systems in other genetically tractable organisms for multiplexed conditional genome engineering.
Yamada, N., Rossi, M. J., Farrell, N., Pugh, B. F. and Mahony, S. (2020). Alignment and quantification of ChIP-exo crosslinking patterns reveal the spatial organization of protein-DNA complexes. Nucleic Acids Res. PubMed ID: 32747934
The ChIP-exo assay precisely delineates protein-DNA crosslinking patterns by combining chromatin immunoprecipitation with 5' to 3' exonuclease digestion. Within a regulatory complex, the physical distance of a regulatory protein to DNA affects crosslinking efficiencies. Therefore, the spatial organization of a protein-DNA complex could potentially be inferred by analyzing how crosslinking signatures vary between its subunits. This study presents a computational framework that aligns ChIP-exo crosslinking patterns from multiple proteins across a set of coordinately bound regulatory regions and detects and quantifies protein-DNA crosslinking events within the aligned profiles. By producing consistent measurements of protein-DNA crosslinking strengths across multiple proteins, this approach enables characterization of relative spatial organization within a regulatory complex. Applying this approach to collections of ChIP-exo data, it is demonstrated that it can recover aspects of regulatory complex spatial organization at yeast ribosomal protein genes and yeast tRNA genes. The ability to quantify changes in protein-DNA complex organization across conditions is demonstrated by applying this approach to analyze Drosophila Pol II transcriptional components. The results suggest that principled analyses of ChIP-exo crosslinking patterns enable inference of spatial organization within protein-DNA complexes.
Tanevski, J., Nguyen, T., ...., Saez-Rodriguez, J. and Meyer, P. (2020). Gene selection for optimal prediction of cell position in tissues from single-cell transcriptomics data. Life Sci Alliance 3(11). PubMed ID: 32972997
Single-cell RNA-sequencing (scRNAseq) technologies are rapidly evolving. Although very informative, in standard scRNAseq experiments, the spatial organization of the cells in the tissue of origin is lost. Conversely, spatial RNA-seq technologies designed to maintain cell localization have limited throughput and gene coverage. Mapping scRNAseq to genes with spatial information increases coverage while providing spatial location. However, methods to perform such mapping have not yet been benchmarked. To fill this gap, this study organized the DREAM Single-Cell Transcriptomics challenge focused on the spatial reconstruction of cells from the Drosophila embryo from scRNAseq data, leveraging as silver standard, genes with in situ hybridization data from the Berkeley Drosophila Transcription Network Project reference atlas. The 34 participating teams used diverse algorithms for gene selection and location prediction, while being able to correctly localize clusters of cells. Selection of predictor genes was essential for this task. Predictor genes showed a relatively high expression entropy, high spatial clustering and included prominent developmental genes such as gap and pair-rule genes and tissue markers. Application of the top 10 methods to a zebra fish embryo dataset yielded similar performance and statistical properties of the selected genes as found in the Drosophila data. This suggests that methods developed in this challenge are able to extract generalizable properties of genes that are useful to accurately reconstruct the spatial arrangement of cells in tissues.
Scheffer, L. K., Xu, C. S., Januszewski, M., ...., Jain, V. and Plaza, S. M. (2020). A connectome and analysis of the adult Drosophila central brain. Elife 9. PubMed ID: 32880371
The neural circuits responsible for animal behavior remain largely unknown. This paper summarize new methods and presents the circuitry of a large fraction of the brain of the fruit fly Drosophila melanogaster. Improved methods include new procedures to prepare, image, align, segment, find synapses in, and proofread such large data sets. This study defines cell types, refine computational compartments, and provide an exhaustive atlas of cell examples and types, many of them novel. Detailed circuits are provided consisting of neurons and their chemical synapses for most of the central brain. The data is made public and access is simplified, reducing the effort needed to answer circuit questions, and procedures are provided linking the neurons defined by this analysis with genetic reagents. Biologically, distributions were examined of connection strengths, neural motifs on different scales, electrical consequences of compartmentalization, and evidence is provided that maximizing packing density is an important criterion in the evolution of the fly's brain.
Champer, J., Yang, E., Lee, E., Liu, J., Clark, A. G. and Messer, P. W. (2020). A CRISPR homing gene drive targeting a haplolethal gene removes resistance alleles and successfully spreads through a cage population. Proc Natl Acad Sci U S A 117(39): 24377-24383. PubMed ID: 32929034
Engineered gene drives are being explored as a new strategy in the fight against vector-borne diseases due to their potential for rapidly spreading genetic modifications through a population. However, CRISPR-based homing gene drives proposed for this purpose have faced a major obstacle in the formation of resistance alleles that prevent Cas9 cleavage. This paper presents a homing drive in Drosophila melanogaster that reduces the prevalence of resistance alleles below detectable levels by targeting a haplolethal gene with two guide RNAs (gRNAs) while also providing a rescue allele. Resistance alleles that form by end-joining repair typically disrupt the haplolethal target gene and are thus removed from the population because individuals that carry them are nonviable. This drive is highly efficient, with 91% of the progeny of drive heterozygotes inheriting the drive allele and with no functional resistance alleles observed in the remainder. In a large cage experiment, the drive allele successfully spread to all individuals within a few generations. These results show that a haplolethal homing drive can provide an effective tool for targeted genetic modification of entire populations.

Wednesday, November 18th - Evolution

Hsu, S. K., Belmouaden, C., Nolte, V. and Schlotterer, C. (2020). Parallel gene expression evolution in natural and laboratory evolved populations. Mol Ecol. PubMed ID: 32979867
Ecological adaptation is frequently inferred by the comparison of natural populations from different environments. Nevertheless, the inference of the selective forces suffers the challenge that many environmental factors covary. With well-controlled environmental conditions, experimental evolution provides a powerful approach to complement the analysis of natural populations. On the other hand, it is apparent that laboratory conditions differ in many ways from natural environments, which raises the question to what extent selection responses in experimental evolution studies can inform about adaptation processes in the wild. This study compared the expression profiles of replicated Drosophila melanogaster populations which have been exposed to two distinct temperature regimes (18/28 °C and 10/20 °C) in the laboratory for more than 80 generations. Using gene-wise differential expression analysis and co-expression network analysis, 541 genes and three co-regulated gene modules were identified that evolved in the same direction in both temperature regimes, and most of these changes probably reflect an adaptation to the space constrain or diurnal temperature fluctuation that is common in both selection regimes. 203 genes and seven modules evolved temperature-specific expression changes. Remarkably, a significant overlap was detected of these temperature-adaptive genes/modules from experimental evolution with temperature-adaptive genes inferred from natural Drosophila populations covering two different temperature clines. It is concluded that well-designed experimental evolution studies are a powerful tool to dissect evolutionary responses.
Garlovsky, M. D., Evans, C., Rosenow, M. A., Karr, T. L. and Snook, R. R. (2020). Seminal fluid protein divergence among populations exhibiting postmating prezygotic reproductive isolation. Mol Ecol. PubMed ID: 32939895
Despite holding a central role in fertilization, reproductive traits often show elevated rates of evolution and diversification. The rapid evolution of seminal fluid proteins (Sfps) within populations is predicted to cause mis-signalling between the male ejaculate and the female during and after mating resulting in postmating prezygotic (PMPZ) isolation between populations. Crosses between Drosophila montana populations show PMPZ isolation in the form of reduced fertilization success in both noncompetitive and competitive contexts. This study tested whether male ejaculate proteins produced in the accessory glands or ejaculatory bulb differ between populations using liquid chromatography tandem mass spectrometry. More than 150 differentially abundant proteins were found between populations that may contribute to PMPZ isolation, including a number of proteases, peptidases and several orthologues of Drosophila melanogaster Sfps known to mediate fertilization success. Males from the population that elicit the stronger PMPZ isolation after mating with foreign females typically produced greater quantities of Sfps. The accessory glands and ejaculatory bulb show enrichment for different gene ontology (GO) terms and the ejaculatory bulb contributes more differentially abundant proteins. Proteins with a predicted secretory signal evolve faster than nonsecretory proteins. Finally, advantage was taken of quantitative proteomics data for three Drosophila species to determine shared and unique GO enrichments of Sfps between taxa and which potentially mediate PMPZ isolation. This study provides the first high-throughput quantitative proteomic evidence showing divergence of reproductive proteins between populations that exhibit PMPZ isolation.
Lee, J. H., Lee, N. R., Kim, D. H. and Kim, Y. J. (2020). Molecular characterization of ligand selectivity of the sex peptide receptors of Drosophila melanogaster and Aedes aegypti. Insect Biochem Mol Biol: 103472. PubMed ID: 32971207
Drosophila melanogaster sex peptide receptor (DrmSPR) is a G protein-coupled receptor (GPCR) with 'dual ligand selectivity' towards sex peptide (SP) and myoinhibitory peptides (MIPs), which are only remotely related to one another. SPR is conserved in almost all the sequenced lophotrochozoan and ecdysozoan genomes. SPRs from non-drosophilid taxa, such as those from the mosquitoes Aedes aegypti (AeaSPR), Anopheles gambiae (AngSPR), and the sea slug Aplysia californica (ApcSPR), are highly sensitive to MIP, but not to SP. To understand how Drosophila SPRs evolved their SP sensitivity while maintaining MIP sensitivity, ligand selectivity was examined in a series of chimeric GPCRs that combine domains from the SP-sensitive DrmSPR and the SP-insensitive AeaSPR. Replacement of Pro 238 (P238) in DrmSPR with the corresponding residue from AeaSPR (L310) reduced its SP sensitivity 2.7 fold without altering its MIP sensitivity. The P238 residue located in the third extracellular loop (ECL3) is conserved in Drosophila SPRs and in SPR from the moth Bombyx mori (BomSPR), which is considerably more sensitive to SP than AeaSPR, AngSPR, or ApcSPR. It was found, however, that rather than improving AeaSPR's sensitivity to SP, replacement of L310 in AeaSPR with Pro significantly reduces its MIP sensitivity. Thus, the identification of a single amino acid residue critical for SP sensitivity, but not for MIP sensitivity, is an important step in clarifying how DrmSPR evolved the ability to detect SP.
Sarikaya, D. P., Cridland, J., Tarakji, A., Sheehy, H., Davis, S., Kochummen, A., Hatmaker, R., Khan, N., Chiu, J. and Begun, D. J. (2020). Phenotypic coupling of sleep and starvation resistance evolves in D. melanogaster. BMC Evol Biol 20(1): 126. PubMed ID: 32962630
One hypothesis for the function of sleep is that it serves as a mechanism to conserve energy. Recent studies have suggested that increased sleep can be an adaptive mechanism to improve survival under food deprivation in Drosophila melanogaster. To test the generality of this hypothesis, Sleep and its plastic response to starvation was compared in a temperate and tropical population of Drosophila melanogaster. Flies from the temperate population were found to be more starvation resistant, and it was hypothesized that they would engage in behaviors that are considered to conserve energy, including increased sleep and reduced movement. Surprisingly, temperate flies slept less and moved more when they were awake compared to tropical flies, both under fed and starved conditions, therefore sleep did not correlate with population-level differences in starvation resistance. In contrast, total sleep and percent change in sleep when starved were strongly positively correlated with starvation resistance within the tropical population, but not within the temperate population. Thus, unexpectedly complex relationships between starvation and sleep were observed that vary both within and across populations. These observations falsify the simple hypothesis of a straightforward relationship between sleep and energy conservation. The hypothesis that starvation is correlated with metabolic phenotypes was tested by investigating stored lipid and carbohydrate levels; stored metabolites were found to partially contributed towards variation starvation resistance. These findings demonstrate that the function of sleep under starvation can rapidly evolve on short timescales and raise new questions about the physiological correlates of sleep and the extent to which variation in sleep is shaped by natural selection.
Minekawa, K., Amino, K. and Matsuo, T. (2020). A courtship behavior that makes monandrous females polyandrous. Evolution. PubMed ID: 32944943
Females of many animal species mate several times with different males (polyandry), whereas females of some species mate with a single male (monandry) only once. Little is known about the mechanisms by which these different mating systems evolve. Females of Drosophila prolongata mate serially, unlike Drosophila melanogaster females that refuse to remate for several days after their first mating (remating suppression [RS]). Nevertheless, interestingly, nonvirgin D. prolongata females refuse to remate with males that are prohibited from performing their species-specific courtship behavior, leg vibration (LV), suggesting that LV overrides RS making it cryptic in D. prolongata. This study examined how long this cryptic RS persists. Surprisingly, it was sustained for at least 2 weeks, showing that RS is substantially augmented in D. prolongata compared to that of D. melanogaster. The two most closely related species to D. prolongata, Drosophila rhopaloa and Drosophila carrolli, do not perform LV and showed augmented RS, supporting the idea that augmented RS could have evolved before LV was acquired. These results suggested that D. prolongata females are intrinsically monandrous, whereas the newly evolved courtship behavior makes them polyandrous. This is a rare case in which a proximate mechanism of polyandry evolution from monandry is demonstrated.
Liu, J., Frochaux, M., Gardeux, V., Deplancke, B. and Robinson-Rechavi, M. (2020). Inter-embryo gene expression variability recapitulates the hourglass pattern of evo-devo. BMC Biol 18(1): 129. PubMed ID: 32950053
The evolution of embryological development has long been characterized by deep conservation. In animal development, the phylotypic stage in mid-embryogenesis is more conserved than either early or late stages among species within the same phylum. Hypotheses to explain this hourglass pattern have focused on purifying the selection of gene regulation. This paper proposes an alternative-genes are regulated in different ways at different stages and have different intrinsic capacities to respond to perturbations on gene expression. To eliminate the influence of natural selection, the expression variability of isogenetic single embryo transcriptomes was quantified throughout fly Drosophila melanogaster embryogenesis. The expression variability is lower at the phylotypic stage, supporting that the underlying regulatory architecture in this stage is more robust to stochastic variation on gene expression. Evidence is presented that the phylotypic stage is also robust to genetic variations on gene expression. Moreover, chromatin regulation appears to play a key role in the variation and evolution of gene expression. It is suggested that a phylum-level pattern of embryonic conservation can be explained by the intrinsic difference of gene regulatory mechanisms in different stages.

Tuesday, November 17 - Chromatin

You, Q., Cheng, A. Y., Gu, X., Harada, B. T., Yu, M., Wu, T., Ren, B., Ouyang, Z. and He, C. (2020). Direct DNA crosslinking with CAP-C uncovers transcription-dependent chromatin organization at high resolution. Nat Biotechnol. PubMed ID: 32839564
Determining the spatial organization of chromatin in cells mainly relies on crosslinking-based chromosome conformation capture techniques, but resolution and signal-to-noise ratio of these approaches is limited by interference from DNA-bound proteins. This study introduces chemical-crosslinking assisted proximity capture (CAP-C), a method that uses multifunctional chemical crosslinkers with defined sizes to capture chromatin contacts. CAP-C generates chromatin contact maps at subkilobase (sub-kb) resolution with low background noise. CAP-C was applied to formaldehyde prefixed mouse embryonic stem cells (mESCs), and loop domains (median size of 200 kb) and nonloop domains (median size of 9 kb) were investigated. Transcription inhibition caused a greater loss of contacts in nonloop domains than loop domains. Conserved, transcription-state-dependent chromatin compartmentalization was uncovered at high resolution that is shared from Drosophila to human, and a transcription-initiation-dependent nuclear subcompartment was uncovered that brings multiple nonloop domains in close proximity. It was also shown that CAP-C could be used to detect native chromatin conformation without formaldehyde prefixing.
Miller, D. E., Kahsai, L., Buddika, K., Dixon, M. J., Kim, B. Y., Calvi, B. R., Sokol, N. S., Hawley, R. S. and Cook, K. R. (2020). Identification and Characterization of Breakpoints and Mutations on Drosophila melanogaster Balancer Chromosomes. G3 (Bethesda). PubMed ID: 32972999
Balancers are rearranged chromosomes used in Drosophila to maintain deleterious mutations in stable populations, preserve sets of linked genetic elements and construct complex experimental stocks. This study assessed the phenotypes associated with breakpoint-induced mutations on commonly used third chromosome balancers and showed remarkably few deleterious effects. A breakpoint in p53 causes loss of radiation-induced apoptosis and a breakpoint in Fucosyltransferase A causes loss of fucosylation in nervous and intestinal tissue-the latter study providing new markers for intestinal cell identity and challenging previous conclusions about the regulation of fucosylation. Thousands of potentially harmful mutations are described, shared among X or third chromosome balancers, or unique to specific balancers, including an Ankyrin 2 mutation present on most TM3 balancers, and the risks of using balancers as experimental controls is reiterated. Long-read sequencing was used to confirm or refine the positions of two inversions with breakpoints lying in repetitive sequences. Finally, evidence is presented that extremely distal mutations on balancers can add to the stability of stocks whose purpose is to maintain homologous chromosomes carrying mutations in distal genes. Overall, these studies add to understanding of the structure, diversity and effectiveness of balancer chromosomes.
Schmidt, J. M. and Bleichert, F. (2020). Structural mechanism for replication origin binding and remodeling by a metazoan origin recognition complex and its co-loader Cdc6. Nat Commun 11(1): 4263. PubMed ID: 32848132
Eukaryotic DNA replication initiation relies on the origin recognition complex (ORC), a DNA-binding ATPase that loads the Mcm2-7 replicative helicase onto replication origins. This study reports cryo-electron microscopy (cryo-EM) structures of DNA-bound Drosophila ORC with and without the co-loader Cdc6. These structures reveal that Orc1 and Orc4 constitute the primary DNA binding site in the ORC ring and cooperate with the winged-helix domains to stabilize DNA bending. A loop region near the catalytic Walker B motif of Orc1 directly contacts DNA, allosterically coupling DNA binding to ORC's ATPase site. Correlating structural and biochemical data show that DNA sequence modulates DNA binding and remodeling by ORC, and that DNA bending promotes Mcm2-7 loading in vitro. Together, these findings explain the distinct DNA sequence-dependencies of metazoan and S. cerevisiae initiators in origin recognition and support a model in which DNA geometry and bendability contribute to Mcm2-7 loading site selection in metazoans.
Bondarenko, S. M. and Sharakhov, I. V. (2020). Reorganization of the nuclear architecture in the Drosophila melanogaster Lamin B mutant lacking the CaaX box. Nucleus 11(1): 283-298. PubMed ID: 32960740
Lamins interact with the nuclear membrane and chromatin but the precise players and mechanisms of these interactions are unknown. This study tested whether the removal of the CaaX motif from Lamin B disrupts its attachment to the nuclear membrane and affects chromatin distribution. Drosophila melanogaster Lam(A25) homozygous mutants were used that lack the CaaX box. The mutant Lamin B was not confined to the nuclear periphery but was distributed throughout the nuclear interior, colocalizing with chromosomes in salivary gland and proventriculus. The peripheral position of Lamin C, nuclear pore complex (NPC), heterochromatin protein 1a (HP1a), H3K9me2- and H3K27me3-associated chromatin remained intact. The fluorescence intensity of the DAPI-stained peripheral chromatin significantly decreased and that of the central chromatin significantly increased in the proventriculus nuclei of the mutant flies compared to wild-type. However, the mutation had little effect on chromatin radial distribution inside highly polytenized salivary gland nuclei.
Reinig, J., Ruge, F., Howard, M. and Ringrose, L. (2020). A theoretical model of Polycomb/Trithorax action unites stable epigenetic memory and dynamic regulation. Nat Commun 11(1): 4782. PubMed ID: 32963223
Polycomb and Trithorax group proteins maintain stable epigenetic memory of gene expression states for some genes, but many targets show highly dynamic regulation. This study combined experiment and theory to examine the mechanistic basis of these different modes of regulation. A mathematical model is presented comprising a Polycomb/Trithorax response element (PRE/TRE) coupled to a promoter and including Drosophila developmental timing. The model accurately recapitulates published studies of PRE/TRE mediated epigenetic memory of both silencing and activation. With minimal parameter changes, the same model can also recapitulate experimental data for a different PRE/TRE that allows dynamic regulation of its target gene. The model predicts that both cell cycle length and PRE/TRE identity are critical for determining whether the system gives stable memory or dynamic regulation. This work provides a simple unifying framework for a rich repertoire of PRE/TRE functions, and thus provides insights into genome-wide Polycomb/Trithorax regulation.
De, S., Gehred, N. D., Fujioka, M., Chan, F. W., Jaynes, J. B. and Kassis, J. A. (2020). Defining the Boundaries of Polycomb Domains in Drosophila. Genetics. PubMed ID: 32948625
Polycomb group (PcG) proteins are an important group of transcriptional repressors that act by modifying chromatin. PcG target genes are covered by the repressive chromatin mark H3K27me3. Polycomb repressive complex 2 (PRC2) is a multiprotein complex that is responsible for generating H3K27me3. In Drosophila, PRC2 is recruited by Polycomb Response Elements (PREs) and then tri-methylates flanking nucleosomes, spreading the H3K27me3 mark over large regions of the genome, the "Polycomb domains". What defines the boundary of a Polycomb domain? There is experimental evidence that insulators, PolII, and active transcription can all form the boundaries of Polycomb domains. This study divided the boundaries of larval Polycomb domains into six different categories. In one category, genes are transcribed toward the Polycomb domain, where active transcription is thought to stop the spreading of H3K27me3. In agreement with this, it was shown that introducing a transcriptional terminator into such a transcription unit causes an extension of the Polycomb domain. Additional data suggest that active transcription of a boundary gene may restrict the range of enhancer activity of a Polycomb-regulated gene.

Monday November 16th - Adult Neural Development and function

Earl, J. B., Vanderlinden, L. A., Jacobsen, T. L., Aldrich, J. C., Saba, L. M. and Britt, S. G. (2020). Identification of Genes Involved in the Differentiation of R7y and R7p Photoreceptor Cells in Drosophila. G3 (Bethesda). PubMed ID: 32972998
The R7 and R8 photoreceptor cells of the Drosophila compound eye mediate color vision. Throughout the majority of the eye, these cells occur in two principal types of ommatidia. Approximately 35% of ommatidia are of the pale type and express Rh3 in R7 cells and Rh5 in R8 cells. The remaining 65% are of the yellow type and express Rh4 in R7 cells and Rh6 in R8 cells. The specification of an R8 cell in a pale or yellow ommatidium depends on the fate of the adjacent R7 cell. However, pale and yellow R7 cells are specified by a stochastic process that requires the genes spineless, tango and klumpfuss. To identify additional genes involved in this process genetic screens were performed using a collection of 480 P{EP} transposon insertion strains. Genes were identified in gain of function and loss of function screens that significantly altered the percentage of Rh3 expressing R7 cells (Rh3%) from wild-type. 36 strains resulted in altered Rh3% in the gain of function screen where the P{EP} insertion strains were crossed to a sevEP-GAL4 driver line. 53 strains resulted in altered Rh3 percent in the heterozygous loss of function screen. 4 strains showed effects that differed between the two screens, suggesting that the effect found in the gain of function screen was either larger than, or potentially masked by, the P{EP} insertion alone. Analyses of homozygotes validated many of the candidates identified. These results suggest that R7 cell fate specification is sensitive to perturbations in mRNA transcription, splicing and localization, growth inhibition, post-translational protein modification, cleavage and secretion, hedgehog signaling, ubiquitin protease activity, GTPase activation, actin and cytoskeletal regulation, and Ser/Thr kinase activity, among other diverse signaling and cell biological processes.
Ravenscroft, T. A., Janssens, J., Lee, P. T., Tepe, B., Marcogliese, P. C., Makhzami, S., Holmes, T. C., Aerts, S. and Bellen, H. J. (2020). Drosophila voltage-gated sodium channels are only expressed in active neurons and are localized to distal axonal initial segment-like domains. J Neurosci. PubMed ID: 32928889
In multipolar vertebrate neurons, action potentials (AP) initiate close to the soma, at the axonal initial segment (AIS). Invertebrate neurons are typically unipolar with dendrites integrating directly into the axon. Where APs are initiated in the axons of invertebrate neurons is unclear. Voltage-gated sodium (Na(V)) channels are a functional hallmark of the AIS in vertebrates. This study used an intronic MiMIC to determine the endogenous gene expression and subcellular localization of the sole Na(V) channel in both male and female Drosophila, para. Despite being the only Na(V) channel in the fly, only 23 ±1% of neurons in the embryonic and larval CNS express para, while in the adult CNS para is broadly expressed. A single-cell transcriptomic atlas was generated of the whole 3(rd) instar larval brain to identify para expressing neurons and show that it positively correlates with markers of differentiated, actively firing neurons. Therefore only 23 ±1% of larval neurons may be capable of firing Na(V)-dependent APs. It was then shown that Para is enriched in an axonal segment, distal to the site of dendritic integration into the axon; this segment was named the Distal Axonal Segment (DAS). The DAS is present in multiple neuron classes in both the 3(rd) instar larval and adult CNS. Whole cell patch clamp electrophysiological recordings of adult CNS fly neurons are consistent with the interpretation that Na(v)-dependent APs originate in the DAS. Identification of the distal Na(V) localization in fly neurons will enable more accurate interpretation of electrophysiological recordings in invertebrates.
Li, F., Sami, A., Noristani, H. N., Slattery, K., Qiu, J., Groves, T., Wang, S., Veerasammy, K., Chen, Y. X., Morales, J., Haynes, P., Sehgal, A., He, Y., Li, S. and Song, Y. (2020). Glial Metabolic Rewiring Promotes Axon Regeneration and Functional Recovery in the Central Nervous System. Cell Metab. PubMed ID: 32941799
Axons in the mature central nervous system (CNS) fail to regenerate after axotomy, partly due to the inhibitory environment constituted by reactive glial cells producing astrocytic scars, chondroitin sulfate proteoglycans, and myelin debris. This study investigated this inhibitory milieu, showing that it is reversible and depends on glial metabolic status. Glia can be reprogrammed to promote morphological and functional regeneration after CNS injury in Drosophila via increased glycolysis. This enhancement is mediated by the glia derived metabolites: L-lactate and L-2-hydroxyglutarate (L-2HG). Genetically/pharmacologically increasing or reducing their bioactivity promoted or impeded CNS axon regeneration. L-lactate and L-2HG from glia acted on neuronal metabotropic GABA(B) receptors to boost cAMP signaling. Local application of L-lactate to injured spinal cord promoted corticospinal tract axon regeneration, leading to behavioral recovery in adult mice. These findings revealed a metabolic switch to circumvent the inhibition of glia while amplifying their beneficial effects for treating CNS injuries.
Melnattur, K., Kirszenblat, L., Morgan, E., Militchin, V., Sakran, B., English, D., Patel, R., Chan, D., van Swinderen, B. and Shaw, P. J. (2020). A conserved role for sleep in supporting Spatial Learning in Drosophila. Sleep. PubMed ID: 32959053
Sleep loss and aging impair hippocampus-dependent Spatial Learning in mammalian systems. This study used the fly Drosophila melanogaster to investigate the relationship between sleep and Spatial Learning in healthy and impaired flies. The Spatial Learning assay is modeled after the Morris Water Maze. The assay uses a 'thermal maze' consisting of a 5X5 grid of Peltier plates maintained at 36-37°C and a visual panorama. The first trial begins when a single tile that is associated with a specific visual cue is cooled to 25°C. For subsequent trials, the cold tile is heated, the visual panorama is rotated and the flies must find the new cold-tile by remembering its association with the visual cue. Significant learning was observed with two different wild-type strains - Cs and 2U, validating the design. Sleep deprivation prior to training impaired Spatial Learning. Learning was also impaired in the classic learning mutant rutabaga (rut); enhancing sleep restored learning to rut mutants. Further, flies exhibited dramatic age-dependent cognitive decline in Spatial Learning starting at 20-24 days of age. These impairments could be reversed by enhancing sleep. Finally, Spatial Learning was found to requires dopaminergic signaling and that enhancing dopaminergic signaling in aged flies restored learning. These results are consistent with the impairments seen in rodents and humans. These results thus demonstrate a critical conserved role for sleep in supporting Spatial Learning, and suggest potential avenues for therapeutic intervention during aging.
Li, J., Mahoney, B. D., Jacob, M. S. and Caron, S. J. C. (2020). Visual Input into the Drosophila melanogaster Mushroom Body. Cell Rep 32(11): 108138. PubMed ID: 32937130
The patterns of neuronal connectivity underlying multisensory integration, a fundamental property of many brains, remain poorly characterized. The Drosophila melanogaster mushroom body-an associative center-is an ideal system to investigate how different sensory channels converge in higher order brain centers. The neurons connecting the mushroom body to the olfactory system have been described in great detail, but input from other sensory systems remains poorly defined. This study used a range of anatomical and genetic techniques to identify two types of input neurons that connect visual processing centers-the lobula and the posterior lateral protocerebrum-to the dorsal accessory calyx of the mushroom body. Together with previous work that described a pathway conveying visual information from the medulla to the ventral accessory calyx of the mushroom body, this study defines a second, parallel pathway that is anatomically poised to convey information from the visual system to the dorsal accessory calyx.
Kinare, V., Iyer, A., Padmanabhan, H., Godbole, G., Khan, T., Khatri, Z., Maheshwari, U., Muralidharan, B. and Tole, S. (2020). An evolutionarily conserved Lhx2-Ldb1 interaction regulates the acquisition of hippocampal cell fate and regional identity. Development. PubMed ID: 32994168
Protein cofactor Ldb1 regulates cell fate specification by interacting with LIM-homeodomain (LIM-HD) proteins in a tetrameric complex consisting of an LDB:LDB dimer that bridges two LIM-HD molecules, a mechanism first demonstrated in the Drosophila wing disc. This study demonstrates conservation of this interaction in the regulation of mammalian hippocampal development, which is profoundly defective upon loss of either Lhx2 (see Drosophila Apterous) or Ldb1 (see Drosophila Chip). Electroporation of a chimeric construct that encodes the Lhx2-HD and Ldb1-DD (dimerization domain) in a single transcript cell-autonomously rescues a comprehensive range of hippocampal deficits in the mouse Ldb1 mutant, including the acquisition of field-specific molecular identity and the regulation of the neuron-glia cell fate switch. This demonstrates that the LHX:LDB complex is an evolutionarily conserved molecular regulatory device that controls complex aspects of regional cell identity in the developing brain.

Friday, November 13th - Adult Development

Mo, D., Chen, Y., Jiang, N., Shen, J. and Zhang, J. (2020). Investigation of Isoform Specific Functions of the V-ATPase a Subunit During Drosophila Wing Development. Front Genet 11: 723. PubMed ID: 32754202
The vacuolar ATPases (V-ATPases) are ATP-dependent proton pumps that play vital roles in eukaryotic cells. Insect V-ATPases are required in nearly all epithelial tissues to regulate a multiplicity of processes including receptor-mediated endocytosis, protein degradation, fluid secretion, and neurotransmission. Composed of fourteen different subunits, several V-ATPase subunits exist in distinct isoforms to perform cell type specific functions. The 100 kD a subunit (see Vha100) of V-ATPases are encoded by a family of five genes in Drosophila, but their assignments are not fully understood. This study reports an experimental survey of the Vha100 gene family during Drosophila wing development. A combination of CRISPR-Cas9 mutagenesis, somatic clonal analysis and in vivo RNAi assays is used to characterize the requirement of Vha100 isoforms, and mutants of Vha100-2, Vha100-3, Vha100-4, and Vha100-5 genes were generated. Vha100-3 and Vha100-5 were shown to be dispensable for fly development, while Vha100-1 is not critically required in the wing. As for the other two isoforms, Vha100-2 was found to regulate wing cuticle maturation, while Vha100-4 is the single isoform involved in developmental patterning. More specifically, Vha100-4 is required for proper activation of the Wingless signaling pathway during fly wing development. Interestingly, a specific genetic interaction was found between Vha100-1 and Vha100-4 during wing development. These results revealed the distinct roles of Vha100 isoforms during insect wing development, providing a rationale for understanding the diverse roles of V-ATPases.
Zhou, F., Green, S. R., Tsay, M., Hsu, S., Dibbs, R. and Beckingham, K. M. (2020). The roles of jim lovell and uninflatable in different endopolyploid larval tissues of Drosophila melanogaster. PLoS One 15(8): e0237662. PubMed ID: 32822370
The larvae of Drosophila melanogaster grow rapidly through use of a highly truncated cell cycle in which mitosis is entirely eliminated. The Drosophila homolog of the protooncogene transcription factor Myc plays a major role in promoting this endopolyploid (EP) growth. It was previously determined that the gene jim lovell (lov), which encodes a member of the BTB/POZ (Bric-a-brac, Tramtrack, Broad/Pox virus zinc finger) domain family of transcription factors, is also required for EP growth in one larval tissue, the trachea. This study shows that lov promotes EP growth in three further tissues indicating a fundamental role in this process. However, epistasis experiments revealed heterogeneity in lov's action in these tissues. Whereas in the tracheae and salivary glands lov acts downstream of Myc, in the fat body, reduced expression of lov does not impede the action of Myc, indicating an upstream action for the gene. This study shows that lov's regulation of the gene uninflatable (uif) in the tracheae is a component of this difference. uif is required for tracheal EP growth downstream of Myc and lov but has no equivalent role in the fat body. Although Uif is a transmembrane component of the plasma membrane in the tracheae, its action downstream of Myc suggests an intracellular role for the protein in the tracheae. In addition to regulating uif expression in some tissues it was also shown that lov locates to the nucleolus, indicating it can function in both polymerase I and polymerase II transcriptional events. The major finding of this study is that tissue-specific mechanisms can interact with universal growth promotion by Myc to generate the individual endopolyploid organs of the larvae.
Duffraisse, M., Paul, R., Carnesecchi, J., Hudry, B., Banreti, A., Reboulet, J., Ajuria, L., Lohmann, I. and Merabet, S. (2020). Role of a versatile peptide motif controlling Hox nuclear export and autophagy in the Drosophila fat body. J Cell Sci 133(18). PubMed ID: 32878938
Hox proteins are major regulators of embryonic development, acting in the nucleus to regulate the expression of their numerous downstream target genes. By analyzing deletion forms of the Drosophila Hox protein Ultrabithorax (Ubx), the presence of an unconventional nuclear export signal (NES) was identified that overlaps with a highly conserved motif originally described as mediating the interaction with the PBC proteins, a generic and crucial class of Hox transcriptional cofactors that act in development and cancer. This unconventional NES is involved in the interaction with the major exportin protein CRM1 (also known as Embargoed in flies) in vivo and in vitro. This interaction is tightly regulated in the Drosophila fat body to control the autophagy-repressive activity of Ubx during larval development. The role of the PBC interaction motif as part of an unconventional NES was also uncovered in other Drosophila and human Hox proteins, highlighting the evolutionary conservation of this novel function. Together, these results reveal the extreme molecular versatility of a unique short peptide motif for controlling the context-dependent activity of Hox proteins both at transcriptional and non-transcriptional levels.
Nagel, A. C., Maier, D., Scharpf, J., Ketelhut, M. and Preiss, A. (2020). Limited Availability of General Co-Repressors Uncovered in an Overexpression Context during Wing Venation in Drosophila melanogaster. Genes (Basel) 11(10). PubMed ID: 32998295
Cell fate is determined by the coordinated activity of different pathways, including the conserved Notch pathway. Activation of Notch results in the transcription of Notch targets that are otherwise silenced by repressor complexes. In Drosophila, the repressor complex comprises the transcription factor Suppressor of Hairless (Su(H)) bound to the Notch antagonist Hairless (H) and the general co-repressors Groucho (Gro) and C-terminal binding protein (CtBP). The latter two are shared by different repressors from numerous pathways, raising the possibility that they are rate-limiting. It was noted that the overexpression during wing development of H mutants H(dNT) and H(LD) compromised in Su(H)-binding induced ectopic veins. On the basis of the role of H as Notch antagonist, overexpression of Su(H)-binding defective H isoforms should be without consequence, implying different mechanisms but repression of Notch signaling activity. Perhaps excess H protein curbs general co-repressor availability. Supporting this model, nearly normal wings developed upon overexpression of H mutant isoforms that bound neither Su(H) nor co-repressor Gro and CtBP. Excessive H protein appeared to sequester general co-repressors, resulting in specific vein defects, indicating their limited availability during wing vein development. In conclusion, interpretation of overexpression phenotypes requires careful consideration of possible dominant negative effects from interception of limiting factors.
Zhang, Y., Liu, J. and Liu, J. L. (2020). The atlas of cytoophidia in Drosophila larvae. J Genet Genomics. PubMed ID: 32912804
In 2010, cytidine 5'-triphosphate synthase (CTPS) was reported to form the filamentous or serpentine structure in Drosophila, which was termed the cytoophidium. In the last decade, CTPS filaments/cytoophidia have been found in bacteria, budding yeast, human cells, mice, fission yeast, plants, and archaea, indicating that this mechanism is highly conserved in evolution. In addition to CTPS, other metabolic enzymes have been identified to have the characteristic of forming cytoophidia or similar advanced structures, demonstrating that this is a basic strategy of cells. Nevertheless, understanding of the physiological function of the cytoophidium remains incomplete and elusive. This study took the larva of Drosophila melanogaster as a model to systematically describe the localization and distribution of cytoophidia in different tissues during larval development. The distribution pattern of CTPS cytoophidia was found to be dynamic and heterogenic in larval tissues. This study provides a road map for further understanding of the function and regulatory mechanism of cytoophidia.
Fujisawa, Y., Shinoda, N., Chihara, T. and Miura, M. (2020). ROS Regulate Caspase-Dependent Cell Delamination without Apoptosis in the Drosophila Pupal Notum. iScience 23(8): 101413. PubMed ID: 32791328
Thorax fusion occurs in the midline of the Drosophila pupal notum and involves epithelial cell delamination requiring apoptotic signaling. By genetic screening, this study found that NADPH oxidases (Nox and Duox) associated with superoxide anion (O-2) are responsible for caspase-3 activation and delamination. Nox is upregulated in cells that undergo delamination and that delamination depends on caspase activation. However, the cell morphology and the almost complete lack of propidium iodide incorporation suggested little membrane disruption and signified apoptotic modulation. These results demonstrate that most delaminating cells undergo caspase activation, but this activation is not sufficient for apoptosis. The expression of Catalase, encoding an H2O2 scavenger in the cytosol, increases delamination and induces apoptotic nuclear fragmentation in caspase-3-activated cells. These findings suggest that the roles of O-2 and intracellular H2O2 for delamination differs before and after caspase-3 activation, which involves live cell delamination.

Thursday, November 12th - Disease Models

Brengdahl, M. I., Kimber, C. M., Elias, P., Thompson, J. and Friberg, U. (2020). Deleterious mutations show increasing negative effects with age in Drosophila melanogaster. BMC Biol 18(1): 128. PubMed ID: 32993647
In order for aging to evolve in response to a declining strength of selection with age, a genetic architecture that allows for mutations with age-specific effects on organismal performance is required. Understanding of how selective effects of individual mutations are distributed across ages is however poor. Established evolutionary theories assume that mutations causing aging have negative late-life effects, coupled to either positive or neutral effects early in life. New theory now suggests evolution of aging may also result from deleterious mutations with increasing negative effects with age, a possibility that has not yet been empirically explored. To directly test how the effects of deleterious mutations are distributed across ages, age-specific effects on fecundity were separately measure for each of 20 mutations in Drosophila melanogaster. Deleterious mutations in general were found to have a negative effect that increases with age, and the rate of increase depends on how deleterious a mutation is early in life. These findings suggest that aging does not exclusively depend on genetic variants assumed by the established evolutionary theories of aging. Instead, aging can result from deleterious mutations with negative effects that amplify with age. If increasing negative effect with age is a general property of deleterious mutations, the proportion of mutations with the capacity to contribute towards aging may be considerably larger than previously believed.
Mangleburg, C. G., Wu, T., Yalamanchili, H. K., Guo, C., Hsieh, Y. C., Duong, D. M., Dammer, E. B., De Jager, P. L., Seyfried, N. T., Liu, Z. and Shulman, J. M. (2020). Integrated analysis of the aging brain transcriptome and proteome in tauopathy. Mol Neurodegener 15(1): 56. PubMed ID: 32993812
Tau (see Drosophila Tau) neurofibrillary tangle pathology characterizes Alzheimer's disease and other neurodegenerative tauopathies. This study performed paired, longitudinal RNA-sequencing and mass-spectrometry were performed in a Drosophila model of tauopathy, based on pan-neuronal expression of human wildtype Tau (Tau(WT)) or a mutant form causing frontotemporal dementia (Tau(R406W)). Tau-induced, differentially expressed transcripts and proteins were examined cross-sectionally or using linear regression and adjusting for age. Overlaps with human brain gene expression profiles in tauopathy were examined. Tau(WT) induced 1514 and 213 differentially expressed transcripts and proteins, respectively. Tau(R406W) had a substantially greater impact, causing changes in 5494 transcripts and 697 proteins. There was a ~ 70% overlap between age- and Tau-induced changes and the analyses reveal pervasive bi-directional interactions. Strikingly, 42% of Tau-induced transcripts were discordant in the proteome, showing opposite direction of change. Tau-responsive gene expression networks strongly implicate innate immune activation. Cross-species analyses pinpoint human brain gene perturbations specifically triggered by Tau pathology and/or aging, and further differentiate between disease amplifying and protective changes. These results comprise a powerful, cross-species functional genomics resource for tauopathy, revealing Tau-mediated disruption of gene expression, including dynamic, age-dependent interactions between the brain transcriptome and proteome.
Chatterjee, M., Steffan, J. S., Lukacsovich, T., Marsh, J. L. and Agrawal, N. (2020). Serine residues 13 and 16 are key modulators of mutant huntingtin induced toxicity in Drosophila. Exp Neurol: 113463. PubMed ID: 32941796
Poly-glutamine expansion near the N-terminus of the huntingtin protein (HTT) is the prime determinant of Huntington's disease (HD) pathology; however, post-translational modifications and protein context are also reported to influence poly-glutamine induced HD toxicity. The impact of phosphorylating serine 13/16 of mutant HTT (mHTT) on HD has been documented in cell culture and murine models. However, endogenous processing of the human protein in mammalian systems complicates the interpretations. Therefore, to study the impact of S13/16 phosphorylation on the subcellular behavior of HTT under a controlled genetic background with minimal proteolytic processing of the human protein, Drosophila was employed as the model system. Full-length (FL) and exon1 fragment of human HTT with phosphomimetic and resistant mutations at serines 13 and 16 were ectopically expressed in different neuronal populations. Phosphomimetic mHTT aggravates and the phosphoresistant mutation ameliorates mHTT-induced toxicity in the context of both FL- and exon1- mHTT in Drosophila although in all cases FL appears less toxic than exon1. These observations strongly indicate that the phosphorylation status of S13/16 can affect HD pathology in Drosophila and these residues can be potential targets for affecting HD pathogenesis.
Johnson, S. L., Ranxhi, B., Libohova, K., Tsou, W. L. and Todi, S. V. (2020). Ubiquitin-interacting motifs of ataxin-3 regulate its polyglutamine toxicity through Hsc70-4-dependent aggregation. Elife 9. PubMed ID: 32955441
Spinocerebellar ataxia type 3 (SCA3) belongs to the family of polyglutamine neurodegenerations. Each disorder stems from the abnormal lengthening of a glutamine repeat in a different protein. Although caused by a similar mutation, polyglutamine disorders are distinct, implicating non-polyglutamine regions of disease proteins as regulators of pathogenesis. SCA3 is caused by polyglutamine expansion in ataxin-3. To determine the role of ataxin-3's non-polyglutamine domains in disease, a new, allelic series of Drosophila melanogaster was utilized. Ataxin-3 pathogenicity was found to be saliently controlled by polyglutamine-adjacent ubiquitin-interacting motifs (UIMs) that enhance aggregation and toxicity. UIMs function by interacting with the heat shock protein, Hsc70-4, whose reduction diminishes ataxin-3 toxicity in a UIM-dependent manner. Hsc70-4 also enhances pathogenicity of other polyglutamine proteins. These studies provide a unique insight into the impact of ataxin-3 domains in SCA3, identify Hsc70-4 as a SCA3 enhancer, and indicate pleiotropic effects from HSP70 chaperones, which are generally thought to suppress polyglutamine degeneration.
Kim, Y. Y., Yoon, J. H., Um, J. H., Jeong, D. J., Shin, D. J., Hong, Y. B., Kim, J. K., Kim, D. H., Kim, C., Chung, C. G., Lee, S. B., Koh, H. and Yun, J. (2020). PINK1 alleviates thermal hypersensitivity in a paclitaxel-induced Drosophila model of peripheral neuropathy. PLoS One 15(9): e0239126. PubMed ID: 32941465
Paclitaxel is a representative anticancer drug that induces chemotherapy-induced peripheral neuropathy (CIPN), a common side effect that limits many anticancer chemotherapies. Although PINK1, a key mediator of mitochondrial quality control, has been shown to protect neuronal cells from various toxic treatments, the role of PINK1 in CIPN has not been investigated. This study examined the effect of PINK1 expression on CIPN using a recently established paclitaxel-induced peripheral neuropathy model in Drosophila larvae. The class IV dendritic arborization (C4da) sensory neuron-specific expression of PINK1 significantly ameliorated the paclitaxel-induced thermal hyperalgesia phenotype. In contrast, knockdown of PINK1 resulted in an increase in thermal hypersensitivity, suggesting a critical role for PINK1 in sensory neuron-mediated thermal nociceptive sensitivity. Interestingly, analysis of the C4da neuron morphology suggests that PINK1 expression alleviates paclitaxel-induced thermal hypersensitivity by means other than preventing alterations in sensory dendrites in C4da neurons. Paclitaxel was found to induce mitochondrial dysfunction in C4da neurons and PINK1 expression suppressed the paclitaxel-induced increase in mitophagy in C4da neurons. These results suggest that PINK1 mitigates paclitaxel-induced sensory dendrite alterations and restores mitochondrial homeostasis in C4da neurons and that improvement in mitochondrial quality control could be a promising strategy for the treatment of CIPN.
Losev, Y., Frenkel-Pinter, M., Abu-Hussien, M., Viswanathan, G. K., Elyashiv-Revivo, D., Geries, R., Khalaila, I., Gazit, E. and Segal, D. (2020). Differential effects of putative N-glycosylation sites in human Tau on Alzheimer's disease-related neurodegeneration. Cell Mol Life Sci. PubMed ID: 32926180
Amyloid assemblies of Tau (see Drosophila Tau) are associated with Alzheimer's disease (AD). In AD Tau undergoes several abnormal post-translational modifications, including hyperphosphorylation and glycosylation, which impact disease progression. N-glycosylated Tau was reported to be found in AD brain tissues but not in healthy counterparts. This is surprising since Tau is a cytosolic protein whereas N-glycosylation occurs in the ER-Golgi. Previous in vitro studies indicated that N-glycosylation of Tau facilitated its phosphorylation and contributed to maintenance of its Paired Helical Filament structure. However, the specific Tau residue(s) that undergo N-glycosylation and their effect on Tau-engendered pathology are unknown. High-performance liquid chromatography and mass spectrometry (LC-MS) analysis indicated that both N359 and N410 were N-glycosylated in wild-type (WT) human Tau (hTau) expressed in human SH-SY5Y cells. Asparagine to glutamine mutants, which cannot undergo N-glycosylation, at each of three putative N-glycosylation sites in hTau (N167Q, N359Q, and N410Q) were generated and expressed in SH-SY5Y cells and in transgenic Drosophila. The mutants modulated the levels of hTau phosphorylation in a site-dependent manner in both cell and fly models. Additionally, N359Q ameliorated, whereas N410Q exacerbated various aspects of hTau-engendered neurodegeneration in transgenic flies.

Wednesday, November 11th - RNA

Carrasco, J., Rauer, M., Hummel, B., Grzejda, D., Alfonso-Gonzalez, C., Lee, Y., Wang, Q., Puchalska, M., Mittler, G. and Hilgers, V. (2020). ELAV and FNE Determine Neuronal Transcript Signatures through EXon-Activated Rescue. Mol Cell 80(1): 156-163 PubMed ID: 33007255
The production of alternative RNA variants contributes to the tissue-specific regulation of gene expression. In the animal nervous system, a systematic shift toward distal sites of transcription termination produces transcript signatures that are crucial for neuron development and function. This study reports that, in Drosophila, the highly conserved protein ELAV globally regulates all sites of neuronal 3' end processing and directly binds to proximal polyadenylation sites of target mRNAs in vivo. An endogenous strategy of functional gene rescue was uncovered that safeguards neuronal RNA signatures in an ELAV loss-of-function context. When not directly repressed by ELAV, the transcript encoding the ELAV paralog FNE acquires a mini-exon, generating a new protein able to translocate to the nucleus and rescue ELAV-mediated alternative polyadenylation and alternative splicing. It is proposed that exon-activated functional rescue is a more widespread mechanism that ensures robustness of processes regulated by a hierarchy, rather than redundancy, of effectors.
Jung, J. E., Lee, J. Y., Kim, I. R., Park, S. M., Kang, J. W., Kim, Y. H., Park, H. R. and Lee, J. H. (2020). MicroRNA-31 Regulates Expression of Wntless in Both Drosophila melanogaster and Human Oral Cancer Cells. Int J Mol Sci 21(19). PubMed ID: 33007980
Recent comparative studies have indicated distinct expression profiles of short, non-coding microRNAs (miRNAs) in various types of cancer, including oral squamous cell carcinoma (OSCC). This study employed a hybrid approach using Drosophila melanogaster as well as OSCC cell lines to validate putative targets of oral cancer-related miRNAs both in vivo and in vitro. Following overexpression of Drosophila miR-31, a significant decrease was found in the size of the imaginal wing discs and downregulation of a subset of putative targets, including wntless (wls), an important regulator of the Wnt signaling pathway. Parallel experiments performed in OSCC cells have also confirmed a similar miR-31-dependent regulation of human WLS that was not initially predicted as targets of human miR-31. Furthermore, subsequent downregulation was found of cyclin D1 and c-MYC, two of the main transcriptional targets of Wnt signaling, suggesting a potential role of miR-31 in regulating the cell cycle and proliferation of OSCC cells. Taken together, the Drosophila-based in vivo system in conjunction with the human in vitro platform will thus provide a novel insight into a mammal-to-Drosophila-to-mammal approach to validate putative targets of human miRNA and to better understand the miRNA-target relationships that play an important role in the pathophysiology of oral cancer.
Yu, X., Li, M., Cui, M., Sun, B. and Zhou, Z. (2020). Silence of yki by miR-7 regulates the Hippo pathway. Biochem Biophys Res Commun. PubMed ID: 32888651
The Hippo signaling pathway governs organ size via coordinating cell proliferation and apoptosis, and its dysregulation causes congenital diseases and cancers. The homeostasis of Hippo pathway is achieved through multiple post translational modifications. Through Drosophila genetic screening, this study found that miRNAs were also involved in Hippo pathway regulation. Overexpression of miR-7 resulted in small wings, which were neutralized by miR-7-sponge (miR-7-sp) co-expression. Mechanistically, miR-7 inhibited the expression of Hippo pathway target genes. Epistatic analyses revealed that miR-7 modulated Hippo pathway through the transcriptional cofactor Yorkie (Yki). Consistently, overexpression of miR-7 decreased Yki protein. A seed sequence of miR-7 in the yki 3'-UTR region. In addition, it was discovered that miR-7 was a transcriptional target of Yki. Thus, a negative feedback loop existed for fine tuning Hippo pathway activity. Taken together, these findings uncovered a novel mechanism by which Yki was silenced by miR-7 for Hippo pathway regulation.
Lyu, J., Chen, Y., Yang, W., Guo, T., Xu, X., Xi, Y., Yang, X. and Ge, W. (2020). The conserved microRNA miR-210 regulates lipid metabolism and photoreceptor maintenance in the Drosophila retina. Cell Death Differ. PubMed ID: 32913227
Increasing evidence suggests that miRNAs play important regulatory roles in the nervous system. However, the molecular mechanisms of how specific miRNAs affect neuronal development and functions remain less well understood. The present study provides evidence that the conserved microRNA miR-210 regulates lipid metabolism and prevents neurodegeneration in the Drosophila retina. miR-210 is specifically expressed in the photoreceptor neurons and other sensory organs. Genetic deletion of miR-210 leads to lipid droplet accumulation and photoreceptor degeneration in the retina. These effects are associated with abnormal activation of the Drosophila sterol regulatory element-binding protein signaling. The acetyl-coenzyme A synthetase (ACS) was identified as one functionally important target of miR-210 in this context. Reduction of ACS in the miR-210 mutant background suppresses the neurodegeneration defects, suggesting that miR-210 acts through regulation of the ACS transcript. Together, these results reveal an unexpected role of miR-210 in controlling lipid metabolism and neuronal functions.
Pandey, R. R., Delfino, E., Homolka, D., Roithova, A., Chen, K. M., Li, L., Franco, G., Vagbo, C. B., Taillebourg, E., Fauvarque, M. O. and Pillai, R. S. (2020). The Mammalian Cap-Specific m(6)Am RNA Methyltransferase PCIF1 Regulates Transcript Levels in Mouse Tissues. Cell Rep 32(7): 108038. PubMed ID: 32814042
The 5' end of eukaryotic mRNAs is protected by the m(7)G-cap structure. The transcription start site nucleotide is ribose methylated (Nm) in many eukaryotes, whereas an adenosine at this position is further methylated at the N(6) position (m(6)A) by the mammalian Phosphorylated C-terminal domain (CTD)-interacting Factor 1 (PCIF1 see Drosophila Pcif1) to generate m(6)Am. This study shows that although the loss of cap-specific m(6)Am in mice does not affect viability or fertility, the Pcif1 mutants display reduced body weight. Transcriptome analyses of mutant mouse tissues support a role for the cap-specific m(6)Am modification in stabilizing transcripts. In contrast, the Drosophila Pcif1 is catalytically dead, but like its mammalian counterpart, it retains the ability to associate with the Ser5-phosphorylated CTD of RNA polymerase II (RNA Pol II). Finally, this study shows that the Trypanosoma Pcif1 is an m(6)Am methylase that contributes to the N(6),N(6),2'-O-trimethyladenosine (m(6)(2)Am) in the hypermethylated cap4 structure of trypanosomatids. Thus, PCIF1 has evolved to function in catalytic and non-catalytic roles.
Seong, K. M., Coates, B. S. and Pittendrigh, B. R. (2020). Post-transcriptional modulation of cytochrome P450s, Cyp6g1 and Cyp6g2, by miR-310s cluster is associated with DDT-resistant Drosophila melanogaster strain 91-R. Sci Rep 10(1): 14394. PubMed ID: 32873850
The role of miRNAs in mediating insecticide resistance remains largely unknown, even for the model species Drosophila melanogaster. Building on prior research, this study used microinjection of synthetic miR-310s mimics into DDT-resistant 91-R flies and observed both a significant transcriptional repression of computationally-predicted endogenous target P450 detoxification genes, Cyp6g1 and Cyp6g2, and also a concomitant increase in DDT susceptibility. Additionally, co-transfection of D. melanogaster S2 cells with dual luciferase reporter constructs validated predictions that miR-310s bind to target binding sites in the 3' untranslated regions (3'-UTR) of both Cyp6g1 and Cyp6g2 in vitro. Findings in the current study provide empirical evidence for a link between reduced miRNA expression and an insecticidal resistance phenotype through reduced targeted post-transcriptional suppression of transcripts encoding proteins involved in xenobiotic detoxification. These insights are important for understanding the breadth of adaptive molecular changes that have contributed to the evolution of DDT resistance in D. melanogaster.

Tuesday, November 11th - Embryonic Development

Stern, T., Shvartsman, S. Y. and Wieschaus, E. F. (2020). Template-based mapping of dynamic motifs in tissue morphogenesis. PLoS Comput Biol 16(8): e1008049. PubMed ID: 32822341
Tissue morphogenesis relies on repeated use of dynamic behaviors at the levels of intracellular structures, individual cells, and cell groups. Rapidly accumulating live imaging datasets make it increasingly important to formalize and automate the task of mapping recurrent dynamic behaviors (motifs), as it is done in speech recognition and other data mining applications. This study presents a "template-based search" approach for accurate mapping of sub- to multi-cellular morphogenetic motifs using a time series data mining framework. The task of motif mapping was formulated as a subsequence matching problem, and it was solved using dynamic time warping, while relying on high throughput graph-theoretic algorithms for efficient exploration of the search space. This formulation allows the algorithm to accurately identify the complete duration of each instance and automatically label different stages throughout its progress, such as cell cycle phases during cell division. To illustrate this approach, cell intercalations were mapped during germband extension in the early Drosophila embryo. This framework enabled statistical analysis of intercalary cell behaviors in wild-type and mutant embryos, comparison of temporal dynamics in contracting and growing junctions in different genotypes, and the identification of a novel mode of iterative cell intercalation. This formulation of tissue morphogenesis using time series opens new avenues for systematic decomposition of tissue morphogenesis.
Chowdhary, S., Madan, S., Tomer, D., Mavrakis, M. and Rikhy, R. (2020). Mitochondrial morphology and activity regulate furrow ingression and contractile ring dynamics in Drosophila cellularization. Mol Biol Cell 31(21): 2331-2347. PubMed ID: 32755438
Mitochondria are maternally inherited in many organisms. Mitochondrial morphology and activity regulation is essential for cell survival, differentiation, and migration. An analysis of mitochondrial dynamics and function in morphogenetic events in early metazoan embryogenesis has not been carried out. This study found a crucial role of mitochondrial morphology regulation in cell formation in Drosophila embryogenesis. Mitochondria are small and fragmented and translocate apically on microtubules and distribute progressively along the cell length during cellularization. Embryos mutant for the mitochondrial fission protein, Drp1 (dynamin-related protein 1), die in embryogenesis and show an accumulation of clustered mitochondria on the basal side in cellularization. Additionally, Drp1 mutant embryos contain lower levels of reactive oxygen species (ROS). ROS depletion was previously shown to decrease myosin II activity. Drp1 loss also leads to myosin II depletion at the membrane furrow, thereby resulting in decreased cell height and larger contractile ring area in cellularization similar to that in myosin II mutants. The mitochondrial morphology and cellularization defects in Drp1 mutants are suppressed by reducing mitochondrial fusion and increasing cytoplasmic ROS in superoxide dismutase mutants. These data show a key role for mitochondrial morphology and activity in supporting the morphogenetic events that drive cellularization in Drosophila embryos.
Khadilkar, R. J., Ho, K. Y. L., Venkatesh, B. and Tanentzapf, G. (2020). Integrins Modulate Extracellular Matrix Organization to Control Cell Signaling during Hematopoiesis. Curr Biol. PubMed ID: 32649911
During hematopoiesis, progenitor cells receive and interpret a diverse array of regulatory signals from their environment. These signals control the maintenance of the progenitors and regulate the production of mature blood cells. Integrins (see Myospheroid) are well known in vertebrates for their roles in hematopoiesis, particularly in assisting in the migration to, as well as the physical attachment of, progenitors to the niche. However, whether and how integrins are also involved in the signaling mechanisms that control hematopoiesis remains to be resolved. This study shows that integrins play a key role during fly hematopoiesis in regulating cell signals that control the behavior of hematopoietic progenitors. Integrins can regulate hematopoiesis directly, via focal adhesion kinase (FAK) signaling, and indirectly, by directing extracellular matrix (ECM) assembly and/or maintenance. ECM organization and density controls blood progenitor behavior by modulating multiple signaling pathways, including bone morphogenetic protein (BMP) and Hedgehog (Hh). Furthermore, this study shows that integrins and the ECM are reduced following infection, which may assist in activating the immune response. These results provide mechanistic insight into how integrins can shape the signaling environment around hematopoietic progenitors.
Howard, A. M., Milner, H., Hupp, M., Willett, C., Palermino-Rowland, K. and Nowak, S. J. (2020). Akirin is critical for early tinman induction and subsequent formation of the heart in Drosophila melanogaster. Dev Biol. PubMed ID: 32950464
The regulation of formation of the Drosophila heart by the Nkx 2.5 homologue Tinman is a key event during embryonic development. This study identified the highly conserved transcription cofactor Akirin as a key factor in the earliest induction of tinman by the Twist transcription cofactor. akirin mutant embryos display a variety of morphological defects in the heart, including abnormal spacing between rows of aortic cells and abnormal patterning of the aortic outflow tract. akirin mutant embryos have a greatly reduced level of tinman transcripts, together with a reduction of Tinman protein in the earliest stages of cardiac patterning. Further, akirin mutants have reduced numbers of Tinman-positive cardiomyoblasts, concomitant with disrupted patterning and organization of the heart. Finally, despite the apparent formation of the heart in akirin mutants, these mutant hearts exhibit fewer coordinated contractions in akirin mutants compared with wild-type hearts. These results indicate that Akirin is crucial for the first induction of tinman by the Twist transcription factor, and that the success of the cardiac patterning program is highly dependent upon establishing the proper level of tinman at the earliest steps of the cardiac developmental pathway.
Zappia, M. P., de Castro, L., Ariss, M. M., Jefferson, H., Islam, A. B. and Frolov, M. V. (2020). A cell atlas of adult muscle precursors uncovers early events in fibre-type divergence in Drosophila. EMBO Rep: e49555. PubMed ID: 32815271
In Drosophila, the wing disc-associated muscle precursor cells give rise to the fibrillar indirect flight muscles (IFM) and the tubular direct flight muscles (DFM). To understand early transcriptional events underlying this muscle diversification, single-cell RNA-sequencing experiments were performed and a cell atlas of myoblasts associated with third instar larval wing disc was built. The analysis identified distinct transcriptional signatures for IFM and DFM myoblasts that underlie the molecular basis of their divergence. The atlas further revealed various states of differentiation of myoblasts, thus illustrating previously unappreciated spatial and temporal heterogeneity among them. Novel markers were identified and validated for both IFM and DFM myoblasts at various states of differentiation by immunofluorescence and genetic cell-tracing experiments. Finally, a systematic genetic screen was performed using a panel of markers from the reference cell atlas as an entry point and it was found that Amalgam is functionally important in muscle development. This work provides a framework for leveraging scRNA-seq for gene discovery and details a strategy that can be applied to other scRNA-seq datasets.
Hsu, J., Huang, H. T., Lee, ..., Speck, N. A. and Zon, L. I. (2020). CHD7 and Runx1 interaction provides a braking mechanism for hematopoietic differentiation. Proc Natl Acad Sci U S A. PubMed ID: 32883883
Hematopoietic stem and progenitor cell (HSPC) formation and lineage differentiation involve gene expression programs orchestrated by transcription factors and epigenetic regulators. Genetic disruption of the chromatin remodeler chromodomain-helicase-DNA-binding protein 7 (CHD7; see Drosophila Kismet) expanded phenotypic HSPCs, erythroid, and myeloid lineages in zebrafish and mouse embryos. CHD7 acts to suppress hematopoietic differentiation. Binding motifs for RUNX and other hematopoietic transcription factors are enriched at sites occupied by CHD7, and decreased RUNX1 (see Drosophila Runt) occupancy correlated with loss of CHD7 localization. CHD7 physically interacts with RUNX1 and suppresses RUNX1-induced expansion of HSPCs during development through modulation of RUNX1 activity. Consequently, the RUNX1:CHD7 axis provides proper timing and function of HSPCs as they emerge during hematopoietic development or mature in adults, representing a distinct and evolutionarily conserved control mechanism to ensure accurate hematopoietic lineage differentiation.

Monday, November 9th - Adult Physiology

Simard, C., Lebel, A., Allain, E. P., Touaibia, M., Hebert-Chatelain, E. and Pichaud, N. (2020). Metabolic Characterization and Consequences of Mitochondrial Pyruvate Carrier Deficiency in Drosophila melanogaster. Metabolites 10(9). PubMed ID: 32899962
In insect, pyruvate is generally the predominant oxidative substrate for mitochondria. This metabolite is transported inside mitochondria via the mitochondrial pyruvate carrier (MPC), but whether and how this transporter controls mitochondrial oxidative capacities in insects is still relatively unknown. This study characterized the importance of pyruvate transport as a metabolic control point for mitochondrial substrate oxidation in two genotypes of an insect model, Drosophila melanogaster, differently expressing MPC1, an essential protein for the MPC function. The kinetics were evaluated of pyruvate oxidation, mitochondrial oxygen consumption, metabolic profile, activities of metabolic enzymes, and climbing abilities of wild-type (WT) flies and flies harboring a deficiency in MPC1 (MPC1(def)). It was hypothesized that MPC1 deficiency would cause a metabolic reprogramming that would favor the oxidation of alternative substrates. The results show that the MPC1(def) flies display significantly reduced climbing capacity, pyruvate-induced oxygen consumption, and enzymatic activities of pyruvate kinase, alanine aminotransferase, and citrate synthase. Moreover, increased proline oxidation capacity was detected in MPC1(def) flies, which was associated with generally lower levels of several metabolites, and particularly those involved in amino acid catabolism such as ornithine, citrulline, and arginosuccinate. This study therefore reveals the flexibility of mitochondrial substrate oxidation allowing Drosophila to maintain cellular homeostasis.
Abdu-Allah, G. A. M., Seong, K. M., Mittapalli, O., Ojo, J. A., Sun, W., Posos-Parra, O., Mota-Sanchez, D., Clark, J. M. and Pittendrigh, B. R. (2020). Dietary antioxidants impact DDT resistance in Drosophila melanogaster. PLoS One 15(8): e0237986. PubMed ID: 32841282
Insects experience a diversity of subtoxic and/or toxic xenobiotics through exposure to pesticides and, in the case of herbivorous insects, through plant defensive compounds in their diets. Many insects are also concurrently exposed to antioxidants in their diets. The impact of dietary antioxidants on the toxicity of xenobiotics in insects is not well understood, in part due to the challenge of developing appropriate systems in which doses and exposure times (of both the antioxidants and the xenobiotics) can be controlled and outcomes can be easily measured. However, in Drosophila melanogaster, a well-established insect model system, both dietary factors and pesticide exposure can be easily controlled. Additionally, the mode of action and xenobiotic metabolism of dichlorodiphenyltrichloroethane (DDT), a highly persistent neurotoxic organochlorine insecticide that is detected widely in the environment, have been well studied in DDT-susceptible and -resistant strains. Using a glass-vial bioassay system with blue diet as the food source, seven compounds with known antioxidant effects (ascorbic acid, β-carotene, glutathione, α-lipoic acid, melatonin, minocycline, and serotonin) were orally tested for their impact on DDT toxicity across three strains of D. melanogaster: one highly susceptible to DDT (Canton-S), one mildly susceptible (91-C), and one highly resistant (91-R). Three of the antioxidants (serotonin, ascorbic acid, and β-carotene) significantly impacted the toxicity of DDT in one or more strains. Fly strain and gender, antioxidant type, and antioxidant dose all affected the relative toxicity of DDT. This work demonstrates that dietary antioxidants can potentially alter the toxicity of a xenobiotic in an insect population.
Rezende, E. L., Bozinovic, F., Szilagyi, A. and Santos, M. (2020). Predicting temperature mortality and selection in natural Drosophila populations. Science 369(6508): 1242-1245. PubMed ID: 32883867
Average and extreme temperatures will increase in the near future, but how such shifts will affect mortality in natural populations is still unclear. This study used a dynamic model to predict mortality under variable temperatures on the basis of heat tolerance laboratory measurements. Theoretical lethal temperatures for 11 Drosophila species under different warming conditions were virtually indistinguishable from empirical results. For Drosophila in the field, daily mortality predicted from ambient temperature records accumulate over weeks or months, consistent with observed seasonal fluctuations and population collapse in nature. This model quantifies temperature-induced mortality in nature, which is crucial to study the effects of global warming on natural populations, and analyses highlight that critical temperatures are unreliable predictors of mortality.
Carvalho-Santos, Z., Cardoso-Figueiredo, R., Elias, A. P., Tastekin, I., Baltazar, C. and Ribeiro, C. (2020). Cellular metabolic reprogramming controls sugar appetite in Drosophila. Nat Metab 2(9): 958-973. PubMed ID: 32868922
Cellular metabolic reprogramming is an important mechanism by which cells rewire their metabolism to promote proliferation and cell growth. This process has been mostly studied in the context of tumorigenesis, but less is known about its relevance for nonpathological processes and how it affects whole-animal physiology. This study shows that metabolic reprogramming in Drosophila female germline cells affects nutrient preferences of animals. Egg production depends on the upregulation of the activity of the pentose phosphate pathway in the germline, which also specifically increases the animal's appetite for sugar, the key nutrient fuelling this metabolic pathway. Functional evidence is provided that the germline alters sugar appetite by regulating the expression of the fat-body-secreted satiety factor Fit. These findings demonstrate that the cellular metabolic program of a small set of cells is able to increase the animal's preference for specific nutrients through inter-organ communication to promote specific metabolic and cellular outcomes.
Zugasti, O., Tavignot, R. and Royet, J. (2020). Gut bacteria-derived peptidoglycan induces a metabolic syndrome-like phenotype via NF-kappaB-dependent insulin/PI3K signaling reduction in Drosophila renal system. Sci Rep 10(1): 14097. PubMed ID: 32839462
Although microbiome-host interactions are usual at steady state, gut microbiota dysbiosis can unbalance the physiological and behavioral parameters of the host, mostly via yet not understood mechanisms. Using the Drosophila model, this study investigated the consequences of a gut chronic dysbiosis on the host physiology. The results show that adult flies chronically infected with the non-pathogenic Erwinia carotorova caotovora bacteria displayed organ degeneration resembling wasting-like phenotypes reminiscent of Metabolic Syndrome associated pathologies. Genetic manipulations demonstrate that a local reduction of insulin signaling consecutive to a peptidoglycan-dependent NF-κB (Relish) activation in the excretory system of the flies is responsible for several of the observed phenotypes. This work establishes a functional crosstalk between bacteria-derived peptidoglycan and the immune NF-κB cascade that contributes to the onset of metabolic disorders by reducing insulin signal transduction. Giving the high degree of evolutionary conservation of the mechanisms and pathways involved, this study is likely to provide a helpful model to elucidate the contribution of altered intestinal microbiota in triggering human chronic kidney diseases.
Wang, Y., Farine, J. P., Yang, Y., Yang, J., Tang, W., Gehring, N., Ferveur, J. F. and Moussian, B. (2020). Transcriptional Control of Quality Differences in the Lipid-Based Cuticle Barrier in Drosophila suzukii and Drosophila melanogaster. Front Genet 11: 887. PubMed ID: 32849846
Cuticle barrier efficiency in insects depends largely on cuticular lipids. This study compared the basic properties of the cuticle inward and outward barrier function in adults of Drosophila suzukii (Ds) and Drosophila melanogaster (Dm) that live on fruits sharing a similar habitat. At low air humidity, Ds flies desiccate faster than Dm flies. In this respect males are less robust than females in both species. Xenobiotics penetration occurs at lower temperatures in Ds than in Dm. Likewise, Ds flies are more susceptible to contact insecticides than Dm flies. Thus, both the inward and outward barriers of Ds are less efficient. Consistently, Ds flies have less cuticular hydrocarbons (CHC) that participate as key components of the cuticle barrier. Especially, the relative amounts of branched and desaturated CHCs, known to enhance desiccation resistance, show reduced levels in Ds. Moreover, the expression of snustorr (snu) that encodes an ABC transporter involved in barrier construction and CHC externalization is strongly suppressed in Ds. Hence, species-specific genetic programs regulate the quality of the lipid-based cuticle barrier in these two Drosophilae. Together, it is concluded that the weaker inward and outward barriers of Ds may be partly explained by differences in CHC composition and by a reduced Snu-dependent transport rate of CHCs to the surface. In turn, this suggests that snu is an ecologically adjustable and therefore relevant gene in cuticle barrier efficiency.

Friday, November 6th - Evolution

Cartwright, E. L. and Lott, S. E. (2020). Evolved Differences in cis and trans Regulation Between the Maternal and Zygotic mRNA Complements in the Drosophila Embryo. Genetics. PubMed ID: 32928902
How gene expression can evolve depends on the mechanisms driving gene expression. Gene expression is controlled in different ways in different developmental stages; this study asked whether different developmental stages show different patterns of regulatory evolution. To explore the mode of regulatory evolution, this study used the early stages of embryonic development controlled by two different genomes, that of the mother and that of the zygote. During embryogenesis in all animals, initial developmental processes are driven entirely by maternally provided gene products deposited into the oocyte. The zygotic genome is activated later, when developmental control is handed off from maternal gene products to the zygote during the maternal-to-zygotic transition. Using hybrid crosses between sister species of Drosophila (D. simulans, D. sechellia, and D. mauritiana) and transcriptomics, this study finds that the regulation of maternal transcript deposition and zygotic transcription evolve through different mechanisms. Patterns of transcript level inheritance in hybrids, relative to parental species, were found to differ between maternal and zygotic transcripts, and maternal transcript levels are more likely to be conserved. Changes in transcript levels occur predominantly through differences in trans regulation for maternal genes, while changes in zygotic transcription occur through a combination of both cis and trans regulatory changes. Differences in the underlying regulatory landscape in the mother and the zygote are likely the primary determinants for how maternal and zygotic transcripts evolve.
Dowle, E. J., Powell, T. H. Q., Doellman, M. M., Meyers, P. J., Calvert, M. B., Walden, K. K. O., Robertson, H. M., Berlocher, S. H., Feder, J. L., Hahn, D. A. and Ragland, G. J. (2020). Genome-wide variation and transcriptional changes in diverse developmental processes underlie the rapid evolution of seasonal adaptation. Proc Natl Acad Sci U S A 117(38): 23960-23969. PubMed ID: 32900926
Many organisms enter a dormant state in their life cycle to deal with predictable changes in environments over the course of a year. The timing of dormancy is therefore a key seasonal adaptation, and it evolves rapidly with changing environments. The hypothesis that differences in the timing of seasonal activity are driven by differences in the rate of development during diapause was tested in Rhagoletis pomonella, a fly specialized to feed on fruits of seasonally limited host plants. Transcriptomes from the central nervous system across a time series during diapause show consistent and progressive changes in transcripts participating in diverse developmental processes, despite a lack of gross morphological change. Moreover, population genomic analyses suggested that many genes of small effect enriched in developmental functional categories underlie variation in dormancy timing and overlap with gene sets associated with development rate in Drosophila melanogaster. These transcriptional data also suggested that a recent evolutionary shift from a seasonally late to a seasonally early host plant drove more rapid development during diapause in the early fly population. Moreover, genetic variants that diverged during the evolutionary shift were also enriched in putative cis regulatory regions of genes differentially expressed during diapause development. Overall, these data suggest polygenic variation in the rate of developmental progression during diapause contributes to the evolution of seasonality in R. pomonella. Patterns that suggest hourglass-like developmental divergence early and late in diapause development are discussed along with an important role for hub genes in the evolution of transcriptional divergence.
de Lima, L. G., Hanlon, S. L. and Gerton, J. L. (2020). Origins and Evolutionary Patterns of the 1.688 Satellite DNA Family in Drosophila Phylogeny. G3 (Bethesda). PubMed ID: 32934018
Satellite DNAs (satDNAs) are a ubiquitous feature of eukaryotic genomes and are usually the major components of constitutive heterochromatin. The 1.688 satDNA, also known as the 359 bp satellite, is one of the most abundant repetitive sequences in Drosophila melanogaster and has been linked to several different biological functions. This study investigated the presence and evolution of the 1.688 satDNA in 16 Drosophila genomes. The 1.688 satDNA family was found to be much more ancient than previously appreciated, being shared amongst part of the melanogaster group that diverged from a common ancestor ∼27 Mya. The 1.688 satDNA family has two major subfamilies spread throughout Drosophila phylogeny (∼360 bp and ∼190 bp). Phylogenetic analysis of ∼10,000 repeats extracted from 14 of the species revealed that the 1.688 satDNA family is present within heterochromatin and euchromatin. A high number of euchromatic repeats are gene proximal, suggesting the potential for local gene regulation. Notably, heterochromatic copies display concerted evolution and a species-specific pattern, whereas euchromatic repeats display a more typical evolutionary pattern, suggesting that chromatin domains may influence the evolution of these sequences. Overall, these data indicate the 1.688 satDNA as the most perduring satDNA family described in Drosophila phylogeny to date. This study provides a strong foundation for future work on the functional roles of 1.688 satDNA across many Drosophila species.
Fitzpatrick, J. L., Bridge, C. D. and Snook, R. R. (2020). Repeated evidence that the accelerated evolution of sperm is associated with their fertilization function. Proc Biol Sci 287(1932): 20201286. PubMed ID: 32752988
Spermatozoa are the most morphologically diverse cell type, leading to the widespread assumption that they evolve rapidly. However, there is no direct evidence that sperm evolve faster than other male traits. Such a test requires comparing male traits that operate in the same selective environment, ideally produced from the same tissue, yet vary in function. This study examined rates of phenotypic evolution in sperm morphology using two insect groups where males produce fertile and non-fertile sperm types (Drosophila species from the obscura group and a subset of Lepidoptera species), where these constraints are solved. Moreover, in Drosophila, the relationship between rates of sperm evolution and the link with the putative selective pressures of fertilization function and postcopulatory sexual selection exerted by female reproductive organs were tested. Repeated evolutionary patterns were found across these insect groups-lengths of fertile sperm evolve faster than non-fertile sperm. In Drosophila, fertile sperm length evolved faster than body size, but at the same rate as female reproductive organ length. Rates of evolution of different sperm components were also compared, showing that head length evolves faster in fertile sperm while flagellum length evolves faster in non-fertile sperm. This study provides direct evidence that sperm length evolves more rapidly in fertile sperm, probably because of their functional role in securing male fertility and in response to selection imposed by female reproductive organs.
Maselko, M., Feltman, N., Upadhyay, A., Hayward, A., Das, S., Myslicki, N., Peterson, A. J., O'Connor, M. B. and Smanski, M. J. (2020). Engineering multiple species-like genetic incompatibilities in insects. Nat Commun 11(1): 4468. PubMed ID: 32901021
Speciation constrains the flow of genetic information between populations of sexually reproducing organisms. Gaining control over mechanisms of speciation would enable new strategies to manage wild populations of disease vectors, agricultural pests, and invasive species. Additionally, such control would provide safe biocontainment of transgenes and gene drives. This study demonstrates a general approach to create engineered genetic incompatibilities (EGIs) in the model insect Drosophila melanogaster. EGI couples a dominant lethal transgene with a recessive resistance allele. Strains homozygous for both elements are fertile and fecund when they mate with similarly engineered strains, but incompatible with wild-type strains that lack resistant alleles. EGI genotypes can also be tuned to cause hybrid lethality at different developmental life-stages. Further, it was demonstrated that multiple orthogonal EGI strains of D. melanogaster can be engineered to be mutually incompatible with wild-type and with each other. EGI is a simple and robust approach in multiple sexually reproducing organisms.
Cheatle Jarvela, A. M., Trelstad, C. S. and Pick, L. (2020). Regulatory gene function handoff allows essential gene loss in mosquitoes. Commun Biol 3(1): 540. PubMed ID: 32999445
Regulatory genes are often multifunctional and constrained, which results in evolutionary conservation. It is difficult to understand how a regulatory gene could be lost from one species' genome when it is essential for viability in closely related species. The gene paired is a classic Drosophila pair-rule gene, required for formation of alternate body segments in diverse insect species. Surprisingly, paired was lost in mosquitoes without disrupting body patterning. This study demonstrates that a paired family member, gooseberry, has acquired paired-like expression in the malaria mosquito Anopheles stephensi. Anopheles-gooseberry CRISPR-Cas9 knock-out mutants display pair-rule phenotypes and alteration of target gene expression similar to what is seen in Drosophila and beetle paired mutants. Thus, paired was functionally replaced by the related gene, gooseberry, in mosquitoes. These findings document a rare example of a functional replacement of an essential regulatory gene and provide a mechanistic explanation of how such loss can occur.

Thursday, November 5th - Cytoskeleton

Vaziri, P., Ryan, D., Johnston, C. A. and Cripps, R. M. (2020). A Novel Mechanism for Activation of Myosin Regulatory Light Chain by Protein Kinase C-Delta in Drosophila. Genetics 216(1): 177-190. PubMed ID: 32753389
Myosin is an essential motor protein, which in muscle is comprised of two molecules each of myosin heavy-chain (MHC), the essential or alkali myosin light-chain 1 (MLC1), and the regulatory myosin light-chain 2 (MLC2). It has been shown previously that MLC2 phosphorylation at two canonical serine residues is essential for proper flight muscle function in Drosophila; however, MLC2 is also phosphorylated at additional residues for which the mechanism and functional significance is not known. This study found that a hypomorphic allele of Pkcδ causes a flightless phenotype; therefore, it was hypothesized that PKCδ phosphorylates MLC2. Flight disability was rescued by duplication of the wild-type Pkcδ gene. Moreover, MLC2 is hypophosphorylated in Pkcδ mutant flies, but it is phosphorylated in rescued animals. Myosin isolated from Pkcδ mutant flies shows a reduced actin-activated ATPase activity, and MLC2 in these myosin preparations can be phosphorylated directly by recombinant human PKCδ. The flightless phenotype is characterized by a shortened and disorganized sarcomere phenotype that becomes apparent following eclosion. It is concluded that MLC2 is a direct target of phosphorylation by PKCδ, and that this modification is necessary for flight muscle maturation and function.
Wittek, A., Hollmann, M., Schleutker, R. and Luschnig, S. (2020). The Transmembrane Proteins M6 and Anakonda Cooperate to Initiate Tricellular Junction Assembly in Epithelia of Drosophila. Curr Biol. PubMed ID: 32857972
Cell vertices in epithelia comprise specialized tricellular tricellular junctions (TCJs) that seal the paracellular space between three adjoining cells. Although TCJs play fundamental roles in tissue homeostasis, pathogen defense, and in sensing tension and cell shape, how they are assembled, maintained, and remodeled is poorly understood. In Drosophila, the transmembrane proteins Anakonda (Aka) and Gliotactin (Gli) are TCJ components essential for epithelial barrier formation. Additionally, the conserved four-transmembrane-domain protein M6, the only myelin proteolipid protein (PLP) family member in Drosophila, localizes to TCJs. PLPs associate with cholesterol-rich membrane domains and induce filopodia formation and membrane curvature, and Drosophila M6 acts as a tumor suppressor, but its role in TCJ formation remained unknown. This study shows that M6 is essential for the assembly of tricellular, but not bicellular, occluding junctions, and for barrier function in embryonic epithelia. M6 and Aka localize to TCJs in a mutually dependent manner and are jointly required for TCJ localization of Gli, whereas Aka and M6 localize to TCJs independently of Gli. Aka acts instructively and is sufficient to direct M6 to cell vertices in the absence of septate junctions, while M6 is required permissively to maintain Aka at TCJs. Furthermore, M6 and Aka are mutually dependent for their accumulation in a low-mobility pool at TCJs. These findings suggest a hierarchical model for TCJ assembly, where Aka and M6 promote TCJ formation through synergistic interactions that demarcate a distinct plasma membrane microdomain at cell vertices.
Nematbakhsh, A., Levis, M., Kumar, N., Chen, W., Zartman, J. J. and Alber, M. (2020). Epithelial organ shape is generated by patterned actomyosin contractility and maintained by the extracellular matrix. PLoS Comput Biol 16(8): e1008105. PubMed ID: 32817654
Epithelial sheets define organ architecture during development. This study employed an iterative multiscale computational modeling and quantitative experimental approach to decouple direct and indirect effects of actomyosin-generated forces, nuclear positioning, extracellular matrix, and cell-cell adhesion in shaping Drosophila wing imaginal discs. Basally generated actomyosin forces generate epithelial bending of the wing disc pouch. Surprisingly, acute pharmacological inhibition of ROCK-driven actomyosin contractility does not impact the maintenance of tissue height or curved shape. Computational simulations show that ECM tautness provides only a minor contribution to modulating tissue shape. Instead, passive ECM pre-strain serves to maintain the shape independent from actomyosin contractility. These results provide general insight into how the subcellular forces are generated and maintained within individual cells to induce tissue curvature. Thus, the results suggest an important design principle of separable contributions from ECM prestrain and actomyosin tension during epithelial organogenesis and homeostasis.
Pollitt, S. L., Myers, K. R., Yoo, J. and Zheng, J. Q. (2020). LIM and SH3 Protein 1 Localizes to the Leading Edge of Protruding Lamellipodia and Regulates Axon Development. Mol Biol Cell: mbcE20060366. PubMed ID: 32997597
The actin cytoskeleton drives cell motility and is essential for neuronal development and function. LIM and SH3 Protein 1 (LASP1) is a unique actin-binding protein that is expressed in a wide range of cells including neurons, but its roles in cellular motility and neuronal development are not well understood. LASP1 is expressed in rat hippocampus early in development, and this expression is maintained through adulthood. High-resolution imaging reveals that LASP1 is selectively concentrated at the leading edge of lamellipodia in migrating cells and axonal growth cones. This local enrichment of LASP1 is dynamically associated with the protrusive activity of lamellipodia, depends on the barbed ends of actin filaments, and requires both the LIM domain and nebulin repeats of LASP1. Knockdown of LASP1 in cultured rat hippocampal neurons results in a substantial reduction in axonal outgrowth and arborization. Finally, loss of the Drosophila homolog Lasp from a subset of commissural neurons in the developing ventral nerve cord produces defasciculated axon bundles that do not reach their targets. Together, these data support a novel role for LASP1 in actin-based lamellipodial protrusion and establish LASP1 as a positive regulator of both in vitro and in vivo axon development.
Sysoev, V. O., Kato, M., Sutherland, L., Hu, R., McKnight, S. L. and Murray, D. T. (2020). Dynamic structural order of a low-complexity domain facilitates assembly of intermediate filaments. Proc Natl Acad Sci U S A 117(38): 23510-23518. PubMed ID: 32907935
The coiled-coil domains of intermediate filament (IF) proteins are flanked by regions of low sequence complexity. Whereas IF coiled-coil domains assume dimeric and tetrameric conformations on their own, maturation of eight tetramers into cylindrical IFs is dependent on either "head" or "tail" domains of low sequence complexity. This study confirms that the tail domain required for assembly of Drosophila Tropomyosin 1 (Tm1-I/C) IFs functions by forming labile cross-β interactions. These interactions are seen in polymers made from the tail domain alone, as well as in assembled IFs formed by the intact Tm1-I/C protein. The ability to visualize such interactions in situ within the context of a discrete cellular assembly lends support to the concept that equivalent interactions may be used in organizing other dynamic aspects of cell morphology.
Deng, S., Silimon, R. L., Balakrishnan, M., Bothe, I., Juros, D., Soffar, D. B. and Baylies, M. K. (2020). The actin polymerization factor diaphanous and the actin severing protein flightless I collaborate to regulate sarcomere size. Dev Biol. PubMed ID: 32980309
The sarcomere is the basic contractile unit of muscle, composed of repeated sets of actin thin filaments and myosin thick filaments. During muscle development, sarcomeres grow in size to accommodate the growth and function of muscle fibers. Failure in regulating sarcomere size results in muscle dysfunction; yet, it is unclear how the size and uniformity of sarcomeres are controlled. This study shows that the formin Diaphanous is critical for the growth and maintenance of sarcomere size: Dia sets sarcomere length and width through regulation of the number and length of the actin thin filaments in the Drosophila flight muscle. To regulate thin filament length and sarcomere size, Dia interacts with the Gelsolin superfamily member Flightless I (FliI). It is suggested that, through controlling actin dynamics and turnover, that these actin regulators generate uniformly sized sarcomeres tuned for the muscle contractions required for flight.

Wednesday, November 4th - Signaling

Vuong, L. T., Won, J. H., Nguyen, M. B. and Choi, K. W. (2020). Role of Armadillo repeat 2 and kinesin-II motor subunit Klp64D for wingless signaling in Drosophila. Sci Rep 10(1): 13864. PubMed ID: 32807823
Armadillo (Arm) is crucial for transducing Wingless (Wg) signaling. Previous work has shown that Klp64D, a motor subunit of Drosophila kinesin-II, interacts with Arm for Wg signaling. Molecular basis for this interaction has remained unknown. This study has identified a critical Arm repeat (AR) required for binding Klp64D and Wg signaling. Arm/β-catenin family proteins contain a conserved domain of 12 Arm repeats (ARs). Five of these ARs can interact with Klp64D, but only the second AR (AR2) binds to the cargo/tail domain of Klp64D. Overexpression of AR2 in wing imaginal disc is sufficient to cause notched wing margin. This phenotype by AR2 is enhanced or suppressed by reducing or increasing Klp64D expression, respectively. AR2 overexpression inhibits Wg signaling activity in TopFlash assay, consistent with its dominant-negative effects on Klp64D-dependent Wg signaling. Overexpression of the Klp64D cargo domain also results in dominant-negative wing notching. Genetic rescue data indicate that both AR2 and Klp64D cargo regions are required for the function of Arm and Klp64D, respectively. AR2 overexpression leads to an accumulation of Arm with GM130 Golgi marker in Klp64D knockdown. This study suggests that Wg signaling for wing development is regulated by specific interaction between AR2 and the cargo domain of Klp64D.
Zhang, M., Nagaosa, K., Nakai, Y., Yasugi, T., Kushihiki, M., Rahmatika, D., Sato, M., Shiratsuchi, A. and Nakanishi, Y. (2020). Role for phagocytosis in the prevention of neoplastic transformation in Drosophila. Genes Cells. PubMed ID: 32865275
Immunity is considered to be involved in the prevention of cancer. Although both humoral and cellular immune reactions may participate, underlying mechanisms have yet to be clarified. The present study was conducted to clarify this issue using a Drosophila model, in which neoplastic transformation was induced through the simultaneous inhibition of cell-cycle checkpoints and apoptosis. First, the location was determined of hemocytes, blood cells of Drosophila playing a role of immune cells, in neoplasia-induced and normal larvae, but there was no significant difference between the two groups. When gene expression pattern in larval hemocytes was determined, the expression of immunity-related genes including those necessary for phagocytosis was reduced in the neoplasia model. Then the involvement of phagocytosis was determined in the prevention of neoplasia, examining animals where the expression of engulfment receptors (Draper and integrin αPS3/βν) instead of apoptosis was retarded. It was found that the inhibition of engulfment receptor expression augmented the occurrence of neoplasia induced by a defect in cell-cycle checkpoints. This suggested a role for phagocytosis in the prevention of neoplastic transformation in Drosophila.
Yang, S., Zhang, Y., Ting, C. Y., Bettedi, L., Kim, K., Ghaniam, E. and Lilly, M. A. (2020). The Rag GTPase Regulates the Dynamic Behavior of TSC Downstream of Both Amino Acid and Growth Factor Restriction. Dev Cell. PubMed ID: 32898476
The dysregulation of the metabolic regulator TOR complex I (TORC1) contributes to a wide array of human pathologies. Tuberous sclerosis complex (TSC) is a potent inhibitor of TORC1. This study demonstrates that the Rag GTPase acts in both the amino-acid-sensing and growth factor signaling pathways to control TORC1 activity through the regulation of TSC dynamics in HeLa cells and Drosophila. TSC lysosomal-cytosolic exchange increases in response to both amino acid and growth factor restriction. Moreover, the rate of exchange mirrors TSC function, with depletions of the Rag GTPase blocking TSC lysosomal mobility and rescuing TORC1 activity. Finally, this study shows that the GATOR2 complex controls the phosphorylation of TSC2, which is essential for TSC exchange. These data support the model that the amino acid and growth factor signaling pathways converge on the Rag GTPase to inhibit TORC1 activity through the regulation of TSC dynamics.
Zipper, L., Jassmann, D., Burgmer, S., Gorlich, B. and Reiff, T. (2020). Ecdysone steroid hormone remote controls intestinal stem cell fate decisions via the PPARγ-homolog Eip75B in Drosophila. Elife 9. PubMed ID: 32773037
Developmental studies revealed fundamental principles on how organ size and function is achieved, but less is known about organ adaptation to new physiological demands. In fruit flies, juvenile hormone (JH) induces intestinal stem cell (ISC) driven absorptive epithelial expansion balancing energy uptake with increased energy demands of pregnancy. This study showa 20-Hydroxy-Ecdysone (20HE)-signaling controlling organ homeostasis with physiological and pathological implications. Upon mating, 20HE titer in ovaries and hemolymph are increased and act on nearby midgut progenitors inducing Ecdysone-induced-protein-75B (Eip75B). Strikingly, the PPARγ-homologue Eip75B drives ISC daughter cells towards absorptive enterocyte lineage ensuring epithelial growth. This is the first time a systemic hormone is shown to direct local stem cell fate decisions. Given the protective, but mechanistically unclear role of steroid hormones in female colorectal cancer patients, these findings suggest a tumor-suppressive role for steroidal signaling by promoting postmitotic fate when local signaling is deteriorated.
Yu, J. J. S., Maugarny-Cales, A., Pelletier, S., Alexandre, C., Bellaiche, Y., Vincent, J. P. and McGough, I. J. (2020). Frizzled-Dependent Planar Cell Polarity without Secreted Wnt Ligands. Dev Cell 54(5): 583-592.e585. PubMed ID: 32888416
Planar cell polarity (PCP) organizes the orientation of cellular protrusions and migratory activity within the tissue plane. PCP establishment involves the subcellular polarization of core PCP components. It has been suggested that Wnt gradients could provide a global cue that coordinates local PCP with tissue axes. This study dissected the role of Wnt ligands in the orientation of hairs of Drosophila wings, an established system for the study of PCP. PCP was normal in quintuple mutant wings that rely solely on the membrane-tethered Wingless for Wnt signaling, suggesting that a Wnt gradient is not required. A nanobody-based approach to was used to trap Wntless in the endoplasmic reticulum, and hence prevent all Wnt secretion, specifically during the period of PCP establishment. PCP was still established. It is concluded that, even though Wnt ligands could contribute to PCP, they are not essential, and another global cue must exist for tissue-wide polarization.
De Jamblinne, C. V., Decelle, B., Dehghani, M., Joseph, M., Sriskandarajah, N., Leguay, K., Rambaud, B., Lemieux, S., Roux, P. P., Hipfner, D. R. and Carreno, S. (2020). STRIPAK regulates Slik localization to control mitotic morphogenesis and epithelial integrity. J Cell Biol 219(11). PubMed ID: 32960945
Proteins of the ezrin, radixin, and moesin (ERM) family control cell and tissue morphogenesis. A previous study reported that moesin, the only ERM in Drosophila, controls mitotic morphogenesis and epithelial integrity. This study also found that the Pp1-87B phosphatase dephosphorylates moesin, counteracting its activation by the Ste20-like kinase Slik. To understand how this signaling pathway is itself regulated, a genome-wide RNAi screen was conducted, looking for new regulators of moesin activity. Slik was identified as a new member of the striatin-interacting phosphatase and kinase complex (STRIPAK). The phosphatase activity of STRIPAK reduces Slik phosphorylation to promote its cortical association and proper activation of moesin. Consistent with this finding, inhibition of STRIPAK phosphatase activity causes cell morphology defects in mitosis and impairs epithelial tissue integrity. These results implicate the Slik-STRIPAK complex in the control of multiple morphogenetic processes.

3 November 2020 - Adult Neural Function

Dannhauser, S., Lux, T. J., Hu, C., Selcho, M., Chen, J. T., Ehmann, N., Sachidanandan, D., Stopp, S., Pauls, D., Pawlak, M., Langenhan, T., Soba, P., Rittner, H. L. and Kittel, R. J. (2020). Antinociceptive modulation by the adhesion GPCR CIRL promotes mechanosensory signal discrimination. Elife 9. PubMed ID: 32996461
Adhesion-type GPCRs (aGPCRs) participate in a vast range of physiological processes. Their frequent association with mechanosensitive functions suggests that processing of mechanical stimuli may be a common feature of this receptor family. Previous studies reported that the Drosophila aGPCR CIRL sensitizes sensory responses to gentle touch and sound by amplifying signal transduction in low-threshold mechanoreceptors. This study shows that Cirl is also expressed in high-threshold mechanical nociceptors where it adjusts nocifensive behaviour under physiological and pathological conditions. Optogenetic in vivo experiments indicate that CIRL lowers cAMP levels in both mechanosensory submodalities. However, contrasting its role in touch-sensitive neurons, CIRL dampens the response of nociceptors to mechanical stimulation. Consistent with this finding, rat nociceptors display decreased Cirl1 expression during allodynia. Thus, cAMP-downregulation by CIRL exerts opposing effects on low-threshold mechanosensors and high-threshold nociceptors. This intriguing bipolar action facilitates the separation of mechanosensory signals carrying different physiological information.
Brent, A. E. and Rajan, A. (2020). Insulin and Leptin/Upd2 Exert Opposing Influences on Synapse Number in Fat-Sensing Neurons. Cell Metab. PubMed ID: 32976758
Energy-sensing neural circuits decide to expend or conserve resources based, in part, on the tonic, steady-state, energy-store information they receive. Tonic signals, in the form of adipose tissue-derived adipokines, set the baseline level of activity in the energy-sensing neurons, thereby providing context for interpretation of additional inputs. However, the mechanism by which tonic adipokine information establishes steady-state neuronal function has heretofore been unclear. This study shows that under conditions of nutrient surplus, Upd2, a Drosophila leptin ortholog, regulates actin-based synapse reorganization to reduce bouton number in an inhibitory circuit, thus establishing a neural tone that is permissive for insulin release. Unexpectedly, this study found that insulin feeds back on these same inhibitory neurons to conversely increase bouton number, resulting in maintenance of negative tone. These results point to a mechanism by which two surplus-sensing hormonal systems, Upd2/leptin and insulin, converge on a neuronal circuit with opposing outcomes to establish energy-store-dependent neuron activity.
Versteven, M., Ernst, K. M. and Stanewsky, R. (2020). A Robust and Self-Sustained Peripheral Circadian Oscillator Reveals Differences in Temperature Compensation Properties with Central Brain Clocks. iScience 23(8): 101388. PubMed ID: 32798967
Circadian clocks are characterized by three properties: they run in constant conditions with a period of ∼24 h, synchronize to the environmental cycles of light and temperature, and are temperature compensated, meaning they do not speed up with temperature. Central brain clocks regulate daily activity rhythms, whereas peripheral clocks are dispersed throughout the body of insects and vertebrates. Using a set of luciferase reporter genes, this study shows that Drosophila peripheral clocks are self-sustained but over-compensated, i.e., they slow down with increasing temperature. In contrast, central clock neurons in the fly brain, both in intact flies and in cultured brains, show accurate temperature compensation. Although this suggests that neural network properties contribute to temperature compensation, the circadian neuropeptide Pigment Dispersing Factor (PDF) is not required for temperature-compensated oscillations in brain clock neurons. These findings reveal a fundamental difference between central and peripheral clocks, which likely also applies for vertebrate clocks.
Wang, P., Jia, Y., Liu, T., Jan, Y. N. and Zhang, W. (2020). Visceral Mechano-sensing Neurons Control Drosophila Feeding by Using Piezo as a Sensor. Neuron. PubMed ID: 32910893
Animal feeding is controlled by external sensory cues and internal metabolic states. Does it also depend on enteric neurons that sense mechanical cues to signal fullness of the digestive tract? This study identified a group of piezo-expressing neurons innervating the Drosophila crop (the fly equivalent of the stomach) that monitor crop volume to avoid food overconsumption. These neurons reside in the pars intercerebralis (PI), a neuro-secretory center in the brain involved in homeostatic control, and express insulin-like peptides with well-established roles in regulating food intake and metabolism. Piezo knockdown in these neurons of wild-type flies phenocopies the food overconsumption phenotype of piezo-null mutant flies. Conversely, expression of either fly Piezo or mammalian Piezo1 in these neurons of piezo-null mutants suppresses the overconsumption phenotype. Importantly, Piezo(+) neurons at the PI are activated directly by crop distension, thus conveying a rapid satiety signal along the "brain-gut axis" to control feeding.
Davla, S., Artiushin, G., Li, Y., Chitsaz, D., Li, S., Sehgal, A. and van Meyel, D. J. (2020). AANAT1 functions in astrocytes to regulate sleep homeostasis. Elife 9. PubMed ID: 32955431
How the brain controls the need and acquisition of recovery sleep after prolonged wakefulness is an important issue in sleep research. The monoamines serotonin and dopamine are key regulators of sleep in mammals and in Drosophila. This study found that the enzyme arylalkylamine N-acetyltransferase 1 (AANAT1) is expressed by Drosophila astrocytes and specific subsets of neurons in the adult brain. AANAT1 acetylates monoamines and inactivates them, and it was found that AANAT1 limited the accumulation of serotonin and dopamine in the brain upon sleep deprivation. Loss of AANAT1 from astrocytes, but not from neurons, caused flies to increase their daytime recovery sleep following overnight sleep deprivation. Together, these findings demonstrate a crucial role for AANAT1 and astrocytes in the regulation of monoamine bioavailability and homeostatic sleep.
Amin, H., Apostolopoulou, A. A., Suarez-Grimalt, R., Vrontou, E. and Lin, A. C. (2020). Localized inhibition in the Drosophila mushroom body. Elife 9. PubMed ID: 32955437
Many neurons show compartmentalized activity, in which activity does not spread readily across the cell, allowing input and output to occur locally. However, the functional implications of compartmentalized activity for the wider neural circuit are often unclear. This problem was addressed in the Drosophila mushroom body, whose principal neurons, Kenyon cells, receive feedback inhibition from a non-spiking interneuron called APL. This study used local stimulation and volumetric calcium imaging to show that APL inhibits Kenyon cells' dendrites and axons, and that both activity in APL and APL's inhibitory effect on Kenyon cells are spatially localized (the latter somewhat less so), allowing APL to differentially inhibit different mushroom body compartments. Applying these results to the Drosophila hemibrain connectome predicts that individual Kenyon cells inhibit themselves via APL more strongly than they inhibit other individual Kenyon cells. These findings reveal how cellular physiology and detailed network anatomy can combine to influence circuit function.

Monday, September 2nd - Enhancers and gene regulation

Ford, D. J., Zraly, C. B., Perez, J. H. and Dingwall, A. K. (2020). The Drosophila MLR COMPASS-like complex regulates bantam miRNA expression differentially in the context of cell fate. Dev Biol 468(1-2): 41-53. PubMed ID: 32946789
The conserved MLR COMPASS-like complexes (Complex of Proteins Associated with Set1) are histone modifiers that are recruited by a variety of transcription factors to enhancer regions where they act as necessary epigenetic tools for enhancer establishment and function. A critical in vivo target of the Drosophila MLR complex is the bantam miRNA that regulates cell survival and functions in feedback regulation of cellular signaling pathways during development. Loss of Drosophila MLR complex function in developing wing and eye imaginal discs results in growth and patterning defects that are sensitive to bantam levels. Consistent with an essential regulatory role in modulating bantam transcription, the MLR complex binds to tissue-specific bantam enhancers and contributes to fine-tuning expression levels during larval tissue development. In wing imaginal discs, the MLR complex attenuates bantam enhancer activity by negatively regulating expression; whereas, in differentiating eye discs, the complex exerts either positive or negative regulatory activity on bantam transcription depending on cell fate. Furthermore, while the MLR complex is not required to control bantam levels in undifferentiated eye cells anterior to the morphogenetic furrow, it serves to prepare critical enhancer control of bantam transcription for later regulation upon differentiation. This investigation into the transcriptional regulation of a single target in a developmental context has provided novel insights as to how the MLR complex contributes to the precise timing of gene expression, and how the complex functions to help orchestrate the regulatory output of conserved signaling pathways during animal development.
Ariyapala, I. S., Holsopple, J. M., Popodi, E. M., Hartwick, D. G., Kahsai, L., Cook, K. R. and Sokol, N. S. (2020). Identification of Split-GAL4 Drivers and Enhancers That Allow Regional Cell Type Manipulations of the Drosophila melanogaster Intestine. Genetics. PubMed ID: 32988987
The Drosophila adult midgut is a model epithelial tissue composed of a few major cell types with distinct regional identities. One of the limitations to its analysis is the lack of tools to manipulate gene expression based on these regional identities. To overcome this obstacle, the intersectional split-GAL4 system was applied to the adult midgut; 653 driver combinations are reported that label cells by region and cell type. 424 split-GAL4 drivers with midgut expression were identified from over 7,300 drivers screened, and then the expression patterns of each of these 424 were evaluated when paired with three reference drivers that report activity specifically in progenitor cells, enteroendocrine cells, or enterocytes. A subset of the drivers expressed in progenitor cells was evaluated for expression in enteroblasts using another reference driver. It was possible to show that driver combinations can define novel cell populations by identifying a driver that marks a distinct subset of enteroendocrine cells expressing genes usually associated with progenitor cells. The regional cell type patterns associated with the entire set of driver combinations are documented in a freely available website, providing information for the design of thousands of additional driver combinations to experimentally manipulate small subsets of intestinal cells. In addition, it was shown that intestinal enhancers identified with the split-GAL4 system can confer equivalent expression patterns on other transgenic reporters. Altogether, the resource reported here will enable more precisely targeted gene expression for studying intestinal processes, epithelial cell functions, and diseases affecting self-renewing tissues.
Sigalova, O. M., Shaeiri, A., Forneris, M., Furlong, E. E. and Zaugg, J. B. (2020). Predictive features of gene expression variation reveal mechanistic link with differential expression. Mol Syst Biol 16(8): e9539. PubMed ID: 32767663
For most biological processes, organisms must respond to extrinsic cues, while maintaining essential gene expression programmes. Although studied extensively in single cells, it is still unclear how variation is controlled in multicellular organisms. This study used a machine-learning approach to identify genomic features that are predictive of genes with high versus low variation in their expression across individuals, using bulk data to remove stochastic cell-to-cell variation. Using embryonic gene expression across 75 Drosophila isogenic lines, features predictive of expression variation (controlling for expression level) were identified, many of which are promoter-related. Genes with low variation fall into two classes reflecting different mechanisms to maintain robust expression, while genes with high variation seem to lack both types of stabilizing mechanisms. Applying this framework to humans revealed similar predictive features, indicating that promoter architecture is an ancient mechanism to control expression variation. Remarkably, expression variation features could also partially predict differential expression after diverse perturbations in both Drosophila and humans. Differential gene expression signatures may therefore be partially explained by genetically encoded gene-specific features, unrelated to the studied treatment.
Xin, Y., Le Poul, Y., Ling, L., Museridze, M., Muhling, B., Jaenichen, R., Osipova, E. and Gompel, N. (2020). Enhancer evolutionary co-option through shared chromatin accessibility input. Proc Natl Acad Sci U S A 117(34): 20636-20644. PubMed ID: 32778581
The diversity of forms in multicellular organisms originates largely from the spatial redeployment of developmental genes [S. B. Carroll, Cell 134, 25-36 (2008)]. Several scenarios can explain the emergence of cis-regulatory elements that govern novel aspects of a gene expression pattern. One scenario, enhancer co-option, holds that a DNA sequence producing an ancestral regulatory activity also becomes the template for a new regulatory activity, sharing regulatory information. While enhancer co-option might fuel morphological diversification, it has rarely been documented. Moreover, if two regulatory activities are borne from the same sequence, their modularity, considered a defining feature of enhancers, might be affected by pleiotropy. Sequence overlap may thereby play a determinant role in enhancer function and evolution. This study investigated this problem with two regulatory activities of the Drosophila gene yellow, the novel spot enhancer and the ancestral wing blade enhancer. Precise and comprehensive quantification was used of each activity in Drosophila wings to systematically map their sequences along the locus.The spot enhancer has co-opted the sequences of the wing blade enhancer. A pleiotropic site was identified necessary for DNA accessibility of a shared regulatory region. While the evolutionary steps leading to the derived activity are still unknown, such pleiotropy suggests that enhancer accessibility could be one of the molecular mechanisms seeding evolutionary co-option.
Hoppe, C., Bowles, J. R., Minchington, T. G., Sutcliffe, C., Upadhyai, P., Rattray, M. and Ashe, H. L. (2020). Modulation of the Promoter Activation Rate Dictates the Transcriptional Response to Graded BMP Signaling Levels in the Drosophila Embryo. Dev Cell 54(6): 727-741. PubMed ID: 32758422
Morphogen gradients specify cell fates during development, with a classic example being the bone morphogenetic protein (BMP) gradient's conserved role in embryonic dorsal-ventral axis patterning. This study elucidates how the gradient of Dpp is interpreted in the Drosophila embryo by combining live imaging with computational modeling to infer transcriptional burst parameters at single-cell resolution. By comparing burst kinetics in cells receiving different levels of BMP signaling, this study shows that BMP signaling controls burst frequency by regulating the promoter activation rate. Evidence is provided that the promoter activation rate is influenced by both enhancer and promoter sequences, whereas Pol II loading rate is primarily modulated by the enhancer. Consistent with BMP-dependent regulation of burst frequency, the numbers of BMP target gene transcripts per cell are graded across their expression domains. It is suggested that graded mRNA output is a general feature of morphogen gradient interpretation and discuss how this can impact on cell-fate decisions.
Waymack, R., Fletcher, A., Enciso, G. and Wunderlich, Z. (2020). Shadow enhancers can suppress input transcription factor noise through distinct regulatory logic. Elife 9. PubMed ID: 32804082
Shadow enhancers, groups of seemingly redundant enhancers, are found in a wide range of organisms and are critical for robust developmental patterning. However, their mechanism of action is unknown. It is hypothesized that shadow enhancers drive consistent expression levels by buffering upstream noise through a separation of transcription factor (TF) inputs at the individual enhancers. By measuring the transcriptional dynamics of several Kruppel shadow enhancer configurations in live Drosophila embryos, this study showed that individual member enhancers act largely independently. TF fluctuations were found to be an appreciable source of noise that the shadow enhancer pair can better buffer than duplicated enhancers. The shadow enhancer pair is also uniquely able to maintain low levels of expression noise across a wide range of temperatures. A stochastic model demonstrated the separation of TF inputs is sufficient to explain these findings. These results suggest the widespread use of shadow enhancers is partially due to their noise suppressing ability.
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