What's hot today:
Current papers in developmental biology and gene function


Thursday, April 30th, 2020 - Behavior

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Smith, B. R. and Macdonald, S. J. (2020). Dissecting the Genetic Basis of Variation in Drosophila Sleep Using a Multiparental QTL Mapping Resource. Genes (Basel) 11(3). PubMed ID: 32168738
There is considerable variation in sleep duration, timing and quality in human populations, and sleep dysregulation has been implicated as a risk factor for a range of health problems. Human sleep traits are known to be regulated by genetic factors, but also by an array of environmental and social factors. These uncontrolled, non-genetic effects complicate powerful identification of the loci contributing to sleep directly in humans. The model system, Drosophila melanogaster, exhibits a behavior that shows the hallmarks of mammalian sleep, and this study used a multitiered approach, encompassing high-resolution QTL mapping, expression QTL data, and functional validation with RNAi to investigate the genetic basis of sleep under highly controlled environmental conditions. A battery of sleep phenotypes was measured in >750 genotypes derived from a multiparental mapping panel and identified several, modest-effect QTL contributing to natural variation for sleep. Merging sleep QTL data with a large head transcriptome eQTL mapping dataset from the same population allowed refining the list of plausible candidate causative sleep loci. This set includes genes with previously characterized effects on sleep and circadian rhythms, in addition to novel candidates. Finally, adult, nervous system-specific RNAi was used on the Dopa decarboxylase, dyschronic, and timeless genes, finding significant effects on sleep phenotypes for all three. The genes resolved in this study are strong candidates to harbor causative, regulatory variation contributing to sleep.
Singh, P. and Donlea, J. M. (2020). Bidirectional regulation of sleep and synapse pruning after neural injury. Curr Biol. PubMed ID: 32142703
Following acute neural injury, severed axons undergo programmed Wallerian degeneration over several following days. While sleep has been linked with synaptic reorganization under other conditions, the role of sleep in responses to neural injuries remains poorly understood. To study the relationship between sleep and neural injury responses, Drosophila melanogaster was examined following the removal of antennae or other sensory tissues. Daytime sleep is elevated after antennal or wing injury, but sleep returns to baseline levels within 24 h after injury. Similar increases in sleep are not observed when olfactory receptor neurons are silenced or when other sensory organs are severed, suggesting that increased sleep after injury is not attributed to sensory deprivation, nociception, or generalized inflammatory responses. Neuroprotective disruptions of the E3 ubiquitin ligase highwire and c-Jun N-terminal kinase basket in olfactory receptor neurons weaken the sleep-promoting effects of antennal injury, suggesting that post-injury sleep may be influenced by the clearance of damaged neurons. Finally, pre-synaptic active zones were shown to be preferentially removed from severed axons within hours after injury, and depriving recently injured flies of sleep slows the removal of both active zones and damaged axons. These data support a bidirectional interaction between sleep and synapse pruning after antennal injury: locally increasing the need to clear neural debris is associated with increased sleep, which is required for efficient active zone removal after injury.
Palavalli-Nettimi, R. and Theobald, J. C. (2020). Small eyes in dim light: Implications to spatio-temporal visual abilities in Drosophila melanogaster. Vision Res 169: 33-40. PubMed ID: 32163744
Fruit flies, Drosophila melanogaster, are active over a range of light intensities in the wild, but lab-reared flies are often tested only in bright light. Similarly, scarce feeding during larval stages-common in nature-generates smaller adults, and a wide range of eye sizes not found in well-fed lab colonies. Both dimmer light and smaller eyes limit light capture and have undetermined effects on visual behaviors such as flight. This study used moving sinusoidal gratings to test spatial acuity, temporal acuity, and contrast threshold of female flies of varying eye sizes at different light intensities. Vision was also tested in the smaller and often neglected male fruit flies. As light intensity drops from 50.1 lx to 0.3 lx, flies have a reduced spatial acuity (females: from 0.1 to 0.06 cycles per degree, CPD, males: 0.1 to 0.04 CPD) and temporal acuity (females: from 50 Hz to 10 Hz, males: 25 Hz to 10 Hz), and an increased contrast detection threshold (females: from 10% to 29%, males: 19% to 48%). No major sex-specific differences were found after accounting for eye size. Visual abilities in both small (eye area of 0.1-0.17 mm(2)) and large flies (0.17-0.23 mm(2)) suffer at 0.3 lx compared to 50.1 lx, but small flies suffer more (spatial acuity: 0.03 vs 0.06 CPD, contrast threshold: 76% vs 57%, temporal acuity: 5 Hz vs 10 Hz). These results suggest visual abilities of small flies suffer more than large flies at low light levels, possibly leading to size- and light intensity-dependent effects on foraging, navigation, and flight.
Brown, E. B., Shah, K. D., Faville, R., Kottler, B. and Keene, A. C. (2020). Drosophila insulin-like peptide 2 mediates dietary regulation of sleep intensity. PLoS Genet 16(3): e1008270. PubMed ID: 32160200
Sleep is a nearly universal behavior that is regulated by diverse environmental stimuli and physiological states. A defining feature of sleep is a homeostatic rebound following deprivation, where animals compensate for lost sleep by increasing sleep duration and/or sleep depth. The fruit fly, Drosophila melanogaster, exhibits robust recovery sleep following deprivation and represents a powerful model to study neural circuits regulating sleep homeostasis. Numerous neuronal populations have been identified in modulating sleep homeostasis as well as depth, raising the possibility that the duration and quality of recovery sleep is dependent on the environmental or physiological processes that induce sleep deprivation. This study finds that unlike most pharmacological and environmental manipulations commonly used to restrict sleep, starvation potently induces sleep loss without a subsequent rebound in sleep duration or depth. Both starvation and a sucrose-only diet result in increased sleep depth, suggesting that dietary protein is essential for normal sleep depth and homeostasis. Finally, Drosophila insulin like peptide 2 (Dilp2) was found to be acutely required for starvation-induced changes in sleep depth without regulating the duration of sleep. Flies lacking Dilp2 exhibit a compensatory sleep rebound following starvation-induced sleep deprivation, suggesting Dilp2 promotes resiliency to sleep loss. Together, these findings reveal innate resilience to starvation-induced sleep loss and identify distinct mechanisms that underlie starvation-induced changes in sleep duration and depth.
Slankster, E., Kollala, S., Baria, D., Dailey-Krempel, B., Jain, R., Odell, S. R. and Mathew, D. (2020). Mechanism underlying starvation-dependent modulation of olfactory behavior in Drosophila larva. Sci Rep 10(1): 3119. PubMed ID: 32080342
Starvation enhances olfactory sensitivity that encourage animals to search for food. The molecular mechanisms that enable sensory neurons to remain flexible and adapt to a particular internal state remain poorly understood. The roles of GABA and insulin signaling in starvation-dependent modulation of olfactory sensory neuron (OSN) function was studied in the Drosophila larva. The GABAB-receptor and insulin-receptor play important roles during OSN modulation. Using an OSN-specific gene expression analysis, this study explored downstream targets of insulin signaling in OSNs. The results suggest that insulin and GABA signaling pathways interact within OSNs and modulate OSN function by impacting olfactory information processing. It was further shown that manipulating these signaling pathways specifically in the OSNs impact larval feeding behavior and its body weight. These results challenge the prevailing model of OSN modulation and highlight opportunities to better understand OSN modulation mechanisms and their relationship to animal physiology.
Kerwin, P., Yuan, J. and von Philipsborn, A. C. (2020). Female copulation song is modulated by seminal fluid. Nat Commun 11(1): 1430. PubMed ID: 32188855
In most animal species, males and females communicate during sexual behavior to negotiate reproductive investments. Pre-copulatory courtship may settle if copulation takes place, but often information exchange and decision-making continue beyond that point. This study shows that female Drosophila sing by wing vibration in copula. This copulation song is distinct from male courtship song and requires neurons expressing the female sex determination factor DoublesexF. Copulation song depends on transfer of seminal fluid components of the male accessory gland. Hearing female copulation song increases the reproductive success of a male when he is challenged by competition, suggesting that auditory cues from the female modulate male ejaculate allocation. These findings reveal an unexpected fine-tuning of reproductive decisions during a multimodal copulatory dialog. The discovery of a female-specific acoustic behavior sheds new light on Drosophila mating, sexual dimorphisms of neuronal circuits and the impact of seminal fluid molecules on nervous system and behavior.

Wednesday, April 29th, Gonads

Jevitt, A., Chatterjee, D., Xie, G., Wang, X. F., Otwell, T., Huang, Y. C. and Deng, W. M. (2020). A single-cell atlas of adult Drosophila ovary identifies transcriptional programs and somatic cell lineage regulating oogenesis. PLoS Biol 18(4): e3000538. PubMed ID: 32339165
Oogenesis is a complex developmental process that involves spatiotemporally regulated coordination between the germline and supporting, somatic cell populations. This process has been modeled extensively using the Drosophila ovary. Although different ovarian cell types have been identified through traditional means, the large-scale expression profiles underlying each cell type remain unknown. Using single-cell RNA sequencing technology, this study has built a transcriptomic data set for the adult Drosophila ovary and connected tissues. Using this data set, the transcriptional trajectory was identified of the entire follicle-cell population over the course of their development from stem cells to the oogenesis-to-ovulation transition. This study further identified expression patterns during essential developmental events that take place in somatic and germline cell types such as differentiation, cell-cycle switching, migration, symmetry breaking, nurse-cell engulfment, egg-shell formation, and corpus luteum signaling. Extensive experimental validation of unique expression patterns in both ovarian and nearby, nonovarian cells also led to the identification of many new cell type-and stage-specific markers. The inclusion of several nearby tissue types in this data set also led to identification of functional convergence in expression between distantly related cell types such as the immune-related genes that were similarly expressed in immune cells (hemocytes) and ovarian somatic cells (stretched cells) during their brief phagocytic role in nurse-cell engulfment. Taken together, these findings provide new insight into the temporal regulation of genes in a cell-type specific manner during oogenesis and begin to reveal the relatedness in expression between cell and tissues types.
Schotthofer, S. K. and Bohrmann, J. (2020). Bioelectrical and cytoskeletal patterns correlate with altered axial polarity in the follicular epithelium of the Drosophila mutant gurken. BMC Dev Biol 20(1): 5. PubMed ID: 32169045
Bioelectrical signals are known to be involved in the generation of cell and tissue polarity as well as in cytoskeletal dynamics. The epithelium of Drosophila ovarian follicles is a suitable model system for studying connections between electrochemical gradients, patterns of cytoskeletal elements and axial polarity. By interactions between soma and germline cells, the transforming growth factor-alpha homolog Gurken (Grk) establishes both the anteroposterior and the dorsoventral axis during oogenesis. In the follicular epithelium of the wild-type (wt) and the polarity mutant grk, stage-specific gradients of membrane potentials (Vmem) and intracellular pH (pHi) were analyzed using the potentiometric dye DiBAC4(3) and the fluorescent pH-indicator 5-CFDA,AM, respectively. Corresponding to impaired polarity in grk, the slope of the anteroposterior Vmem-gradient in stage S9 is significantly reduced compared to wt. Even more striking differences in Vmem- and pHi-patterns become obvious during stage S10B, when the respective dorsoventral gradients are established in wt but not in grk. Concurrent with bioelectrical differences, wt and grk exhibit differences concerning cytoskeletal patterns in the follicular epithelium. During all vitellogenic stages, basal microfilaments in grk are characterised by transversal alignment, while wt-typical condensations in centripetal follicle cells (S9) and in dorsal centripetal follicle cells (S10B) are absent. Moreover, in grk, longitudinal alignment of microtubules occurs throughout vitellogenesis in all follicle cells, whereas in wt, microtubules in mainbody and posterior follicle cells exhibit a more cell-autonomous organisation. Therefore, in contrast to wt, the follicular epithelium in grk is characterised by missing or shallower electrochemical gradients and by more coordinated transcellular cytoskeletal patterns. These results show that bioelectrical polarity and cytoskeletal polarity are closely linked to axial polarity in both wt and grk.
Thestrup, J., Tipold, M., Kindred, A., Stark, K., Curry, T. and Lewellyn, L. (2020). The Arp2/3 complex and the formin, Diaphanous, are both required to regulate the size of germline ring canals in the developing egg chamber. Dev Biol. PubMed ID: 31945342
Intercellular bridges are an essential structural feature found in both germline and somatic cells throughout the animal kingdom. Because of their large size, the germline intercellular bridges, or ring canals, in the developing fruit fly egg chamber are an excellent model to study the formation, stabilization, and growth of these structures. Within the egg chamber, the germline ring canals connect 15 supporting nurse cells to the developing oocyte, facilitating the transfer of materials required for successful oogenesis. The ring canals are derived from a stalled actomyosin contractile ring; once formed, additional actin and actin-binding proteins are recruited to the ring to support the 20-fold growth that accompanies oogenesis. These behaviors provide a unique model system to study the actin regulators that control incomplete cytokinesis, intercellular bridge formation, and growth. By temporally controlling their expression in the germline, this study has demonstrated that the Arp2/3 complex and the formin, Diaphanous (Dia), coordinately regulate ring canal size and growth throughout oogenesis. Dia is required for successful incomplete cytokinesis and the initial stabilization of the germline ring canals. Once ring canals have formed, the Arp2/3 complex and Dia cooperate to determine ring canal size and maintain stability. These data suggest that nurse cells must maintain a precise balance between the activity of these two nucleators during oogenesis.
Lu, D., Sin, H. S., Lu, C. and Fuller, M. T. (2020). Developmental regulation of cell type-specific transcription by novel promoter-proximal sequence elements. Genes Dev. PubMed ID: 32217666
Cell type-specific transcriptional programs that drive differentiation of specialized cell types are key players in development and tissue regeneration. One of the most dramatic changes in the transcription program in Drosophila occurs with the transition from proliferating spermatogonia to differentiating spermatocytes, with >3000 genes either newly expressed or expressed from new alternative promoters in spermatocytes. This study shows that opening of these promoters from their closed state in precursor cells requires function of the spermatocyte-specific tMAC complex (see always early ), localized at the promoters. The spermatocyte-specific promoters lack the previously identified canonical core promoter elements except for the Inr. Instead, these promoters are enriched for the binding site for the TALE-class homeodomain transcription factors Achi/Vis and for a motif originally identified under tMAC ChIP-seq peaks. The tMAC motif resembles part of the previously identified 14-bp beta2UE1 element critical for spermatocyte-specific expression. Analysis of downstream sequences relative to transcription start site usage suggested that ACA and CNAAATT motifs at specific positions can help promote efficient transcription initiation. These results reveal how promoter-proximal sequence elements that recruit and are acted upon by cell type-specific chromatin binding complexes help establish a robust, cell type-specific transcription program for terminal differentiation.
Shi, Z., Lim, C., Tran, V., Cui, K., Zhao, K. and Chen, X. (2020). Single-cyst transcriptome analysis of Drosophila male germline stem cell lineage. Development. PubMed ID: 32122991
The Drosophila male germline stem cell (GSC) lineage provides a great model to understand stem cell maintenance, proliferation, differentiation, and dedifferentiation. This study used Drosophila GSC lineage to systematically analyze transcriptome of discrete but continuous differentiating germline cysts. First single cysts were isolated at each recognizable stage from wild-type testes, which were subsequently applied for RNA-seq analyses. The data delineate a high-resolution transcriptome atlas in the entire male GSC lineage: The most dramatic switch occurs from early to late spermatocyte, followed by the change from the mitotic spermatogonia to early meiotic spermatocyte. By contrast, the transit-amplifying spermatogonia cysts display similar transcriptomes, suggesting common molecular features among these stages, which may underlie their similar behavior during both differentiation and dedifferentiation processes. Finally, distinct differentiating germ cell cyst samples do not exhibit obvious dosage compensation of X-chromosomal genes, even with consideration of the paucity of X-chromosomal gene expression during meiosis, which is different from somatic cells. Together, the single cyst-resolution, genome-wide transcriptional profile analyses provide an unprecedented resource to understand many questions in both germ cell biology and stem cell biology fields.
Hafezi, Y., Sruba, S. R., Tarrash, S. R., Wolfner, M. F. and Clark, A. G. (2020). Dissecting Fertility Functions of Drosophila Y Chromosome Genes with CRISPR. Genetics. PubMed ID: 32098759
Gene-poor, repeat-rich regions of the genome are poorly understood and have been understudied due to technical challenges and the misconception that they are degenerating "junk". The approximately 40 Mb Y chromosome of Drosophila melanogaster contains only 16 known protein-coding genes and is highly repetitive and entirely heterochromatic. Most of the genes originated from duplication of autosomal genes and have reduced nonsynonymous substitution rates, suggesting functional constraint. This study devised a genetic strategy for recovering and retaining stocks with sterile Y-linked mutations and combined it with CRISPR to create mutants with deletions that disrupt three Y-linked genes. Two genes, PRY and FDY, had no previously identified functions. PRY mutant males are sub-fertile, but FDY mutant males had no detectable fertility defects. FDY, the newest known gene on the Y chromosome, may have fertility effects that are conditional or too subtle to detect. The third gene, CCY, had been predicted but never formally shown to be required for male fertility. CRISPR-targeting and RNAi of CCY caused male sterility. Surprisingly, however, the CCY mutants were sterile even in the presence of an extra wild-type Y chromosome, suggesting that perturbation of the Y chromosome can lead to dominant sterility. This approach provides an important step toward understanding the complex functions of the Y chromosome and parsing which functions are accomplished by genes versus repeat elements.

Tuesday, April 28th - Adult Neural Function

Silva, B., Hidalgo, S. and Campusano, J. M. (2020). Dop1R1, a type 1 dopaminergic receptor expressed in Mushroom Bodies, modulates Drosophila larval locomotion. PLoS One 15(2): e0229671. PubMed ID: 32101569
As in vertebrates, dopaminergic neural systems are key regulators of motor programs in insects, including the fly Drosophila melanogaster. Dopaminergic systems innervate the Mushroom Bodies (MB), an important association area in the insect brain primarily associated to olfactory learning and memory, but that has been also implicated with the execution of motor programs. The main objectives of this work is to assess the idea that dopaminergic systems contribute to the execution of motor programs in Drosophila larvae, and then, to evaluate the contribution of specific dopaminergic receptors expressed in MB to these programs. The results show that animals bearing a mutation in the dopamine transporter show reduced locomotion, while mutants for the dopaminergic biosynthetic enzymes or the dopamine receptor Dop1R1 exhibit increased locomotion. Pan-neuronal expression of an RNAi for the Dop1R1 confirmed these results. Further studies show that animals expressing the RNAi for Dop1R1 in the entire MB neuronal population or only in the MB gamma-lobe forming neurons, exhibit an increased motor output, as well. Interestingly, these results also suggest that other dopaminergic receptors do not contribute to larval motor behavior. Thus, the data support the proposition that CNS dopamine systems innervating MB neurons modulate larval locomotion and that Dop1R1 mediates this effect.
Ly, P. T. and Wang, H. (2020). Fzr/Cdh1 Promotes the Differentiation of Neural Stem Cell Lineages in Drosophila. Front Cell Dev Biol 8: 60. PubMed ID: 32117986
How stem cells and progenitors balance between self-renewal and differentiation is a central issue of stem cell biology. This study describes a novel and essential function of Drosophila Fzr/Cdh1, an evolutionary conserved protein, during the differentiation of neural stem cell (NSC) lineages in the central nervous system. This study shows that Fzr, a known co-activator of Anaphase Promoting Complex/Cyclosome (APC/C) ubiquitin ligase, promotes the production of neurons from neural progenitors called ganglion mother cells (GMCs). However, knockdown of APC/C subunit Ida or another APC/C co-activator CDC20 does not similarly impair GMC-neuron transition. A concomitant loss of differentiation factor Prospero expression was observed, and ectopic accumulation of mitotic kinase Polo in fzr mutant clones, strongly supporting the impairment of GMC to neuron differentiation. Besides functioning in GMCs, Fzr is also present in NSCs to facilitate the production of intermediate neural progenitors from NSCs. Taken together, Fzr plays a novel function in promoting differentiation programs during Drosophila NSC lineage development. Given that human Fzr is inactivated in multiple types of human cancers including brain tumors and that Fzr regulates neurotoxicity in various models of neurodegenerative diseases, this study on the role of Fzr in turning off proliferation in neuronal cells may provide insights into how Fzr deficits may contribute to human neurodegenerative diseases and tumors.
Petruccelli, E., Brown, T., Waterman, A., Ledru, N. and Kaun, K. R. (2020). Alcohol Causes Lasting Differential Transcription in Drosophila Mushroom Body Neurons. Genetics. PubMed ID: 32132098
Repeated alcohol experiences can produce long-lasting memories for sensory cues associated with intoxication. These memories can problematically trigger relapse in individuals recovering from alcohol use disorder (AUD). The molecular mechanisms by which ethanol changes memories to become long-lasting and inflexible remain unclear. New methods to analyze gene expression within precise neuronal cell-types can provide further insight towards AUD prevention and treatment. This study used genetic tools in Drosophila melanogaster to investigate the lasting consequences of ethanol on transcription in memory-encoding neurons. Drosophila rely on mushroom body (MB) neurons to make associative memories, including memories of ethanol-associated sensory cues. Differential expression analyses revealed that distinct transcripts, but not genes, in the MB were associated with experiencing ethanol alone compared to forming a memory of an odor cue associated with ethanol. Adult MB specific knockdown of spliceosome-associated proteins demonstrated the necessity of RNA processing in ethanol memory formation. These findings highlight the dynamic, context-specific regulation of transcription in cue-encoding neurons, and the lasting impact of ethanol on transcript usage during memory formation.
Nye, D. M. R., Albertson, R. M., Weiner, A. T., Hertzler, J. I., Shorey, M., Goberdhan, D. C. I., Wilson, C., Janes, K. A. and Rolls, M. M. (2020). The receptor tyrosine kinase Ror is required for dendrite regeneration in Drosophila neurons. PLoS Biol 18(3): e3000657. PubMed ID: 32163406
While many regulators of axon regeneration have been identified, very little is known about mechanisms that allow dendrites to regenerate after injury. Using a Drosophila model of dendrite regeneration, a candidate screen was performed of receptor tyrosine kinases (RTKs), and a requirement was found for RTK-like orphan receptor (Ror). This study confirmed that Ror was required for regeneration in two different neuron types using RNA interference (RNAi) and mutants. Ror was not required for axon regeneration or normal dendrite development, suggesting a specific role in dendrite regeneration. Ror can act as a Wnt coreceptor with frizzleds (fzs) in other contexts, so this study tested the involvement of Wnt signaling proteins in dendrite regeneration. Knockdown of fz, dishevelled (dsh), Axin, and gilgamesh (gish) also reduced dendrite regeneration. Moreover, Ror was required to position Dsh and Axin in dendrites. Recently studies found that Wnt signaling proteins, including Dsh and Axin, localize microtubule nucleation machinery in dendrites. It is therefore hypothesized that Ror may act by regulating microtubule nucleation at baseline and during dendrite regeneration. Consistent with this hypothesis, localization of the core nucleation protein gammaTubulin was reduced in Ror RNAi neurons, and this effect was strongest during dendrite regeneration. In addition, dendrite regeneration was sensitive to partial reduction of gammaTubulin. It is conclude that Ror promotes dendrite regeneration as part of a Wnt signaling pathway that regulates dendritic microtubule nucleation.

Luo, J., Ting, C. Y., Li, Y., McQueen, P., Lin, T. Y., Hsu, C. P. and Lee, C. H. (2020). Antagonistic regulation by insulin-like peptide and activin ensures the elaboration of appropriate dendritic field sizes of amacrine neurons. Elife 9. PubMed ID: 32175842
Establishing appropriate sizes and shapes of dendritic arbors is critical for proper wiring of the central nervous system. This study reports that Insulin-like Peptide 2 (DILP2) locally activates transiently expressed insulin receptors in the central dendrites of Drosophila Dm8 amacrine neurons to positively regulate dendritic field elaboration. DILP2 is expressed in L5 lamina neurons, which have axonal terminals abutting Dm8 dendrites. Proper Dm8 dendrite morphogenesis and synapse formation required insulin signaling through TOR (target of rapamycin) and SREBP (sterol regulatory element-binding protein), acting in parallel with previously identified negative regulation by Activin signaling to provide robust control of Dm8 dendrite elaboration. A simulation of dendritic growth revealed trade-offs between dendritic field size and robustness when branching and terminating kinetic parameters were constant, but dynamic modulation of the parameters could mitigate these trade-offs. It is suggested that antagonistic DILP2 and Activin signals from different afferents appropriately size Dm8 dendritic fields.
Muha, V., Fenckova, M., Ferenbach, A. T., Catinozzi, M., Eidhof, I., Storkebaum, E., Schenck, A. and van Aalten, D. M. F. (2020). O-GlcNAcase contributes to cognitive function in Drosophila. J Biol Chem. PubMed ID: 32094227
O-GlcNAcylation is an abundant post-translational modification in neurons. In mice, an increase in O-GlcNAcylation leads to defects in hippocampal synaptic plasticity and learning. O-GlcNAcylation is established by two opposing enzymes O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). To investigate the role of OGA in elementary learning, this study generated catalytically inactive and precise knock-out Oga alleles (Oga(D133N) and Oga(KO), respectively) in Drosophila melanogaster. Adult Oga(D133N) and Oga(KO) flies lacking O-GlcNAcase activity showed locomotor phenotypes. Importantly, both Oga lines exhibited deficits in habituation, an evolutionary conserved form of learning, highlighting that the requirement for O-GlcNAcase activity for cognitive function is preserved across species. Loss of O-GlcNAcase affected number of synaptic boutons at the axon terminals of larval neuromuscular junction. Taken together, this study report behavioral and neurodevelopmental phenotypes associated with Oga alleles and show that Oga contributes to cognition and synaptic morphology in Drosophila.

Monday, April 27th - Physiology

Petersen, C. E., Wolf, M. J. and Smyth, J. T. (2020). Suppression of store-operated calcium entry causes dilated cardiomyopathy of the Drosophila heart. Biol Open 9(3). PubMed ID: 32086252
Store-operated Ca(2+) entry (SOCE) is an essential Ca(2+) signaling mechanism present in most animal cells. SOCE refers to Ca(2+) influx that is activated by depletion of sarco/endoplasmic reticulum (S/ER) Ca(2+) stores. The main components of SOCE are STIM and Orai. STIM proteins function as S/ER Ca(2+) sensors, and upon S/ER Ca(2+) depletion STIM rearranges to S/ER-plasma membrane junctions and activates Orai Ca(2+) influx channels. Studies have implicated SOCE in cardiac hypertrophy pathogenesis, but SOCE's role in normal heart physiology remains poorly understood. This study analyzed heart-specific SOCE function in Drosophila, a powerful animal model of cardiac physiology. Heart-specific suppression of Stim and Orai in larvae and adults resulted in reduced contractility consistent with dilated cardiomyopathy. Myofibers were also highly disorganized in Stim and Orai RNAi hearts, reflecting possible decompensation or upregulated stress signaling. Furthermore, this study showed that reduced heart function due to SOCE suppression adversely affected animal viability, as heart specific Stim and Orai RNAi animals exhibited significant delays in post-embryonic development and adults died earlier than controls. Collectively, these results demonstrate that SOCE is essential for physiological heart function, and establish Drosophila as an important model for understanding the role of SOCE in cardiac pathophysiology.
Lepa, C. et al. (2020). LIM and SH3 protein 1 (LASP-1): A novel link between the slit membrane and actin cytoskeleton dynamics in podocytes. FASEB J. PubMed ID: 32086849
The foot processes of podocytes exhibit a dynamic actin cytoskeleton, which maintains their complex cell structure and antagonizes the elastic forces of the glomerular capillary. Interdigitating secondary foot processes form a highly selective filter for proteins in the kidney, the slit membrane. Knockdown of slit membrane components such as Nephrin or Neph1 and cytoskeletal adaptor proteins such as CD2AP in mice leads to breakdown of the filtration barrier with foot process effacement, proteinuria, and early death of the mice. Less is known about the crosstalk between the slit membrane-associated proteins and cytoskeletal components inside the podocyte foot processes. This study shows that LASP-1, an actin-binding protein, is highly expressed in podocytes. Electron microscopy studies demonstrate that LASP-1 is found at the slit membrane suggesting a role in anchoring slit membrane components to the actin cytoskeleton. Live cell imaging experiments with transfected podocytes reveal that LASP-1 is either part of a highly dynamic granular complex or a static, actin cytoskeleton-bound protein. This study identified CD2AP as a novel LASP-1 binding partner that regulates its association with the actin cytoskeleton. Activation of the renin-angiotensin-aldosterone system, which is crucial for podocyte function, leads to phosphorylation and altered localization of LASP-1. In vivo studies using the Drosophila nephrocyte model indicate that Lasp is necessary for the slit membrane integrity and functional filtration.
Kang, D. and Douglas, A. E. (2020). Functional traits of the gut microbiome correlated with host lipid content in a natural population of Drosophila melanogaster. Biol Lett 16(2): 20190803. PubMed ID: 32097599
Most research on the nutritional significance of the gut microbiome is conducted on laboratory animals, and its relevance to wild animals is largely unknown. This study investigated the microbiome correlates of lipid content in individual wild fruit flies, Drosophila melanogaster. Lipid content varied 3.6-fold among the flies and was significantly correlated with the abundance of gut-derived bacterial DNA sequences that were assigned to genes contributing to 16 KEGG pathways. These included genes encoding sugar transporters and enzymes in glycolysis/gluconeogenesis, potentially promoting sugar consumption by the gut microbiome and, thereby, a lean fly phenotype. Furthermore, the lipid content of wild flies was significantly lower than laboratory flies, indicating that, as for some mammalian models, certain laboratory protocols might be obesogenic for Drosophila. This study demonstrates the value of research on natural populations to identify candidate microbial genes that influence ecologically important host traits.
Nagai, H., Kurata, S. and Yano, T. (2020). Immunoglobulin superfamily beat-Ib mediates intestinal regeneration induced by reactive oxygen species in Drosophila. Genes Cells. PubMed ID: 32080940
Reactive oxygen species (ROS) often injure intestinal epithelia that cause loss of damaged cells, which is mainly repaired by proliferation of intestinal stem cells (ISCs). To maintain the homeostatic state, coordination of sensing of the ROS injury and the subsequent epithelial cell loss with the replenishment by cell renewal is crucial. However, little is known about how gut epithelial cells initiate regenerative responses against ROS to maintain the tissue integrity. A genome-wide screen was carried out, by which immunoglobulin superfamily beaten path Ib (beat-Ib) as an essential gene for provoking ISC proliferation against ROS in Drosophila intestine. Interestingly, the beat-Ib function is required in differentiated enterocytes, the main targeted cells by ROS in the intestinal tract, but is dispensable in the stem cells. Moreover, beat-Ib is not involved in enterocyte apoptosis at ROS injury. These findings indicate the essential role of beat-Ib in Drosophila midgut enterocytes for initiating the non-cell-autonomous induction of ISC division in response to environmental ROS stresses.
McCracken, A. W., Adams, G., Hartshorne, L., Tatar, M. and Simons, M. J. P. (2020). The hidden costs of dietary restriction: Implications for its evolutionary and mechanistic origins. Sci Adv 6(8): eaay3047. PubMed ID: 32128403
Dietary restriction (DR) extends life span across taxa. Despite considerable research, universal mechanisms of DR have not been identified, limiting its translational potential. Guided by the conviction that DR evolved as an adaptive, pro-longevity physiological response to food scarcity, biomedical science has interpreted DR as an activator of pro-longevity molecular pathways. Current evolutionary theory predicts that organisms invest in their soma during DR, and thus when resource availability improves, should outcompete rich-fed controls in survival and/or reproduction. Testing this prediction in Drosophila melanogaster (N > 66,000 across 11 genotypes), these experiments revealed substantial, unexpected mortality costs when flies returned to a rich diet following DR. The physiological effects of DR should therefore not be interpreted as intrinsically pro-longevity, acting via somatic maintenance. It is suggested that DR could alternatively be considered an escape from costs incurred under nutrient-rich conditions, in addition to costs associated with DR.
Lucke, J., Kaltofen, S., Hansson, B. S. and Wicher, D. (2020). The role of mitochondria in shaping odor responses in Drosophila melanogaster olfactory sensory neurons. Cell Calcium 87: 102179. PubMed ID: 32070926
Insects detect volatile chemosignals with olfactory sensory neurons (OSNs) that express olfactory receptors. Among them, the most sensitive receptors are the odorant receptors (ORs), which form cation channels passing also Ca(2+). This study investigated if and how odor-induced Ca(2+) signals in Drosophila melanogaster OSNs are controlled by intracellular Ca(2+) stores, especially by mitochondria. Using an open antenna preparation that allows observation and pharmacological manipulation of OSNs Ca(2+) imaging was performed to determine the role of Ca(2+) influx and efflux pathways in OSN mitochondria. The results indicate that mitochondria participate in shaping the OR responses. The major players of this modulation are the mitochondrial calcium uniporter and the mitochondrial permeability transition pore. Intriguingly, OR-induced Ca(2+) signals were only mildly affected by modulating the Ca(2+) management of the endoplasmic reticulum (Lucke, 2020).

Friday, April 24th - Signaling

Malpe, M. S., McSwain, L. F., Kudyba, K., Ng, C. L., Nicholson, J., Brady, M., Qian, Y., Choksi, V., Hudson, A. G., Parrott, B. B. and Schulz, C. (2020). G-protein signaling is required for increasing germline stem cell division frequency in response to mating in Drosophila males. Sci Rep 10(1): 3888. PubMed ID: 32127590
Adult stem cells divide to renew the stem cell pool and replenish specialized cells that are lost due to death or usage. However, little is known about the mechanisms regulating how stem cells adjust to a demand for specialized cells. A failure of the stem cells to respond to this demand can have serious consequences, such as tissue loss, or prolonged recovery post injury. This study challenged the male germline stem cells (GSCs) of Drosophila melanogaster for the production of specialized cells, sperm cells, using mating experiments. Repeated mating reduced the sperm pool and increased the percentage of GSCs in M- and S-phase of the cell cycle. The increase in dividing GSCs depended on the activity of the highly conserved G-proteins. Germline expression of RNA-Interference (RNA-i) constructs against G-proteins, or a dominant negative G-protein eliminated the increase in GSC division frequency in mated males. Consistent with a role for the G-proteins in regulating GSC division frequency, RNA-i against seven out of 35 G-protein coupled receptors (GPCRs) within the germline cells also eliminated the capability of males to increase the numbers of dividing GSCs in response to mating.
Ly, S., Lee, D. A., Strus, E., Prober, D. A. and Naidoo, N. (2020). Evolutionarily Conserved Regulation of Sleep by the Protein Translational Regulator PERK. Curr Biol. PubMed ID: 32169212
Sleep is a cross-species phenomenon whose evolutionary and biological function remain poorly understood. Clinical and animal studies suggest that sleep disturbance is significantly associated with disruptions in protein homeostasis-or proteostasis-in the brain, but the mechanism of this link has not been explored. In the cell, the protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) pathway modulates proteostasis by transiently inhibiting protein synthesis in response to proteostatic stress. This study examined the role of the PERK pathway in sleep regulation and provides the first evidence that PERK signaling is required to regulate normal sleep in both vertebrates and invertebrates. Pharmacological inhibition of PERK reduces sleep in both Drosophila and zebrafish, indicating an evolutionarily conserved requirement for PERK in sleep. Genetic knockdown of PERK activity also reduces sleep in Drosophila, whereas PERK overexpression induces sleep. Finally, changes in PERK signaling were demonstrated to directly impact wake-promoting neuropeptide expression, revealing a mechanism through which proteostatic pathways can affect sleep and wake behavior. Taken together, these results demonstrate that protein synthesis pathways like PERK could represent a general mechanism of sleep and wake regulation and provide greater insight into the relationship between sleep and proteostasis.
Matsumura, T., Uryu, O., Matsuhisa, F., Tajiri, K., Matsumoto, H. and Hayakawa, Y. (2020). N-acetyl-l-tyrosine is an intrinsic triggering factor of mitohormesis in stressed animals. EMBO Rep: e49211. PubMed ID: 32118349
Under stress conditions, mitochondria release low levels of reactive oxygen species (ROS), which triggers a cytoprotective response, called "mitohormesis". It still remains unclear how mitochondria respond to stress-derived stimuli and release a low level of ROS. This study shows that N-acetyl-l-tyrosine (NAT) functions as a plausible intrinsic factor responsible for these tasks in stressed animals. NAT is present in the blood or hemolymph of healthy animals, and its concentrations increase in response to heat stress. Pretreatment with NAT significantly increases the stress tolerance of tested insects and mice. Analyses using Drosophila larvae and cultured cells demonstrate that the hormetic effects are triggered by transient NAT-induced perturbation of mitochondria, which causes a small increase in ROS production and leads to sequential retrograde responses: NAT-dependent FoxO activation increases in the gene expression of antioxidant enzymes and Keap1. Moreover, NAT represses tumor growth, possibly via the activation of Keap1. In sum, it is proposed that NAT is a vital endogenous molecule that could serve as a triggering factor for mitohormesis.
Neal, S. J., Zhou, Q. and Pignoni, F. (2020). STRIPAK-PP2A regulates Hippo-Yorkie signaling to suppress retinal fate in the Drosophila eye disc peripodial epithelium. J Cell Sci. PubMed ID: 32184260
The specification of organs, tissues and cell types results from cell fate restrictions enacted by nuclear transcription factors under the control of conserved signaling pathways. The progenitor epithelium of the Drosophila compound eye, the eye imaginal disc, is a premier model for the study of such processes. Early in development, apposing cells of the eye disc are established as either retinal progenitors or support cells of the peripodial epithelium (PE), in a process whose genetic and mechanistic determinants are poorly understood. This study identified Protein Phosphatase 2A (PP2A), and specifically a STRIPAK-PP2A complex that includes the scaffolding and substrate-specificity components Cka, Strip and SLMAP, as a critical player in the retina-PE fate choice.These factors suppress ectopic retina formation in the presumptive PE and do so via the Hippo signaling axis. STRIPAK-PP2A negatively regulates Hpo kinase, and consequently its substrate Wts, to release the transcriptional co-activator Yki into the nucleus. Thus, a modular higher-order PP2A complex refines the activity of this general phosphatase to act in a precise specification of cell fate.
Nguyen, T. H., Ralbovska, A. and Kugler, J. M. (2020). RhoBTB Proteins Regulate the Hippo Pathway by Antagonizing Ubiquitination of LKB1. G3 (Bethesda). PubMed ID: 32111652
The Hippo pathway regulates growth and apoptosis. This study identifirf RhoBTB proteins as novel regulators of Hippo signaling. RhoBTB depletion in the Drosophila wing disc epithelium cooperated with Yki to drive hyperplasia into neoplasia. Depletion of RhoBTB2 caused elevated YAP activity in human cells. RhoBTB2 deficiency resulted in increased colony formation in assays for anchorage-independent growth. Evidence that RhoBTBs acts on Hippo signaling through regulation of the kinase LKB1. LKB1 protein levels were reduced upon RhoBTB2 depletion which correlated with increased LKB1 ubiquitination. Restoring LKB1 levels rescued loss of RhoBTB in Drosophila. These results suggest that RhoBTB-dependent LKB1 regulation may contribute to its tumor-suppressive function.
Keenan, S. E., Blythe, S. A., Marmion, R. A., Djabrayan, N. J., Wieschaus, E. F. and Shvartsman, S. Y. (2020). Rapid dynamics of signal-dependent transcriptional repression by Capicua. Dev Cell. PubMed ID: 32142631
Optogenetic perturbations, live imaging, and time-resolved ChIP-seq assays in Drosophila embryos were used to dissect the ERK-dependent control of the HMG-box repressor Capicua (Cic), which plays critical roles in development and is deregulated in human spinocerebellar ataxia and cancers. It was established that Cic target genes are activated before significant downregulation of nuclear localization of Cic, and their activation was demonstrated to be preceded by fast dissociation of Cic from the regulatory DNA. Both Cic-DNA binding and repression are rapidly reinstated in the absence of ERK activation, revealing that inductive signaling must be sufficiently sustained to ensure robust transcriptional response. This work provides a quantitative framework for the mechanistic analysis of dynamics and control of transcriptional repression in development.

Thursday, April 23rd - Adult Neural Development and Function

Li, H., Li, T., Horns, F., Li, J., Xie, Q., Xu, C., Wu, B., Kebschull, J. M., McLaughlin, C. N., Kolluru, S. S., Jones, R. C., Vacek, D., Xie, A., Luginbuhl, D. J., Quake, S. R. and Luo, L. (2020). Single-Cell Transcriptomes Reveal Diverse Regulatory Strategies for Olfactory Receptor Expression and Axon Targeting. Curr Biol. PubMed ID: 32059767
The regulatory mechanisms by which neurons coordinate their physiology and connectivity are not well understood. The Drosophila olfactory receptor neurons (ORNs) provide an excellent system to investigate this question. Each ORN type expresses a unique olfactory receptor, or a combination thereof, and sends their axons to a stereotyped glomerulus. Using single-cell RNA sequencing, this study identified 33 transcriptomic clusters for ORNs, and 20 were mapped to their glomerular types, demonstrating that transcriptomic clusters correspond well with anatomically and physiologically defined ORN types. Each ORN type expresses hundreds of transcription factors. Transcriptome-instructed genetic analyses revealed that (1) one broadly expressed transcription factor (Acj6) only regulates olfactory receptor expression in one ORN type and only wiring specificity in another type, (2) one type-restricted transcription factor (Forkhead) only regulates receptor expression, and (3) another type-restricted transcription factor (Unplugged) regulates both events. Thus, ORNs utilize diverse strategies and complex regulatory networks to coordinate their physiology and connectivity.
Lai, Y. W., Chu, S. Y., Li, J. C., Chen, P. L., Chen, C. H. and Yu, H. H. (2020). Visualization of Endogenous Type I TGF-beta Receptor Baboon in the Drosophila Brain. Sci Rep 10(1): 5132. PubMed ID: 32198477
The transforming growth factor beta (TGF-beta) signaling pathway is evolutionarily conserved and widely used in the animal kingdom to regulate diverse developmental processes. Prior studies have shown that Baboon (Babo), a Drosophila type I TGF-beta receptor, plays essential roles in brain development and neural circuit formation. However, the expression pattern for Babo in the developing brain has not been previously reported. This study generated a knock-in fly with a human influenza hemagglutinin (HA) tag at the C-terminus of Babo and assessed its localization. Babo::HA was primarily expressed in brain structures enriched with neurites, including the mushroom body lobe and neuropils of the optic lobe, where Babo has been shown to instruct neuronal morphogenesis. Since the babo 3' untranslated region contains a predicted microRNA-34 (miR-34) target sequence, tests were performed to see whether Babo::HA expression was affected by modulating the level of miR-34. Babo was found to be upregulated by mir-34 deletion and downregulated by miR-34 overexpression, confirming that it is indeed a miR-34 target gene. Taken together, these results demonstrate that the babo(HA) fly permits accurate visualization of endogenous Babo expression during brain development and the construction of functional neural circuits.
Hervas, R., Rau, M. J., Park, Y., Zhang, W., Murzin, A. G., Fitzpatrick, J. A. J., Scheres, S. H. W. and Si, K. (2020). Cryo-EM structure of a neuronal functional amyloid implicated in memory persistence in Drosophila. Science 367(6483): 1230-1234. PubMed ID: 32165583
How long-lived memories withstand molecular turnover is a fundamental question. Aggregates of a prion-like RNA-binding protein, cytoplasmic polyadenylation element-binding (CPEB) protein, is a putative substrate of long-lasting memories. This study isolated aggregated Drosophila CPEB, Orb2, from adult heads and determined its activity and atomic structure, at 2.6-angstrom resolution, using cryo-electron microscopy. Orb2 formed ~75-nanometer-long threefold-symmetric amyloid filaments. Filament formation transformed Orb2 from a translation repressor to an activator and "seed" for further translationally active aggregation. The 31-amino acid protofilament core adopted a cross-beta unit with a single hydrophilic hairpin stabilized through interdigitated glutamine packing. Unlike the hydrophobic core of pathogenic amyloids, the hydrophilic core of Orb2 filaments suggests how some neuronal amyloids could be a stable yet regulatable substrate of memory.
Li, G., Forero, M. G., Wentzell, J. S., Durmus, I., Wolf, R., Anthoney, N. C., Parker, M., Jiang, R., Hasenauer, J., Strausfeld, N. J., Heisenberg, M. and Hidalgo, A. (2020). A Toll-receptor map underlies structural brain plasticity. Elife 9. PubMed ID: 32066523
Experience alters brain structure, but the underlying mechanism remained unknown. Structural plasticity reveals that brain function is encoded in generative changes to cells that compete with destructive processes driving neurodegeneration. At an adult critical period, experience increases fiber number and brain size in Drosophila. This study asked if Toll receptors are involved. Tolls demarcate a map of brain anatomical domains. Focusing on Toll-2, loss of function caused apoptosis, neurite atrophy and impaired behaviour. Toll-2 gain of function and neuronal activity at the critical period increased cell number. Toll-2 induced cycling of adult progenitor cells via a novel pathway, that antagonized MyD88-dependent quiescence, and engaged Weckle and Yorkie downstream. Constant knock-down of multiple Tolls synergistically reduced brain size. Conditional over-expression of Toll-2 and wek at the adult critical period increased brain size. Through their topographic distribution, Toll receptors regulate neuronal number and brain size, modulating structural plasticity in the adult brain.
Linneweber, G. A., Andriatsilavo, M., Dutta, S. B., Bengochea, M., Hellbruegge, L., Liu, G., Ejsmont, R. K., Straw, A. D., Wernet, M., Hiesinger, P. R. and Hassan, B. A. (2020). A neurodevelopmental origin of behavioral individuality in the Drosophila visual system. Science 367(6482): 1112-1119. PubMed ID: 32139539
The genome versus experience dichotomy has dominated understanding of behavioral individuality. By contrast, the role of nonheritable noise during brain development in behavioral variation is understudied. Using Drosophila melanogaster, this study demonstrated a link between stochastic variation in brain wiring and behavioral individuality. A visual system circuit called the dorsal cluster neurons (DCN; ~40 clustered neurons located in the dorso-lateral central brain) shows nonheritable, interindividual variation in right/left wiring asymmetry and controls object orientation in freely walking flies. DCN wiring asymmetry instructs an individual's object responses: The greater the asymmetry, the better the individual orients toward a visual object. Silencing DCNs abolishes correlations between anatomy and behavior, whereas inducing DCN asymmetry suffices to improve object responses.
Keles, M. F., Hardcastle, B. J., Stadele, C., Xiao, Q. and Frye, M. A. (2020). Inhibitory interactions and columnar inputs to an object motion detector in Drosophila. Cell Rep 30(7): 2115-2124. PubMed ID: 32075756
The direction-selective T4/T5 cells innervate optic-flow processing projection neurons in the lobula plate of the fly that mediate the visual control of locomotion. In the lobula, visual projection neurons coordinate complex behavioral responses to visual features, however, the input circuitry and computations that bestow their feature-detecting properties are less clear. A highly specialized small object motion detector, LC11, was studied, and its responses were shown to be suppressed by local background motion. LC11 expresses GABA-A receptors that serve to sculpt responses to small objects but are not responsible for the rejection of background motion. Instead, LC11 is innervated by columnar T2 and T3 neurons that are themselves highly sensitive to small static or moving objects, insensitive to wide-field motion and, unlike T4/T5, respond to both ON and OFF luminance steps.

Wednesday, April 21st - RNA

Kluge, F., Goetze, M. and Wahle, E. (2020). Establishment of 5'-3' interactions in mRNA independent of a continuous ribose-phosphate backbone. RNA. PubMed ID: 32111664
The functions of eukaryotic mRNAs are characterized by intramolecular interactions between their 5' and 3' ends. This study has addressed the question whether such 5'-3' interactions are established by diffusion-controlled encounter of the ends 'through solution' or by some type of scanning along the RNA backbone. For this purpose, in vitro translation system derived from Drosophila embryo extract was used that displays two types of 5'-3' interactions: cap-dependent translation initiation is stimulated by the poly(A) tail and inhibited by Smaug Recognition Elements (SREs) in the 3' UTR. Chimeric RNAs were constructed in which a luciferase open reading frame was separated from SREs and the poly(A) tail by a protein linker. Stimulation of translation by the poly(A) tail was fully functional with such RNAs even when disruption of the RNA backbone was combined with an inversion of the 5'-3' polarity between open reading frame and poly(A) segment. The stimulatory effect of the poly(A) tail also became weaker with increasing distance between the 5' end and the poly(A) segment. Both observations suggest that contacts between the poly(A) tail and the 5' end are established through solution, independently of the RNA backbone. In the same RNA constructs, SRE-dependent inhibition of translation was also insensitive to disruption of the RNA backbone. Thus, tracking of the RNA backbone is excluded as a mechanism for repression of cap-dependent initiation. However, SRE-dependent repression was insensitive to mRNA length, suggesting the possibility that the contact between the SREs in the 3' UTR and the 5' end of the RNA is established in a manner that differs from the contact between poly(A) tail and the cap.
Lim, D. H., Lee, S., Choi, M. S., Han, J. Y., Seong, Y., Na, D., Kwon, Y. S. and Lee, Y. S. (2020). The conserved microRNA miR-8-3p coordinates the expression of V-ATPase subunits to regulate ecdysone biosynthesis for Drosophila metamorphosis. FASEB J. PubMed ID: 32196731
The steroid hormone ecdysone is the central regulator of insect metamorphosis, during which a growing, immature larva is remodeled, through pupal stages, to a reproductive adult. However, the underlying mechanisms of ecdysone-mediated metamorphosis remain to be fully elucidated. This study identified metamorphosis-associated microRNAs (miRNAs) and their potential targets by cross-linking immunoprecipitation coupled with deep sequencing of endogenous Argonaute 1 protein in Drosophila. Interestingly, miR-8-3p targeted five Vha genes encoding distinct subunits of vacuolar H(+) -ATPase (V-ATPase; see Vha68-2), which has a vital role in the organellar acidification. The expression of ecdysone-responsive miR-8-3p is normally downregulated during Drosophila metamorphosis, but temporary overexpression of miR-8-3p in the whole body at the end of larval development led to defects in metamorphosis and survival, hallmarks of aberrant ecdysone signaling. In addition, miR-8-3p was expressed in the prothoracic gland (PG), which produces and releases ecdysone in response to prothoracicotropic hormone (PTTH). Notably, overexpression of miR-8-3p or knockdown of its Vha targets in the PG resulted in larger than normal, ecdysone-deficient larvae that failed to develop into the pupal stage but could be rescued by ecdysone feeding. Moreover, these animals showed defective PTTH signaling with a concomitant decrease in the expression of ecdysone biosynthetic genes. Tnis study also demonstrated that the regulatory network between the conserved miR-8-3p/miR-200 family and V-ATPase was functional in human cells. Consequently, these data indicate that the coordinated regulation of V-ATPase subunits by miR-8-3p is involved in Drosophila metamorphosis by controlling the ecdysone biosynthesis.
Ramat, A., Garcia-Silva, M. R., Jahan, C., Nait-Saidi, R., Dufourt, J., Garret, C., Chartier, A., Cremaschi, J., Patel, V., Decourcelle, M., Bastide, A., Juge, F. and Simonelig, M. (2020). The PIWI protein Aubergine recruits eIF3 to activate translation in the germ plasm. Cell Res. PubMed ID: 32132673
Piwi-interacting RNAs (piRNAs) and PIWI proteins are essential in germ cells to repress transposons and regulate mRNAs. In Drosophila, piRNAs bound to the PIWI protein Aubergine (Aub) are transferred maternally to the embryo and regulate maternal mRNA stability through two opposite roles. They target mRNAs by incomplete base pairing, leading to their destabilization in the soma and stabilization in the germ plasm. This study reports a function of Aub in translation. Aub is required for translational activation of nanos mRNA, a key determinant of the germ plasm. Aub physically interacts with the poly(A)-binding protein (PABP) and the translation initiation factor eIF3. Polysome gradient profiling reveals the role of Aub at the initiation step of translation. In the germ plasm, PABP and eIF3d assemble in foci that surround Aub-containing germ granules, and Aub acts with eIF3d to promote nanos translation. These results identify translational activation as a new mode of mRNA regulation by Aub, highlighting the versatility of PIWI proteins in mRNA regulation.
Kotov, A. A., Godneeva, B. K., Olenkina, O. M., Adashev, V. E., Trostnikov, M. V. and Olenina, L. V. (2020). The Drosophila RNA Helicase Belle (DDX3) Non-Autonomously Suppresses Germline Tumorigenesis Via Regulation of a Specific mRNA Set. Cells 9(3). PubMed ID: 32111103
DDX3 subfamily DEAD-box RNA helicases are essential developmental regulators of RNA metabolism in eukaryotes. Belle, the single DDX3 ortholog in Drosophila, is required for fly viability, fertility, and germline stem cell maintenance. Direct targets of Belle in the testes are essentially unknown. Tnis study showed that belle RNAi knockdown in testis cyst cells caused a disruption of adhesion between germ and cyst cells and generation of tumor-like clusters of stem-like germ cells. Ectopic expression of beta-integrin in cyst cells rescued early stages of spermatogenesis in belle knockdown testes, indicating that integrin adhesion complexes are required for the interaction between somatic and germ cells in a cyst. To address Belle functions in spermatogenesis in detail cross-linking immunoprecipitation and sequencing (CLIP-seq) analysis were performed and multiple mRNAs were identified. that interacted with Belle in the testes. The set of Belle targets includes transcripts of proteins that are essential for preventing the tumor-like clusters of germ cells and for sustaining spermatogenesis. It is hypothesized that failures in the translation of a number of mRNA targets additively contribute to developmental defects observed in the testes with belle knockdowns both in cyst cells and in the germline.
Laver, J. D., Ly, J., Winn, A. K., Karaiskakis, A., Lin, S., Nie, K., Benic, G., Jaberi-Lashkari, N., Cao, W. X., Khademi, A., Westwood, J. T., Sidhu, S. S., Morris, Q., Angers, S., Smibert, C. A. and Lipshitz, H. D. (2020). The RNA-binding protein Rasputin/G3BP enhances the stability and translation of its target mRNAs. Cell Rep 30(10): 3353-3367. PubMed ID: 32160542
G3BP RNA-binding proteins are important components of stress granules (SGs), dense aggregations in the cytosol composed of proteins & RNAs that appear when the cell is under stress. This study analyze the role of the Drosophila G3BP Rasputin (RIN) in unstressed cells, where RIN is not SG associated. Immunoprecipitation followed by microarray analysis identifies over 550 mRNAs that copurify with RIN. The mRNAs found in SGs are long and translationally silent. In contrast, it was found that RIN-bound mRNAs, which encode core components of the transcription, splicing, and translation machinery, are short, stable, and highly translated. RIN was shown to be associated with polysomes and evidence was provided for a direct role for RIN and its human homologs in stabilizing and upregulating the translation of their target mRNAs. It is proposed that when cells are stressed, the resulting incorporation of RIN/G3BPs into SGs sequesters them away from their short target mRNAs. This would downregulate the expression of these transcripts, even though they are not incorporated into stress granules.
Prieto-Sanchez, S., Moreno-Castro, C., Hernandez-Munain, C. and Sune, C. (2020). Drosophila Prp40 localizes to the histone locus body and regulates gene transcription and development. J Cell Sci. PubMed ID: 32094262
In eukaryotes, a large amount of histones must be synthesized during the S phase of the cell cycle to package newly synthesized DNA into chromatin. The transcription and 3' end processing of histone pre-mRNA are controlled by the histone locus body (HLB), which is assembled in the H3/H4 promoter. This study identified the Drosophila Prp40 pre-mRNA processing factor (dPrp40) as a novel HLB component. dPrp40 was shown to be essential for Drosophila development, with functionally conserved activity in vertebrates and invertebrates. dPrp40 was observed to be fundamental in endocycling cells, highlighting a role for this factor in mediating replication efficiency in vivo The depletion of dPrp40 from fly cells inhibited the transcription but not the 3' end processing of histone mRNA in a H3/H4 promoter-dependent manner. These results establish that dPrp40 is an essential gene for Drosophila development that can localize to the HLB and may participate in histone mRNA biosynthesis.

Tuesday, April 21 - Chromatin and DNA Replication

Armstrong, R. L., Das, S., Hill, C. A., Duronio, R. J. and Nordman, J. T. (2020). Rif1 Functions in a Tissue-Specific Manner To Control Replication Timing Through Its PP1-binding Motif. Genetics. PubMed ID: 32144132
Replication initiation in eukaryotic cells occurs asynchronously throughout S phase, yielding early and late replicating regions of the genome, a process known as replication timing (RT). RT changes during development to ensure accurate genome duplication and maintain genome stability. To understand the relative contributions that cell lineage, cell cycle, and replication initiation regulators have on RT, this study used the powerful developmental systems available in Drosophila melanogaster. RT profiles were generated and compared from mitotic cells of different tissues and from mitotic and endocycling cells of the same tissue. The results demonstrate that cell lineage has the largest effect on RT, whereas switching from a mitotic to an endoreplicative cell cycle has little to no effect on RT. Additionally, it was demonstrated that the RT differences that were observed in all cases are largely independent of transcriptional differences. A genetic approach was employed in these same cell types to understand the relative contribution the eukaryotic RT control factor, Rif1, has on RT control. The results demonstrate that Rif1 can function in a tissue-specific manner to control RT. Importantly, the Protein Phosphatase 1 (PP1) binding motif of Rif1 is essential for Rif1 to regulate RT. Together, these data support a model in which the RT program is primarily driven by cell lineage and is further refined by Rif1/PP1 to ultimately generate tissue-specific RT programs. ">
Fasulo, B., Meccariello, A., Morgan, M., Borufka, C., Papathanos, P. A. and Windbichler, N. (2020). A fly model establishes distinct mechanisms for synthetic CRISPR/Cas9 sex distorters. PLoS Genet 16(3): e1008647. PubMed ID: 32168334
Synthetic sex distorters have recently been developed in the malaria mosquito, relying on endonucleases that target the X-chromosome during spermatogenesis. Although inspired by naturally-occurring traits, it has remained unclear how they function and, given their potential for genetic control, how portable this strategy is across species. This study established Drosophila models for two distinct mechanisms for CRISPR/Cas9 sex-ratio distortion-"X-shredding" and "X-poisoning"-and dissected their target-site requirements and repair dynamics. X-shredding resulted in sex distortion when Cas9 endonuclease activity occurred during the meiotic stages of spermatogenesis but not when Cas9 was expressed from the stem cell stages onwards. Tnese results suggest that X-shredding is counteracted by the NHEJ DNA repair pathway and can operate on a single repeat cluster of non-essential sequences, although the targeting of a number of such repeats had no effect on the sex ratio. X-poisoning by contrast, i.e. targeting putative haplolethal genes on the X chromosome, induced a high bias towards males (>92%) when Cas9 cleavage was directed to the X-linked ribosomal target gene RpS6. In the case of X-poisoning sex distortion was coupled to a loss in reproductive output, although a dominant-negative effect appeared to drive the mechanism of female lethality. These model systems will guide the study and the application of sex distorters to medically or agriculturally important insect target species.
Climent-Canto, P., Carbonell, A., Tatarski, M., Reina, O., Bujosa, P., Font-Mateu, J., Bernues, J., Beato, M. and Azorin, F. (2020). The embryonic linker histone dBigH1 alters the functional state of active chromatin. Nucleic Acids Res. PubMed ID: 32103264
Linker histones H1 are principal chromatin components, whose contribution to the epigenetic regulation of chromatin structure and function is not fully understood. In metazoa, specific linker histones are expressed in the germline, with female-specific H1s being normally retained in the early-embryo. Embryonic H1s are present while the zygotic genome is transcriptionally silent and they are replaced by somatic variants upon activation, suggesting a contribution to transcriptional silencing. This study directly address this question by ectopically expressing dBigH1 in Drosophila S2 cells, which lack dBigH1. dBigH1 was shown to bind across chromatin, replaces somatic histon H1 and reduces nucleosome repeat length (NRL). Concomitantly, dBigH1 expression down-regulates gene expression by impairing RNApol II binding and histone acetylation. These effects depend on the acidic N-terminal ED-domain of dBigH1 since a truncated form lacking this domain binds across chromatin and replaces dH1 like full-length dBigH1, but it does not affect NRL either transcription. In vitro reconstitution experiments using Drosophila preblastodermic embryo extracts corroborate these results. Altogether these results suggest that the negatively charged N-terminal tail of dBigH1 alters the functional state of active chromatin compromising transcription.
Dong, Y., Avva, S., Maharjan, M., Jacobi, J. and Hart, C. M. (2020). Promoter-Proximal Chromatin Domain Insulator Protein BEAF Mediates Local and Long-Range Communication with a Transcription Factor and Directly Activates a Housekeeping Promoter in Drosophila. Genetics. PubMed ID: 32179582
BEAF (Boundary Element-Associated Factor) was originally identified as a Drosophila melanogaster chromatin domain insulator binding protein, suggesting a role in gene regulation through chromatin organization and dynamics. Genome-wide mapping found that BEAF usually binds near transcription start sites, often of housekeeping genes, suggesting a role in promoter function. This would be a nontraditional role for an insulator binding protein. To gain insight into molecular mechanisms of BEAF function, interacting proteins were identified using yeast 2-hybrid assays. This study focused on the transcription factor Sry-delta. Interactions were confirmed in pull-down experiments using bacterially expressed proteins, by bimolecular fluorescence complementation, and in a genetic assay in transgenic flies. Sry-delta interacted with promoter-proximal BEAF both when bound to DNA adjacent to BEAF or over 2 kb upstream to activate a reporter gene in transient transfection experiments. The interaction between BEAF and Sry-delta was detected using both a minimal developmental promoter (y) and a housekeeping promoter (RpS12), while BEAF alone strongly activated the housekeeping promoter. These two functions for BEAF implicate it in playing a direct role in gene regulation at hundreds of BEAF-associated promoters.
Kochanova, N. Y., Schauer, T., Mathias, G. P., Lukacs, A., Schmidt, A., Flatley, A., Schepers, A., Thomae, A. W. and Imhof, A. (2020). A multi-layered structure of the interphase chromocenter revealed by proximity-based biotinylation. Nucleic Acids Res. PubMed ID: 32182352
During interphase centromeres often coalesce into a small number of chromocenters, which can be visualized as distinct, DAPI dense nuclear domains. Intact chromocenters play a major role in maintaining genome stability as they stabilize the transcriptionally silent state of repetitive DNA while ensuring centromere function. Despite its biological importance, relatively little is known about the molecular composition of the chromocenter or the processes that mediate chromocenter formation and maintenance. To provide a deeper molecular insight into the composition of the chromocenter and to demonstrate the usefulness of proximity-based biotinylation as a tool to investigate those questions, super resolution microscopy and proximity-based biotinylation experiments were performed of three distinct proteins associated with the chromocenter in Drosophila, CenpA, HMR and HP1a. This work revealed an intricate internal architecture of the chromocenter suggesting a complex multilayered structure of this intranuclear domain.
Johnston, J. S., Zapalac, M. E. and Hjelmen, C. E. (2020). Flying High-Muscle-Specific Underreplication in Drosophila. Genes (Basel) 11(3). PubMed ID: 32111003
Drosophila underreplicate the DNA of thoracic nuclei, stalling during S phase at a point that is proportional to the total genome size in each species. In polytene tissues, such as the Drosophila salivary glands, all of the nuclei initiate multiple rounds of DNA synthesis and underreplicate. Yet, only half of the nuclei isolated from the thorax stall; the other half do not initiate S phase. To address this problem, flow cytometry was used to compare underreplication phenotypes between thoracic tissues. When individual thoracic tissues are dissected and the proportion of stalled DNA synthesis is scored in each tissue type, it was found that underreplication occurs in the indirect flight muscle, with the majority of underreplicated nuclei in the dorsal longitudinal muscles (DLM). Half of the DNA in the DLM nuclei stall at S phase between the unreplicated G0 and fully replicated G1. The dorsal ventral flight muscle provides the other source of underreplication, and yet, there, the replication stall point is earlier (less DNA replicated), and the endocycle is initiated. The differences in underreplication and ploidy in the indirect flight muscles provide a new tool to study heterochromatin, underreplication and endocycle control.

Monday, April 20th - Embryonic Development

Pridie, C. and Simmonds, A. (2020). The role of Peroxin 7 during Drosophila embryonic development. Genome. PubMed ID: 32191843
Peroxisomes are organelles in eukaryotic cells responsible for processing several types of lipids and managing reactive oxygen species. A conserved family of peroxisome biogenesis (Peroxin, Pex) genes encode proteins essential to peroxisome biogenesis and function. In yeast and mammals, PEROXIN7 (PEX7) acts as a cytosolic receptor protein that targets enzymes containing a peroxisome targeting sequence 2 (PTS2) motif for peroxisome matrix import. The PTS2 motif is not present in the Drosophila melanogaster homologs of these enzymes. However, the fly genome contains a Pex7 gene (CG6486) that is very similar to yeast and human PEX7. Pex7 is expressed in tissue-specific patterns analogous to differentiating neuroblasts in D. melanogaster embryos. This is correlated with a requirement for Pex7 in this cell lineage as targeted somatic Pex7 knockout in embryonic neuroblasts reduced survival. Pex7 over-expression in the same cell lineages caused lethality during the larval stage. Targeted somatic over-expression of a Pex7 transgene in neuroblasts of Pex7 homozygous null mutants resulted in a semi-lethal phenotype similar to targeted Pex7 knockout. These findings suggest that D. melanogaster has tissue-specific requirements for Pex7 during embryo development.
Jiyun, O. and ChCoe, C. P. (2020). even-skipped acts as a pair-rule gene in germ band stages of Tribolium development. Dev Biol. PubMed ID: 32179089
The pair-rule gene even-skipped (eve) is essential for insect segmentation, yet its function varies among insect clades. In Tribolium, knock-down of the eve ortholog (Tc-eve) resulted in a graded phenotypic series ranging from strong to weak, the most informative of which was intermediate phenotypes. The strong knock-down embryos displayed asegmental phenotypes and severely disorganized germ bands which have prevented determination of Tc-eve function in later stages. In order to understand the segmentation function of Tc-eve during later germ band elongation stages, intermediate Tc-eve(RNAi) embryos were analyzed in which germ band elongation was less affected. Most intermediate Tc-eve(RNAi) germ bands displayed segmentation defects with a double segmental periodicity in the abdomen. In these intermediate embryos, Tc-engrailed (Tc-en) stripes were ectopically expanded into large bands with a double segmental periodicity, while the remaining Tc-en stripes between the expanded Tc-en stripes were absent or barely formed. The expanded Tc-en stripes seemed to be activated by primary Tc-eve stripes and Tc-paired, both of which failed to resolve into secondary segmental stripes. The absence of Tc-en stripes appeared to be a consequence of the absence of the secondary stripes of Tc-runt that were required for the activation of Tc-en stripes. These results suggest that Tc-eve functions as a pair-rule gene at least in the germ band stages of Tribolium development.
Dold, A., Han, H., Liu, N., Hildebrandt, A., Bruggemann, M., Ruckle, C., Hanel, H., Busch, A., Beli, P., Zarnack, K., Konig, J., Roignant, J. Y. and Lasko, P. (2020). Makorin 1 controls embryonic patterning by alleviating Bruno1-mediated repression of oskar translation. PLoS Genet 16(1): e1008581. PubMed ID: 31978041
Makorins are evolutionary conserved proteins that contain C3H-type zinc finger modules and a RING E3 ubiquitin ligase domain. In Drosophila, maternal Makorin 1 (Mkrn1) has been linked to embryonic patterning but the mechanism remained unsolved. This study shows that Mkrn1 is essential for axis specification and pole plasm assembly by translational activation of oskar (osk). Mkrn1 interacts with poly(A) binding protein (pAbp) and binds specifically to osk 3' UTR in a region adjacent to A-rich sequences. Using Drosophila S2R+ cultured cells this study shows that this binding site overlaps with a Bruno1 (Bru1) responsive element (BREs) that regulates osk translation. Increased association of the translational repressor Bru1 with osk mRNA was observed upon depletion of Mkrn1, indicating that both proteins compete for osk binding. Consistently, reducing Bru1 dosage partially rescues viability and Osk protein level in ovaries from Mkrn1 females. It is concluded that Mkrn1 controls embryonic patterning and germ cell formation by specifically activating osk translation, most likely by competing with Bru1 to bind to osk 3' UTR.
Lavergne, G., Zmojdzian, M., Da Ponte, J. P., Junion, G. and Jagla, K. (2020). Drosophila adult muscle precursor cells contribute to motor axon pathfinding and proper innervation of embryonic muscles. Development. PubMed ID: 32001438
Despites several decades of studies on the neuromuscular system, the relationship between muscle stem cells and motor neurons remains elusive. Using the Drosophila model, evidences are provided that adult muscle precursors (AMPs), the Drosophila muscle stem cells, interact with the motor axons during embryogenesis. AMPs not only hold the capacity to attract the navigating intersegmental (ISN) and segmental a (SNa) nerve branches, but are also mandatory to the innervation of muscles in the lateral field. This so far ignored AMPs role involves their filopodia-based interactions with nerve growth cones. In parallel, the previously undetected expression of encoding guidance molecules sidestep and side IV in AMPs is reported. Altogether, this data supports the view that Drosophila muscle stem cells represent spatial landmarks for navigating motor neurons and reveal that their positioning is critical for the muscles innervation in the lateral region. Furthermore, AMPs and motor axons are interdependent as the genetic ablation of SNa leads to a specific loss of SNa-associated lateral AMPs.
Nielsen, B. F., Nissen, S. B., Sneppen, K., Mathiesen, J. and Trusina, A. (2020). Model to Link Cell Shape and Polarity with Organogenesis. iScience 23(2): 100830. PubMed ID: 31986479
How do flat sheets of cells form gut and neural tubes? Across systems, several mechanisms are at play: cells wedge, form actomyosin cables, or intercalate. As a result, the cell sheet bends, and the tube elongates. It is unclear to what extent each mechanism can drive tube formation on its own. To address this question, this study computationally probe if one mechanism, either cell wedging or intercalation, may suffice for the entire sheet-to-tube transition. Using a physical model with epithelial cells represented by polarized point particles, it was shown that either cell intercalation or wedging alone can be sufficient and that each can both bend the sheet and extend the tube. When working in parallel, the two mechanisms increase the robustness of the tube formation. The successful simulations of the key features in Drosophila salivary gland budding, sea urchin gastrulation, and mammalian neurulation support the generality of these results.
Tariq, A., Green, L., Jeynes, J. C. G., Soeller, C. and Wakefield, J. G. (2020). In vitro reconstitution of branching microtubule nucleation. Elife 9. PubMed ID: 31933481
Eukaryotic cell division requires the mitotic spindle, a microtubule (MT)-based structure which accurately aligns and segregates duplicated chromosomes. The dynamics of spindle formation are determined primarily by correctly localising the MT nucleator, gamma-Tubulin Ring Complex (gamma-TuRC), within the cell. A conserved MT-associated protein complex, Augmin, recruits gamma-TuRC to pre-existing spindle MTs, amplifying their number, in an essential cellular phenomenon termed 'branching' MT nucleation. This study purified endogenous, GFP-tagged Augmin and gamma-TuRC from Drosophila embryos to near homogeneity using a novel one-step affinity technique. In vitro, while Augmin alone does not affect Tubulin polymerisation dynamics, it stimulates gamma-TuRC-dependent MT nucleation in a cell cycle-dependent manner. This study also assembled and visualised the MT-Augmin-gamma-TuRC-MT junction using light microscopy. This work therefore conclusively reconstitutes branching MT nucleation. It also provides a powerful synthetic approach with which to investigate the emergence of cellular phenomena, such as mitotic spindle formation, from component parts.

Friday, April 17th - Signaling

Rui, M., Ng, K. S., Tang, Q., Bu, S. and Yu, F. (2020). Protein phosphatase PP2A regulates microtubule orientation and dendrite pruning in Drosophila. EMBO Rep: e48843. PubMed ID: 32187821
Pruning that selectively eliminates inappropriate projections is crucial for sculpting neural circuits during development. During Drosophila metamorphosis, ddaC sensory neurons undergo dendrite-specific pruning in response to the steroid hormone ecdysone. However, the understanding of the molecular mechanisms underlying dendrite pruning remains incomplete. This study shows that protein phosphatase 2A (PP2A) is required for dendrite pruning. The catalytic (Microtubule star/Mts), scaffolding (PP2A-29B), and two regulatory subunits (Widerborst/Wdb and Twins/Tws) play important roles in dendrite pruning. Functional analyses indicate that PP2A, via Wdb, facilitates the expression of Sox14 and Mical prior to dendrite pruning. Furthermore, PP2A, via Tws, governs the minus-end-out orientation of microtubules (MTs) in the dendrites. Moreover, the levels of Klp10A, a MT depolymerase, increase when PP2A is compromised. Attenuation of Klp10A fully rescues the MT orientation defects in mts or pp2a-29b RNAi ddaC neurons, suggesting that PP2A governs dendritic MT orientation by suppressing Klp10A levels and/or function. Taken together, this study sheds light on a novel function of PP2A in regulating dendrite pruning and dendritic MT polarity in sensory neurons.
Harsh, S., Fu, Y., Kenney, E., Han, Z. and Eleftherianos, I. (2020). Zika virus non-structural protein NS4A restricts eye growth in Drosophila through regulation of JAK/STAT signaling. Dis Model Mech. PubMed ID: 32152180
RNA-seq analysis revealed that Zika virus (ZIKV) infection alters several and diverse biological processes. To explore the interaction between ZIKV infection and JAK/STAT signaling regulation, genetic constructs were generated overexpressing ZIKV-specific non-structural proteins NS2A, NS2B, NS4A and NS4B. Ectopic expression of non-structural proteins in the developing Drosophila eye significantly restricts growth of the larval and adult eye and correlates with a considerable repression of an in vivo JAK/STAT reporter. At the cellular level, eye growth defects are associated with reduced rate of proliferation without affecting the overall rate of apoptosis. In addition, ZIKV NS4A genetically interacts with the JAK/STAT signaling components; co-expression of NS4A along with dominant negative form of domeless or StatRNAi results in aggravated reduction in eye size while co-expression of NS4A in HopTuml mutant background partially rescues the Hop-induced eye overgrowth phenotype. This study provides evidence that ZIKV infection in Drosophila results in restricted growth of the developing eye and wing, wherein eye phenotype is induced through regulation of JAK/STAT signaling while restricted wing growth is through regulation of Notch signaling. The interaction of ZIKV non-structural proteins with the conserved host signaling pathways further advance understanding of ZIKV-induced pathogenesis.
Rallis, A., Navarro, J. A., Rass, M., Hu, A., Birman, S., Schneuwly, S. and Therond, P. P. (2020). Hedgehog Signaling Modulates Glial Proteostasis and Lifespan. Cell Rep 30(8): 2627-2643.e2625. PubMed ID: 32101741
The conserved Hedgehog signaling pathway has well-established roles in development. However, its function during adulthood remains largely unknown. This study investigated whether the Hedgehog signaling pathway is active during adult life in Drosophila melanogaster, and a protective function was uncovered for Hedgehog signaling in coordinating correct proteostasis in glial cells. Adult-specific depletion of Hedgehog reduces lifespan, locomotor activity, and dopaminergic neuron integrity. Conversely, increased expression of Hedgehog extends lifespan and improves fitness. Moreover, Hedgehog pathway activation in glia rescues the lifespan and age-associated defects of hedgehog mutants. The Hedgehog pathway regulates downstream chaperones, whose overexpression in glial cells was sufficient to rescue the shortened lifespan and proteostasis defects of hedgehog mutants. Finally, the protective ability of Hedgehog signaling was demonstrated in a Drosophila Alzheimer's disease model expressing human amyloid beta in the glia. Overall, it is proposed that Hedgehog signaling is requisite for lifespan determination and correct proteostasis in glial cells.
Ponte, S., Carvalho, L., Gagliardi, M., Campos, I., Oliveira, P. J. and Jacinto, A. (2020). Drp1-mediated mitochondrial fission regulates calcium and F-actin dynamics during wound healing. Biol Open. PubMed ID: 32184231
Mitochondria adapt to cellular needs by changes in morphology through fusion and fission events, referred to as mitochondrial dynamics. Mitochondrial function and morphology are intimately connected and the dysregulation of mitochondrial dynamics is linked to several human diseases. This work investigated the role of mitochondrial dynamics in wound healing in the Drosophila embryonic epidermis. Mutants for mitochondrial fusion and fission proteins fail to close their wounds, indicating that the regulation of mitochondrial dynamics is required for wound healing. By live-imaging, loss of function of the mitochondrial fission protein Dynamin-related protein 1 (Drp1) was found to compromise the increase of cytosolic and mitochondrial calcium upon wounding and leads to reduced ROS production and F-actin defects at the wound edge, culminating in wound healing impairment.These results highlight a new role for mitochondrial dynamics in the regulation of calcium, ROS and F-actin during epithelial repair.
Rozbesky, D., Monistrol, J., Jain, V., Hillier, J., Padilla-Parra, S. and Jones, E. Y. (2020). Drosophila OTK Is a Glycosaminoglycan-Binding Protein with High Conformational Flexibility. Structure. PubMed ID: 32187531
The transmembrane protein OTK plays an essential role in plexin and Wnt signaling during Drosophila development. This study has determined a crystal structure of the last three domains of the OTK ectodomain and found that OTK shows high conformational flexibility resulting from mobility at the interdomain interfaces. Detection of direct binding between Drosophila Plexin A (PlexA) and OTK failed, that was suggested previously. Instead of PlexA, OTK directly binds semaphorin 1a. This binding analyses further revealed that glycosaminoglycans, heparin and heparan sulfate, are ligands for OTK and thus may play a role in the Sema1a-PlexA axon guidance system.
Holly, R. W., Jones, K. and Prehoda, K. E. (2020). A Conserved PDZ-Binding Motif in aPKC Interacts with Par-3 and Mediates Cortical Polarity. Curr Biol 30(5): 893-898. PubMed ID: 32084408
Par-3 regulates animal cell polarity by targeting the Par complex proteins Par-6 and atypical protein kinase C (aPKC) to specific cortical sites. Although numerous physical interactions between Par-3 and the Par complex have been identified, this study discovered a novel interaction between Par-3's second PDZ domain and a highly conserved aPKC PDZ-binding motif (PBM) that is required in the context of the full-length, purified Par-6-aPKC complex. This study also found that Par-3 is phosphorylated by the full Par complex and phosphorylation induces dissociation of the Par-3 phosphorylation site from aPKC's kinase domain but does not disrupt the Par-3 PDZ2-aPKC PBM interaction. In asymmetrically dividing Drosophila neuroblasts, the aPKC PBM is required for cortical targeting, consistent with its role in mediating a persistent interaction with Par-3. These results define a physical connection that targets the Par complex to polarized sites on the cell membrane.

Thursday, April 16th - RNA and Transposons

Gamez-Visairas, V., Romero-Soriano, V., Marti-Carreras, J., Segarra-Carrillo, E. and Garcia Guerreiro, M. P. (2020). Drosophila Interspecific Hybridization Causes A Deregulation of the piRNA Pathway Genes. Genes (Basel) 11(2). PubMed ID: 32092860
Almost all eukaryotes have transposable elements (TEs) against which they have developed defense mechanisms. In the Drosophila germline, the main transposable element (TE) regulation pathway is mediated by specific Piwi-interacting small RNAs (piRNAs). Nonetheless, for unknown reasons, TEs sometimes escape cellular control during interspecific hybridization processes. Because the piRNA pathway genes are involved in piRNA biogenesis and TE control, nine key genes from this pathway were sequenced and characterized in Drosophila buzzatii and Drosophila koepferae species, and their expression pattern in ovaries of both species and their F1 hybrids was studied. It was found that gene structure is, in general, maintained between both species and that two genes-armitage and aubergine-are under positive selection. Three genes-krimper, methyltransferase 2, and zucchini-displayed higher expression values in hybrids than both parental species, while others had RNA levels similar to the parental species with the highest expression. This suggests that the overexpression of some piRNA pathway genes can be a primary response to hybrid stress. Therefore, these results reinforce the hypothesis that TE deregulation may be due to the protein incompatibility caused by the rapid evolution of these genes, leading to a TE silencing failure, rather than to an underexpression of piRNA pathway genes.
Hemmer, L. W., Dias, G. B., Smith, B., Van Vaerenberghe, K., Howard, A., Bergman, C. M. and Blumenstiel, J. P. (2020). Hybrid dysgenesis in Drosophila virilis results in clusters of mitotic recombination and loss-of-heterozygosity but leaves meiotic recombination unaltered. Mob DNA 11: 10. PubMed ID: 32082426
Transposable elements (TEs) are endogenous mutagens and their harmful effects are especially evident in syndromes of hybrid dysgenesis. In Drosophila virilis, hybrid dysgenesis is a syndrome of incomplete gonadal atrophy that occurs when males with multiple active TE families fertilize females that lack active copies of the same families. This study sought to determine how the landscape of germline recombination is affected by parental TE asymmetry. Recombination rate and TE density were shown to be negatively correlated in this species. Then recombination events in the germline of dysgenic versus non-dysgenic F1 females were contrasted to show that the landscape of meiotic recombination is hardly perturbed during hybrid dysgenesis. In contrast, hybrid dysgenesis in the female germline increases transmission of chromosomes with mitotic recombination. Clusters of mitotic recombination events in dysgenic females were shown to be associated with genomic regions with transposons implicated in hybrid dysgenesis. Overall, it is concluded that increased mitotic recombination is likely the result of early TE activation in dysgenic progeny, but a stable landscape of meiotic recombination indicates that either transposition is ameliorated in the adult female germline or that regulation of meiotic recombination is robust to ongoing transposition. These results indicate that the effects of parental TE asymmetry on recombination are likely sensitive to the timing of transposition.
Popitsch, N., Huber, C. D., Buchumenski, I., Eisenberg, E., Jantsch, M., von Haeseler, A. and Gallach, M. (2020). A-to-I RNA editing uncovers hidden signals of adaptive genome evolution in animals. Genome Biol Evol. PubMed ID: 32145015
In animals, the most common type of RNA editing is the deamination of adenosines (A) into inosines (I). Because inosines base-pair with cytosines (C), they are interpreted as guanosines (G) by the cellular machinery and genomically encoded G alleles at edited sites mimic the function of edited RNAs. The contribution of this hardwiring effect on genome evolution remains obscure. This study looked for population genomics signatures of adaptive evolution associated with A-to-I RNA edited sites in humans and Drosophila melanogaster. Single nucleotide polymorphisms at edited sites occur 3 (humans) to 15 times (Drosophila) more often than at unedited sites, the nucleotide G is virtually the unique alternative allele at edited sites and G alleles segregate at higher frequency at edited sites than at unedited sites. This study study reveals that a significant fraction of coding synonymous and nonsynonymous as well as silent and intergenic A-to-I RNA editing sites are likely adaptive in the distantly related human and Drosophila lineages.
Bergalet, J., Patel, D., Legendre, F., Lapointe, C., Benoit Bouvrette, L. P., Chin, A., Blanchette, M., Kwon, E. and Lecuyer, E. (2020). Inter-dependent Centrosomal Co-localization of the Cen and Ik2 cis-Natural Antisense mRNAs in Drosophila. Cell Rep 30(10): 3339-3352. PubMed ID: 32160541
Overlapping genes are prevalent in most genomes, but the extent to which this organization influences regulatory events operating at the post-transcriptional level remains unclear. Studying the Cen and Ik2 genes of Drosophila melanogaster, which are convergently transcribed as cis-natural antisense transcripts (cis-NATs) with overlapping 3' UTRs, it was found that their encoded mRNAs strikingly co-localize to centrosomes. These transcripts physically interact in a 3' UTR-dependent manner, and the targeting of Ik2 requires its 3' UTR sequence and the presence of Cen mRNA, which serves as the main driver of centrosomal co-localization. The Cen transcript undergoes localized translation in proximity to centrosomes, and its localization is perturbed by polysome-disrupting drugs. By interrogating global fractionation-sequencing datasets generated from Drosophila and human cellular models, this study found that RNAs expressed as cis-NATs tend to co-localize to specific subcellular fractions. This work suggests that post-transcriptional interactions between RNAs with complementary sequences can dictate their localization fate in the cytoplasm.
Diaz-Gonzalez, J. and Dominguez, A. (2020). Different structural variants of roo retrotransposon are active in Drosophila melanogaster. Gene 741: 144546. PubMed ID: 32165306
Retrotransposon roo is one of the most active elements in Drosophila melanogaster. The level of nucleotide diversity between copies of roo is very low but structural variation in the 5'-UTR is considerable. Transposition of roo at high frequency (around 5 x 10(-2) per generation) has been shown previously in the set of mutation accumulation lines named Oviedo. This study isolated thirteen individual insertions by inverse PCR and sequenced the 5' end of the elements (between 1663 and 2039 nt) including the LTR, the 5'-UTR and a fragment of 661 nucleotides from the ORF, to study whether the new transposed copies come from a unique variant (the master copy model) or different elements are able to move (the transposon model). The elements in the Oviedo lines presented the same structural variants as the reference genome. Different structural variants were active, a behaviour compatible with the "transposon model" in which the copies localized in multiple sites in the genome are able to transpose. At the level of sequence, the copies of roo in these lines are highly similar to the elements in the reference genome. The phylogenetic tree shows a shallow diversification with unsupported nodes denoting that all the elements currently active are very young. This observation together with the great polymorphism in insertion sites implies a rapid turnover of the elements.
Parhad, S. S., Yu, T., Zhang, G., Rice, N. P., Weng, Z. and Theurkauf, W. E. (2020). Adaptive Evolution Targets a piRNA Precursor Transcription Network. Cell Rep 30(8): 2672-2685. PubMed ID: 32101744
In Drosophila, transposon-silencing piRNAs are derived from heterochromatic clusters and a subset of euchromatic transposon insertions, which are bound by the Rhino-Deadlock-Cutoff complex. The HP1 homolog Rhino binds to Deadlock, which recruits TRF2 to promote non-canonical transcription from both genomic strands. Cuff function is less well understood, but this Rai1 homolog shows hallmarks of adaptive evolution, which can remodel functional interactions within host defense systems. Supporting this hypothesis, Drosophila simulans Cutoff is a dominant-negative allele when expressed in Drosophila melanogaster, in which it traps Deadlock, TRF2, and the conserved transcriptional co-repressor CtBP in stable complexes. Cutoff functions with Rhino and Deadlock to drive non-canonical transcription. In contrast, CtBP suppresses canonical transcription of transposons and promoters flanking the major germline clusters, and canonical transcription interferes with downstream non-canonical transcription and piRNA production. Adaptive evolution thus targets interactions among Cutoff, TRF2, and CtBP that balance canonical and non-canonical piRNA precursor transcription.

Wednesday, April 15th- Synapse and vesicles

Huang, S., Piao, C., Beuschel, C. B., Gotz, T. and Sigrist, S. J. (2020). Presynaptic Active Zone Plasticity Encodes Sleep Need in Drosophila. Curr Biol. PubMed ID: 32142702
Sleep is universal across species and essential for quality of life and health, as evidenced by the consequences of sleep loss. Sleep might homeostatically normalize synaptic gains made over wake states in order to reset information processing and storage and support learning, and sleep-associated synaptic (ultra)structural changes have been demonstrated recently. However, causal relationships between the molecular and (ultra)structural status of synapses, sleep homeostatic regulation, and learning processes have yet to be established. This study shows that the status of the presynaptic active zone can directly control sleep in Drosophila. Short sleep mutants showed a brain-wide upregulation of core presynaptic scaffold proteins and release factors. Increasing the gene copy number of ELKS-family scaffold master organizer Bruchpilot (BRP) not only mimicked changes in the active zone scaffold and release proteins but importantly provoked sleep in a dosage-dependent manner, qualitatively and quantitatively reminiscent of sleep deprivation effects. Conversely, reducing the brp copy number decreased sleep in short sleep mutant backgrounds, suggesting a specific role of the active zone plasticity in homeostatic sleep regulation. Finally, elimination of BRP specifically in the sleep-promoting R2 neurons of 4xBRP animals partially restored sleep patterns and rescued learning deficits. These results suggest that the presynaptic active zone plasticity driven by BRP operates as a sleep homeostatic actuator that also restricts periods of effective learning.
Stawarski, M., Hernandez, R. X., Feghhi, T., Borycz, J. A., Lu, Z., Agarwal, A., Reihl, K., Tavora, R., Lau, A. W. C., Meinertzhagen, I. A., Renden, R. and Macleod, G. T. (2020). Neuronal glutamatergic synaptic clefts alkalinize rather than acidify during neurotransmission. J Neurosci. PubMed ID: 31964719
This study used genetically-encoded fluorescent pH indicators to examine synaptic cleft pH at conventional neuronal synapses. At the neuromuscular junction of female Drosophila larvae, alkaline spikes of over 1 log unit were observed during fictive locomotion in vivo. Ex vivo, single action potentials evoked alkalinizing pH transients of only approximately 0.01 log unit, but these transients summated rapidly during burst firing. A chemical pH indicator targeted to the cleft corroborated these findings. Cleft pH transients were dependent on Ca(2+) movement across the postsynaptic membrane, rather than neurotransmitter release per se, a result consistent with cleft alkalinization being driven by the Ca(2+)/H(+) antiporting activity of the plasma membrane Ca(2+)-ATPase at the postsynaptic membrane. Targeting the pH indicators to the microenvironment of the presynaptic voltage-gated Ca(2+) channels revealed that alkalinization also occurred within the cleft proper at the active zone and not just within extra-synaptic regions. Application of the pH indicators at the mouse calyx of Held, a mammalian central synapse, similarly revealed cleft alkalinization during burst firing in both males and females. These findings, made at two quite different non-ribbon type synapses, suggest that cleft alkalinization during neurotransmission, rather than acidification, is a generalizable phenomenon across conventional neuronal synapses.
Titlow, J., Robertson, F., Jarvelin, A., Ish-Horowicz, D., Smith, C., Gratton, E. and Davis, I. (2020). Syncrip/hnRNP Q is required for activity-induced Msp300/Nesprin-1 expression and new synapse formation. J Cell Biol 219(3). PubMed ID: 32040548
Memory and learning involve activity-driven expression of proteins and cytoskeletal reorganization at new synapses, requiring posttranscriptional regulation of localized mRNA a long distance from corresponding nuclei. A key factor expressed early in synapse formation is Msp300/Nesprin-1, which organizes actin filaments around the new synapse. How Msp300 expression is regulated during synaptic plasticity is poorly understood. This study shows that activity-dependent accumulation of Msp300 in the postsynaptic compartment of the Drosophila larval neuromuscular junction is regulated by the conserved RNA binding protein Syncrip/hnRNP Q. Syncrip (Syp) binds to msp300 transcripts and is essential for plasticity. Single-molecule imaging shows that msp300 is associated with Syp in vivo and forms ribosome-rich granules that contain the translation factor eIF4E. Elevated neural activity alters the dynamics of Syp and the number of msp300:Syp:eIF4E RNP granules at the synapse, suggesting that these particles facilitate translation. These results introduce Syp as an important early acting activity-dependent regulator of a plasticity gene that is strongly associated with human ataxias.
Hong, H., Zhao, K., Huang, S., Huang, S., Yao, A., Jiang, Y., Sigrist, S., Zhao, L. and Zhang, Y. Q. (2020). Structural remodeling of active zones is associated with synaptic homeostasis. J Neurosci. PubMed ID: 32122953
Perturbations to postsynaptic glutamate receptors (GluRs) trigger retrograde signaling to precisely increase presynaptic neurotransmitter release, maintaining stable levels of synaptic strength, a process referred to as homeostatic regulation. However, the structural change of homeostatic regulation remains poorly defined. At wild-type Drosophila neuromuscular junction (NMJ) synapse, there is one Bruchpilot (Brp) ring detected by super-resolution microscopy at active zones (AZs). This study reports multiple Brp rings, i.e., multiple T-bars seen by electron microscopy, at AZs of both male and female larvae when GluRs are reduced. At GluRIIC deficient NMJs, quantal size was reduced but quantal content was increased, indicative of homeostatic presynaptic potentiation. Consistently, multiple Brp rings at AZs were observed in the two classic synaptic homeostasis models, i.e., GluRIIA mutant and pharmacological blockade of GluRIIA activity. Furthermore, postsynaptic overexpression of the cell adhesion protein Neuroligin 1 partially rescued multiple Brp rings phenotype. This study thus supports that the formation of multiple Brp rings at AZs might be a structural basis for synaptic homeostasis.
Silva-Rodrigues, J. F., Patricio-Rodrigues, C. F., de Sousa-Xavier, V., Augusto, P. M., Fernandes, A. C., Farinho, A. R., Martins, J. P. and Teodoro, R. O. (2020). Peripheral axonal ensheathment is regulated by RalA GTPase and the exocyst complex. Development 147(3). PubMed ID: 31969325
Axon ensheathment is fundamental for fast impulse conduction and the normal physiological functioning of the nervous system. Defects in axonal insulation lead to debilitating conditions, but, despite its importance, the molecular players responsible are poorly defined. This study identified RalA GTPase as a key player in axon ensheathment in Drosophila larval peripheral nerves. Genetic analysis revealed that RalA action through the exocyst complex is required in wrapping glial cells to regulate their growth and development. It is suggested that the RalA-exocyst pathway controls the targeting of secretory vesicles for membrane growth or for the secretion of a wrapping glia-derived factor that itself regulates growth. In summary, these findings provide a new molecular understanding of the process by which axons are ensheathed in vivo, a process that is crucial for normal neuronal function.
Hoshika, S., Sun, X., Kuranaga, E. and Umetsu, D. (2020). Reduction of endocytic activity accelerates cell elimination during tissue remodeling of the Drosophila epidermal epithelium. Development. PubMed ID: 32156754
Epithelial tissues undergo cell turnover both during development and for homeostatic maintenance. Cells no longer needed are quickly removed without compromising barrier function of the tissue. During metamorphosis, insects undergo developmentally programed tissue remodeling. However, the mechanisms that regulate this rapid tissue remodeling are not precisely understood. This study shows that the temporal dynamics of endocytosis modulate physiological cell properties to potentiate larval epidermal cells for cell elimination. Endocytic activity gradually reduces as tissue remodeling progresses. This reduced endocytic activity accelerates cell elimination through the regulation of Myosin II subcellular reorganization, junctional E-cadherin levels, and caspase activation. Whereas the increased Myosin II dynamics accelerates cell elimination, E-cadherin rather plays a protective role against cell elimination. Reduced E-cadherin is involved in the amplification of caspase activation by forming a positive feedback loop with caspase. These findings reveal the role of endocytosis in preventing cell elimination and in the cell property switching initiated by the temporal dynamics of endocytic activity to achieve rapid cell elimination during tissue remodeling.

Tuesday, April 14th - Physiology

Consuegra, J., Grenier, T., Baa-Puyoulet, P., Rahioui, I., Akherraz, H., Gervais, H., Parisot, N., da Silva, P., Charles, H., Calevro, F. and Leulier, F. (2020). Drosophila-associated bacteria differentially shape the nutritional requirements of their host during juvenile growth. PLoS Biol 18(3): e3000681. PubMed ID: 32196485
The interplay between nutrition and the microbial communities colonizing the gastrointestinal tract (i.e., gut microbiota) determines juvenile growth trajectory. Nutritional deficiencies trigger developmental delays, and an immature gut microbiota is a hallmark of pathologies related to childhood undernutrition. However, how host-associated bacteria modulate the impact of nutrition on juvenile growth remains elusive. Using gnotobiotic Drosophila melanogaster larvae independently associated with Acetobacter pomorumWJL (ApWJL) and Lactobacillus plantarumNC8 (LpNC8), 2 model Drosophila-associated bacteria, a large-scale, systematic nutritional screen was performed based on larval growth in 40 different and precisely controlled nutritional environments. These results were combined with genome-based metabolic network reconstruction to define the biosynthetic capacities of Drosophila germ-free (GF) larvae and its 2 bacterial partners. It was first established that ApWJL and LpNC8 differentially fulfill the nutritional requirements of the ex-GF larvae, and such difference was parsed down to individual amino acids, vitamins, other micronutrients, and trace metals. Drosophila-associated bacteria not only fortify the host's diet with essential nutrients but, in specific instances, functionally compensate for host auxotrophies by either providing a metabolic intermediate or nutrient derivative to the host or by uptaking, concentrating, and delivering contaminant traces of micronutrients. This systematic work reveals that beyond the molecular dialogue engaged between the host and its bacterial partners, Drosophila and its associated bacteria establish an integrated nutritional network relying on nutrient provision and utilization.
Gillette, C. M., Hazegh, K. E., Nemkov, T., Stefanoni, D., D'Alessandro, A., Taliaferro, J. M. and Reis, T. (2020). Gene-Diet Interactions: Dietary Rescue of Metabolic Defects in spen-depleted Drosophila melanogaster. Genetics. PubMed ID: 32107279
Obesity and its co-morbidities are a growing health epidemic. Interactions between genetic background, the environment and behavior (i.e. diet) greatly influence organismal energy balance. Previous work has described obesogenic mutations in the gene Split ends (Spen) in Drosophila melanogaster, and roles for Spen in fat storage and metabolic state. Lipid catabolism is impaired in Spen-deficient fat storage cells, accompanied by a compensatory increase in glycolytic flux and protein catabolism. This study investigated gene-diet interactions to determine if diets supplemented with specific macronutrients can rescue metabolic dysfunction in Spen-depleted animals. A high-yeast diet partially rescues adiposity and developmental defects. High sugar partially improves developmental timing as well as longevity of mated females. Gene-diet interactions were heavily influenced by developmental-stage-specific organismal needs: extra yeast provides benefits early in development (larval stages) but becomes detrimental in adulthood. High sugar confers benefits to Spen-depleted animals at both larval and adult stages, with the caveat of increased adiposity. A high-fat diet is detrimental according to all tested criteria, regardless of genotype. Whereas Spen depletion influenced phenotypic responses to supplemented diets, diet was the dominant factor in directing the whole-organism steady-state metabolome. Obesity is a complex disease of genetic, environmental, and behavioral inputs. These results show that diet customization can ameliorate metabolic dysfunction underpinned by a genetic factor.
Engelhart, D. C., Azad, P., Ali, S., Granados, J. C., Haddad, G. G. and Nigam, S. K. (2020). Drosophila SLC22 Orthologs Related to OATs, OCTs, and OCTNs Regulate Development and Responsiveness to Oxidative Stress. Int J Mol Sci 21(6). PubMed ID: 32183456
The SLC22 family of transporters play a major role in regulating homeostasis by transporting small organic molecules such as metabolites, signaling molecules, and antioxidants. Evolutionary analysis of Drosophila melanogaster putative SLC22 orthologs reveals that, while many of the 25 SLC22 fruit fly orthologs do not fall within previously established SLC22 subclades, at least four members appear orthologous to mammalian SLC22 members (SLC22A16:CG6356, SLC22A15:CG7458, CG7442 and SLC22A18:CG3168). This study functionally evaluated the role of SLC22 transporters in Drosophila melanogaster by knocking down 14 of these genes. Three putative SLC22 ortholog knockdowns-CG3168, CG6356, and CG7442/SLC22A-did not undergo eclosion and were lethal at the pupa stage, indicating the developmental importance of these genes. Additionally, knocking down four SLC22 members increased resistance to oxidative stress via paraquat testing. Consistent with recent evidence that SLC22 is central to a Remote Sensing and Signaling Network (RSSN) involved in signaling and metabolism, these phenotypes support a key role for SLC22 in handling reactive oxygen species.
Hsu, S. K., Jaksic, A. M., Nolte, V., Lirakis, M., Kofler, R., Barghi, N., Versace, E. and Schlotterer, C. (2020). Rapid sex-specific adaptation to high temperature in Drosophila. Elife 9. PubMed ID: 32083552
The pervasive occurrence of sexual dimorphism demonstrates different adaptive strategies of males and females. While different reproductive strategies of the two sexes are well-characterized, very little is known about differential functional requirements of males and females in their natural habitats. The impact environmental change on the selection response was studied in both sexes. Exposing replicated Drosophila populations to a novel temperature regime, sex-specific changes were demonstrated in gene expression, metabolic and behavioral phenotypes in less than 100 generations. This indicates not only different functional requirements of both sexes in the new environment but also rapid sex-specific adaptation. Supported by computer simulations it is proposed that altered sex-biased gene regulation from standing genetic variation, rather than new mutations, is the driver of rapid sex-specific adaptation. This discovery of environmentally driven divergent functional requirements of males and females has important implications-possibly even for gender aware medical treatments.
Camus, M. F., Moore, J. and Reuter, M. (2020). Nutritional geometry of mitochondrial genetic effects on male fertility. Biol Lett 16(2): 20190891. PubMed ID: 32097597
Organismal fitness is partly determined by how well the nutritional intake matches sex-specific metabolic requirements. Metabolism itself is underpinned by complex genomic interactions involving products from both nuclear and mitochondrial genomes. Products from these two genomes must coordinate how nutrients are extracted, used and recycled, processes vital for fuelling reproduction. Given the complicated nature of metabolism, it is not well understood how the functioning of these two genomes is modulated by nutrients. This study used nutritional geometry techniques on Drosophila lines that only differ in their mtDNA, with the aim to understand if there is nutrient-dependent mitochondrial genetic variance for male reproduction. First, genetic variance was found for diet consumption, indicating that flies are consuming different amounts of food to meet new physiological requirements. Then an interaction was found between mtDNA and diet for fitness, suggesting that the mtDNA plays a role in modulating diet-dependent fitness. These results enhance basic understanding of nutritional health and of chimeric genomes.
Champer, J., Lee, E., Yang, E., Liu, C., Clark, A. G. and Messer, P. W. (2020). A toxin-antidote CRISPR gene drive system for regional population modification. Nat Commun 11(1): 1082. PubMed ID: 32109227
Engineered gene drives based on a homing mechanism could rapidly spread genetic alterations through a population. However, such drives face a major obstacle in the form of resistance against the drive. In addition, they are expected to be highly invasive. This study introduce the Toxin-Antidote Recessive Embryo (TARE) drive. It functions by disrupting a target gene, forming recessive lethal alleles, while rescuing drive-carrying individuals with a recoded version of the target. Modeling shows that such drives will have threshold-dependent invasion dynamics, spreading only when introduced above a fitness-dependent frequency. A TARE drive is demonstrated in Drosophila with 88-95% transmission by female heterozygotes. This drive was able to spread through a large cage population in just six generations following introduction at 24% frequency without any apparent evolution of resistance. These results suggest that TARE drives constitute promising candidates for the development of effective, flexible, and regionally confinable drives for population modification.

Monday April 13th - Transcriptional Regulation

Nair, S., Bahn, J. H., Lee, G., Yoo, S. and Park, J. H. (2020). A homeobox transcription factor Scarecrow (SCRO) negatively regulates Pdf neuropeptide expression through binding an identified cis-acting element in Drosophila melanogaster. Mol Neurobiol. PubMed ID: 31950355
In Drosophila, transcriptional feedback loops contribute to intracellular timekeeping mechanisms responsible for daily rhythms. Pigment-dispersing factor (PDF) is the major neuropeptide produced by latero-ventral neurons (LNvs) that function as a central pacemaker for circadian locomotor activity rhythms. PDF synchronizes other clock neurons thereby playing an essential role in the maintenance and coordination of circadian locomotor rhythms. However, the underlying molecular mechanism of the LNvs-specific Pdf expression is not well understood. Using Pdf promoter-bashing experiment, a cis-acting Pdf regulatory element (PRE) was identified that is sufficient for driving Pdf expression in the LNvs. A homeobox transcription factor, scarecrow (SCRO) was identified as a direct binding factor to PRE. Furthermore, transgenic expression of scro in the clock neurons abolished Pdf expression and circadian locomotor activity rhythms, and such repressive function requires DNA-binding homeodomain, but none of the other conserved domains. scro is predominantly expressed in the optic lobe and various clusters of cells in other areas of the central nervous system. A homozygous scro-null mutant generated by CRIPSR is lethal during embryonic and early larval development, suggesting that scro plays a vital role during early development.
Mazina, M. Y., Ziganshin, R. H., Magnitov, M. D., Golovnin, A. K. and Vorobyeva, N. E. (2020). Proximity-dependent biotin labelling reveals CP190 as an EcR/Usp molecular partner. Sci Rep 10(1): 4793. PubMed ID: 32179799
Proximity-dependent biotin labelling revealed undescribed participants of the ecdysone response in Drosophila. Two labelling enzymes (BioID2 and APEX2) were fused to EcR or Usp to biotin label the surrounding proteins. The EcR/Usp heterodimer was found to collaborate with nuclear pore subunits, chromatin remodelers, and architectural proteins. Many proteins identified through proximity-dependent labelling with EcR/Usp were described previously as functional components of an ecdysone response, corroborating the potency of this labelling method. A link to ecdysone response was confirmed for some newly discovered regulators by immunoprecipitation of prepupal nuclear extract with anti-EcR antibodies and functional experiments in Drosophila S2 cells. A more in-depth study was conducted to clarify the association of EcR/Usp with one of the detected proteins, CP190, a well-described cofactor of Drosophila insulators. CP190 was found to co-immunoprecipitate with the EcR subunit of EcR/Usp in a 20E-independent manner. ChIP-Seq experiments revealed only partial overlapping between CP190 and EcR bound sites in the Drosophila genome and complete absence of CP190 binding at 20E-dependent enhancers. Analysis of Hi-C data demonstrated an existence of remote interactions between 20E-dependent enhancers and CP190 sites which suggests formation of a protein complex between EcR/Usp and CP190 through the space. These results support the previous concept that CP190 has a role in stabilization of specific chromatin loops for proper activation of transcription of genes regulated by 20E hormone.
Chen, H. M., Marques, J. G., Sugino, K., Wei, D., Miyares, R. L. and Lee, T. (2020). CAMIO: a transgenic CRISPR pipeline to create diverse targeted genome deletions in Drosophila. Nucleic Acids Res. PubMed ID: 32187363
The genome is the blueprint for an organism. Interrogating the genome, especially locating critical cis-regulatory elements, requires deletion analysis. This is conventionally performed using synthetic constructs, making it cumbersome and non-physiological. Thus, Cas9-mediated Arrayed Mutagenesis of Individual Offspring (CAMIO) was created to achieve comprehensive analysis of a targeted region of native DNA. CAMIO utilizes CRISPR that is spatially restricted to generate independent deletions in the intact Drosophila genome. Controlled by recombination, a single guide RNA is stochastically chosen from a set targeting a specific DNA region. Combining two sets increases variability, leading to either indels at 1-2 target sites or inter-target deletions. Cas9 restriction to male germ cells elicits autonomous double-strand-break repair, consequently creating offspring with diverse mutations. Thus, from a single population cross, it is possible can obtain a deletion matrix covering a large expanse of DNA at both coarse and fine resolution. The ease and power of CAMIO was documented by mapping 5'UTR sequences crucial for chinmo's post-transcriptional regulation.
Carnesecchi, J., Sigismondo, G., Domsch, K., Baader, C. E. P., Rafiee, M. R., Krijgsveld, J. and Lohmann, I. (2020). Multi-level and lineage-specific interactomes of the Hox transcription factor Ubx contribute to its functional specificity. Nat Commun 11(1): 1388. PubMed ID: 32170121
Transcription factors (TFs) control cell fates by precisely orchestrating gene expression. However, how individual TFs promote transcriptional diversity remains unclear. This study used the Hox TF Ultrabithorax (Ubx) as a model to explore how a single TF specifies multiple cell types. Using proximity-dependent Biotin IDentification in Drosophila, Ubx interactomes were identifed in three embryonic tissues. Ubx interacts with largely non-overlapping sets of proteins with few having tissue-specific RNA expression. Instead most interactors are active in many cell types, controlling gene expression from chromatin regulation to the initiation of translation. Genetic interaction assays in vivo confirm that they act strictly lineage- and process-specific. Thus, functional specificity of Ubx seems to play out at several regulatory levels and to result from the controlled restriction of the interaction potential by the cellular environment. Thereby, it challenges long-standing assumptions such as differential RNA expression as determinant for protein complexes.
Giri, R., Papadopoulos, D. K., Posadas, D. M., Potluri, H. K., Tomancak, P., Mani, M. and Carthew, R. W. (2020). Ordered patterning of the sensory system is susceptible to stochastic features of gene expression. Elife 9. PubMed ID: 32101167
Sensory neuron numbers and positions are precisely organized to accurately map environmental signals in the brain. This precision emerges from biochemical processes within and between cells that are inherently stochastic. This study investigated impact of stochastic gene expression on pattern formation, focusing on senseless (sens), a key determinant of sensory fate in Drosophila. Perturbing microRNA regulation or genomic location of sens produced distinct noise signatures. Noise was greatly enhanced when both sens alleles were present in homologous loci such that each allele was regulated in trans by the other allele. This led to disordered patterning. In contrast, loss of microRNA repression of sens increased protein abundance but not sensory pattern disorder. This suggests that gene expression stochasticity is a critical feature that must be constrained during development to allow rapid yet accurate cell fate resolution.
Diaz-de-la-Loza, M. D., Loker, R., Mann, R. S. and Thompson, B. J. (2020). Control of tissue morphogenesis by the HOX gene Ultrabithorax. Development 147(5). PubMed ID: 32122911
Mutations in the Ultrabithorax (Ubx) gene cause homeotic transformation of the normally two-winged Drosophila into a four-winged mutant fly. Ubx encodes a HOX family transcription factor that specifies segment identity, including transformation of the second set of wings into rudimentary halteres. Ubx is known to control the expression of many genes that regulate tissue growth and patterning, but how it regulates tissue morphogenesis to reshape the wing into a haltere is still unclear. This study shows that Ubx acts by repressing the expression of two genes in the haltere, Stubble and Notopleural, both of which encode transmembrane proteases that remodel the apical extracellular matrix to promote wing morphogenesis. In addition, Ubx induces expression of the Tissue inhibitor of metalloproteases in the haltere, which prevents the basal extracellular matrix remodelling necessary for wing morphogenesis. These results provide a long-awaited explanation for how Ubx controls morphogenetic transformation.

Friday, April 10th - Signaling

Gu, Q., Wu, J., Tian, Y., Cheng, S., Zhang, Z. C. and Han, J. (2020). Galphaq splice variants mediate phototransduction, rhodopsin synthesis, and retinal integrity in Drosophila. J Biol Chem. PubMed ID: 32198182
Heterotrimeric G proteins mediate a variety of signaling processes by coupling G protein-coupled receptors to intracellular effector molecules. In Drosophila, the Galphaq gene encodes several Galphaq splice variants, with the Galphaq1 isoform protein playing a major role in fly phototransduction. However, Galphaq1 null mutant flies still exhibit a residual light response, indicating that other Galphaq splice variants or additional Gq alpha subunits are involved in phototransduction. This study isolated a mutant fly with no detectable light responses, decreased rhodopsin (Rh) levels, and rapid retinal degeneration. Using electrophysiological and genetic studies, biochemical assays, immunoblotting, real-time RT-PCR, and EM analysis, it was found that mutations in the Galphaq gene disrupt light responses, and the Galphaq3 isoform protein was demonstrated to be responsible for the residual light response in Galphaq1 null mutants. Moreover, this study report that Galphaq3 mediates rhodopsin synthesis. Depletion of all Galphaq splice variants led to rapid light-dependent retinal degeneration, due to the formation stable Rh1-arrestin 2 (Arr2) complexes. These findings clarify essential roles of several different Galphaq splice variants in phototransduction and retinal integrity in Drosophila and reveal that Galphaq3 functions in rhodopsin synthesis.
Fraichard, S., Legendre, A., Lucas, P., Chauvel, I., Faure, P., Neiers, F., Artur, Y., Briand, L., Ferveur, J. F. and Heydel, J. M. (2020). Modulation of Sex Pheromone Discrimination by A UDP-Glycosyltransferase in Drosophila melanogaster. Genes (Basel) 11(3). PubMed ID: 32106439
The detection and processing of chemical stimuli involve coordinated neuronal networks that process sensory information. In peripheral olfactory tissues, several classes of proteins, including odorant-degrading enzymes (ODEs), are acting to modulate the detection of chemosensory signals. These enzymes, which primarily act to eliminate toxic compounds from the whole organism also modulate chemodetection. ODEs are thought to neutralize the stimulus molecule concurrently to its detection, avoiding receptor saturation thus allowing chemosensory neurons to respond to the next stimulus. This study shows that one UDP-glycosyltransferase (UGT36E1) expressed in D. melanogaster antennal olfactory sensory neurons (OSNs) is involved in sex pheromone discrimination. UGT36E1 overexpression caused by an insertion mutation affected male behavioral ability to discriminate sex pheromones while it increased OSN electrophysiological activity to male pheromones. Reciprocally, the decreased expression of UGT36E1, controlled by an RNAi transgene, improved male ability to discriminate sex pheromones whereas it decreased electrophysiological activity in the relevant OSNs. When two genotypes (mutation and RNAi) were combined, wild-type-like levels were restored both for the behavioral discrimination and UGT36E1 expression. Taken together, these results strongly suggest that this UGT plays a pivotal role in Drosophila pheromonal detection.
Dimitriadou, A., Chatzianastasi, N., Zacharaki, P. I., O'Connor, M., Goldsmith, S. L., O'Connor, M. B., Consoulas, C. and Newfeld, S. J. (2020). Adult Movement Defects Associated with a CORL Mutation in Drosophila Display Behavioral Plasticity. G3 (Bethesda). PubMed ID: 32161085
The CORL family of CNS-specific proteins share a Smad-binding region with mammalian SnoN and c-Ski protooncogenes. In this family Drosophila CORL has two mouse and two human relatives. Roles for the mouse and human CORL proteins are largely unknown. Based on genome-wide association studies linking the human CORL proteins Fussel15 and Fussel18 with ataxia, this study tested the hypothesis that dCORL mutations will cause adult movement disorders. For initial tests, side by side studies were conducted of adults with the small deletion Df(4)dCORL and eight control strains. Deletion mutants exhibit three types of behavioral plasticity. First, significant climbing defects attributable to loss of dCORL are eliminated by age. Second, significant phototaxis defects due to loss of dCORL are partially ameliorated by age and are not due to faulty photoreceptors. Third, Df(4)dCORL males raised in groups have a lower courtship index than males raised as singles though this defect is not due to loss of dCORL. Subsequent tests showed that the climbing and phototaxis defects were phenocpied by dCORL(21B) and dCORL(23C) two CRISPR generated mutations. Overall, the finding that adult movement defects due to loss of dCORL are subject to age-dependent plasticity suggests new hypotheses for CORL functions in flies and mammals.
Basu, U., Balakrishnan, S. S., Janardan, V. and Raghu, P. (2020). A PI4KIIIalpha protein complex is required for cell viability during Drosophila wing development. Dev Biol. PubMed ID: 32194035
Phosphatidylinositol 4 phosphate (PI4P) and phosphatidylinositol 4,5 bisphosphate [PI(4,5)P2] are enriched on the inner leaflet of the plasma membrane and proposed to be key determinants of its function. PI4P is also the biochemical precursor for the synthesis of PI(4,5)P2 but can itself also bind to and regulate protein function. However, the independent function of PI4P at the plasma membrane in supporting cell function in metazoans during development in vivo remains unclear. Conserved components of a multi-protein complex composed of phosphatidylinositol 4-kinase IIIalpha (PI4KIIIalpha), TTC7, and Efr3 were found to required for normal vein patterning and wing development. Depletion of each of these three components of the PI4KIIIalpha, complex in developing wing cells results in altered wing morphology. These effects are associated with an increase in apoptosis and can be rescued by expression of an inhibitor of Drosophila caspase. In contrast to previous reports, PI4KIIIalphaa depletion does not alter key outputs of hedgehog signalling in developing wing discs. Depletion of PI4KIIIalphae results in reduced PI4P levels at the plasma membrane of developing wing disc cells while levels of PI(4,5)P2, the downstream metabolite of PI4P are not altered. Thus, PI4P itself generated by the activity of the PI4KIIIalpha complex plays an essential role in supporting cell viability in the developing Drosophila wing disc.
Biehler, C., Wang, L. T., Sevigny, M., Jette, A., Gamblin, C. L., Catterall, R., Houssin, E., McCaffrey, L. and Laprise, P. (2020). Girdin is a component of the lateral polarity protein network restricting cell dissemination. PLoS Genet 16(3): e1008674. PubMed ID: 32196494
Epithelial cell polarity defects support cancer progression. It is thus crucial to decipher the functional interactions within the polarity protein network. This study shows that Drosophila Girdin and its human ortholog (GIRDIN) sustain the function of crucial lateral polarity proteins by inhibiting the apical kinase aPKC. Loss of GIRDIN expression is also associated with overgrowth of disorganized cell cysts. Moreover, cell dissemination was observed from GIRDIN knockdown cysts and tumorspheres, thereby showing that GIRDIN supports the cohesion of multicellular epithelial structures. Consistent with these observations, alteration of GIRDIN expression is associated with poor overall survival in subtypes of breast and lung cancers. Overall, this study discovered a core mechanism contributing to epithelial cell polarization from flies to humans. These data also indicate that GIRDIN has the potential to impair the progression of epithelial cancers by preserving cell polarity and restricting cell dissemination.
Griffiths, N. W., Del Bel, L. M., Wilk, R. and Brill, J. A. (2020). Cellular homeostasis in the Drosophila retina requires the lipid phosphatase Sac1. Mol Biol Cell: mbcE20020161. PubMed ID: 32186963
The complex functions of cellular membranes, and thus overall cell physiology, depend on the distribution of crucial lipid species. Sac1 is an essential, conserved, ER-localized phosphatase whose substrate, phosphatidylinositol 4-phosphate (PI4P), coordinates secretory trafficking and plasma membrane function. PI4P from multiple pools is delivered to Sac1 by oxysterol binding protein and related proteins in exchange for other lipids and sterols, which places Sac1 at the intersection of multiple lipid distribution pathways. However, much remains unknown about the roles of Sac1 in subcellular homeostasis and organismal development. Using a temperature-sensitive allele (Sac1(ts)), this study shows that Sac1 is required for structural integrity of the Drosophila retinal floor. The βps-integrin Myospheroid, which is necessary for basal cell adhesion, is mislocalized in Sac1(ts) retinas. In addition, the adhesion proteins Roughest and Kirre, which coordinate apical retinal cell patterning at an earlier stage, accumulate within Sac1(ts) retinal cells due to impaired endo-lysosomal degradation. Moreover, Sac1 is required for ER homeostasis in Drosophila retinal cells. Together, these data illustrate the importance of Sac1 in regulating multiple aspects of cellular homeostasis during tissue development.

Thursday, April 9th - Neural Function

Flaven-Pouchon, J., Alvarez, J. V., Rojas, C. and Ewer, J. (2020). The tanning hormone, bursicon, does not act directly on the epidermis to tan the Drosophila exoskeleton. BMC Biol 18(1): 17. PubMed ID: 32075655
In insects, continuous growth requires the periodic replacement of the exoskeleton. Once the remains of the exoskeleton from the previous stage have been shed during ecdysis, the new one is rapidly sclerotized (hardened) and melanized (pigmented), a process collectively known as tanning. The rapid tanning that occurs after ecdysis is critical for insect survival, as it reduces desiccation, and gives the exoskeleton the rigidity needed to support the internal organs and to provide a solid anchor for the muscles. This rapid postecdysial tanning is triggered by the "tanning hormone", Bursicon. Since Bursicon is released into the hemolymph, it has naturally been assumed that it would act on the epidermal cells to cause the tanning of the overlying exoskeleton. This study investigated the site of Bursicon action in Drosophila by examining the consequences on tanning of disabling the bursicon receptor (encoded by the rickets gene) in different tissues. Surprisingly rapid tanning does not require Rickets function in the epidermis but requires it instead in peptidergic neurons of the ventral nervous system (VNS). Although it was not possible to identify the signal that is transmitted from the VNS to the epidermis, neurons that express the Drosophila insulin-like peptide ILP7, but not the ILP7 peptide itself, are involved. In addition, some of the bursicon targets involved in melanization were found to be different from those that cause sclerotization. These findings show that Bursicon does not act directly on the epidermis to cause the tanning of the overlying exoskeleton but instead requires an intermediary messenger produced by peptidergic neurons within the central nervous system. Thus, this work has uncovered an unexpected layer of control in a process that is critical for insect survival, which will significantly alter the direction of future research aimed at understanding how rapid postecdysial tanning occurs.
Werner, J., Arian, J., Bernhardt, I., Ryglewski, S. and Duch, C. (2020). Differential localization of voltage-gated potassium channels during Drosophila metamorphosis. J Neurogenet: 1-18. PubMed ID: 31997675
Neuronal excitability is determined by the combination of different ion channels and their sub-neuronal localization. This study utilizes protein trap fly strains with endogenously tagged channels to analyze the spatial expression patterns of the four Shaker-related voltage-gated potassium channels, Kv1-4, in the larval, pupal, and adult Drosophila ventral nerve cord. All four channels (Shaker, Kv1; Shab, Kv2; Shaw, Kv3; and Shal, Kv4) each show different spatial expression patterns in the Drosophila ventral nerve cord and are predominantly targeted to different sub-neuronal compartments. Shaker is abundantly expressed in axons, Shab also localizes to axons but mostly in commissures, Shaw expression is restricted to distinct parts of neuropils, and Shal is found somatodendritically, but also in axons of identified motoneurons. During early pupal life expression of all four Shaker-related channels is markedly decreased with an almost complete shutdown of expression at early pupal stage 5 (approximately 30% through metamorphosis). Re-expression of Kv1-4 channels at pupal stage 6 starts with abundant channel localization in neuronal somata, followed by channel targeting to the respective sub-neuronal compartments until late pupal life. The developmental time course of tagged Kv1-4 channel expression corresponds with previously published data on developmental changes in single neuron physiology, thus indicating that protein trap fly strains are a useful tool to analyze developmental regulation of potassium channel expression. This study took advantage of the large diameter of the giant fiber (GF) interneuron to map channel expression onto the axon and axon terminals of an identified interneuron. Shaker, Shaw, and Shal but not Shab channels localize to the non-myelinated GF axonal membrane and axon terminals. This study constitutes a first step toward systematically analyzing sub-neuronal potassium channel localization in Drosophila. Functional implications as well as similarities and differences to Kv1-4 channel localization in mammalian neurons are discussed.
Woitkuhn, J., Ender, A., Beuschel, C. B., Maglione, M., Matkovic-Rachid, T., Huang, S., Lehmann, M., Geiger, J. R. P. and Sigrist, S. J. (2020). The Unc13A isoform is important for phasic release and olfactory memory formation at mushroom body synapses. J Neurogenet: 1-9. PubMed ID: 31980003
The cellular analysis of mushroom body (MB)-dependent memory forming processes is far advanced, whereas, the molecular and physiological understanding of their synaptic basis lags behind. Recent analysis of the Drosophila olfactory system showed that Unc13A, a member of the M(Unc13) release factor family, promotes a phasic, high release probability component, while isoform Unc13B supports a slower tonic release component, reflecting their different nanoscopic positioning within individual active zones. This study used STED super-resolution microscopy of MB lobe synapses to show that Unc13A clusters closer to the active zone centre than Unc13B. Unc13A specifically supported phasic transmission and short-term plasticity of Kenyon cell:output neuron synapses, measured by combining electrophysiological recordings of output neurons with optogenetic stimulation. Knockdown of unc13A within Kenyon cells provoked drastic deficits of olfactory aversive short-term and anaesthesia-sensitive middle-term memory. Knockdown of unc13B provoked milder memory deficits. Thus, a low frequency domain transmission component is probably crucial for the proper representation of memory-associated activity patterns, consistent with sparse Kenyon cell activation during memory acquisition and retrieval. Notably, Unc13A/B ratios appeared highly diversified across MB lobes, leaving room for an interplay of activity components in memory encoding and retrieval.
Zavatone-Veth, J. A., Badwan, B. A. and Clark, D. A. (2020). A minimal synaptic model for direction selective neurons in Drosophila. J Vis 20(2): 2. PubMed ID: 32040161
Visual motion estimation is a canonical neural computation. In Drosophila, recent advances have identified anatomic and functional circuitry underlying direction-selective computations. Models with varying levels of abstraction have been proposed to explain specific experimental results but have rarely been compared across experiments. This studys used the wealth of available anatomical and physiological data to construct a minimal, biophysically inspired synaptic model for Drosophila's first-order direction-selective T4 cells. This model relates mathematically to classical models of motion detection, including the Hassenstein-Reichardt correlator model. Numerical simulation was used to test how well this synaptic model could reproduce measurements of T4 cells across many datasets and stimulus modalities. These comparisons include responses to sinusoid gratings, to apparent motion stimuli, to stochastic stimuli, and to natural scenes. Without fine-tuning this model, it sufficed to reproduce many, but not all, response properties of T4 cells. Since this model is flexible and based on straightforward biophysical properties, it provides an extensible framework for developing a mechanistic understanding of T4 neural response properties. Moreover, it can be used to assess the sufficiency of simple biophysical mechanisms to describe features of the direction-selective computation and identify where understanding must be improved.
Kato, K., Orihara-Ono, M. and Awasaki, T. (2020). Multiple lineages enable robust development of the neuropil-glia architecture in adult Drosophila. Development 147(5). PubMed ID: 32051172
Neural remodeling is essential for the development of a functional nervous system and has been extensively studied in the metamorphosis of Drosophila. Despite the crucial roles of glial cells in brain functions, including learning and behavior, little is known of how adult glial cells develop in the context of neural remodeling. This study shows that the architecture of neuropil-glia in the adult Drosophila brain, which is composed of astrocyte-like glia (ALG) and ensheathing glia (EG), robustly develops from two different populations in the larva: the larval EG and glial cell missing-positive (gcm+) cells. Whereas gcm+ cells proliferate and generate adult ALG and EG, larval EG dedifferentiate, proliferate and redifferentiate into the same glial subtypes. Each glial lineage occupies a certain brain area complementary to the other, and together they form the adult neuropil-glia architecture. Both lineages require the FGF receptor Heartless to proliferate, and the homeoprotein Prospero to differentiate into ALG. Lineage-specific inhibition of gliogenesis revealed that each lineage compensates for deficiency in the proliferation of the other. Together, the lineages ensure the robust development of adult neuropil-glia, thereby ensuring a functional brain.
Chung, H. L., Wangler, M. F., Marcogliese, P. C., Jo, J., Ravenscroft, T. A., Zuo, Z., Duraine, L., Sadeghzadeh, S., Li-Kroeger, D., Schmidt, R. E., Pestronk, A., Rosenfeld, J. A., Burrage, L., Herndon, M. J., Chen, S., Shillington, A., Vawter-Lee, M., Hopkin, R., Rodriguez-Smith, J., Henrickson, M., Lee, B., Moser, A. B., Jones, R. O., Watkins, P., Yoo, T., Mar, S., Choi, M., Bucelli, R. C., Yamamoto, S., Lee, H. K., Prada, C. E., Chae, J. H., Vogel, T. P. and Bellen, H. J. (2020). Loss- or Gain-of-Function Mutations in ACOX1 Cause Axonal Loss via Different Mechanisms. Neuron. PubMed ID: 32169171
ACOX1 (acyl-CoA oxidase 1) encodes the first and rate-limiting enzyme of the very-long-chain fatty acid (VLCFA) beta-oxidation pathway in peroxisomes and leads to H2O2 production. Unexpectedly, Drosophila ACOX1 is mostly expressed and required in glia, and loss of ACOX1 leads to developmental delay, pupal death, reduced lifespan, impaired synaptic transmission, and glial and axonal loss. Patients who carry a previously unidentified, de novo, dominant variant in ACOX1 (p.N237S) also exhibit glial loss. However, this mutation causes increased levels of ACOX1 protein and function resulting in elevated levels of reactive oxygen species in glia in flies and murine Schwann cells. ACOX1 (p.N237S) patients exhibit a severe loss of Schwann cells and neurons. However, treatment of flies and primary Schwann cells with an antioxidant suppressed the p.N237S-induced neurodegeneration. In summary, both loss and gain of ACOX1 lead to glial and neuronal loss, but different mechanisms are at play and require different treatments.

Wednesday, April 8th - Disease Models

Casu, M. A., Mocci, I., Isola, R., Pisanu, A., Boi, L., Mulas, G., Greig, N. H., Setzu, M. D. and Carta, A. R. (2020). Neuroprotection by the Immunomodulatory Drug Pomalidomide in the Drosophila LRRK2(WD40) Genetic Model of Parkinson's Disease. Front Aging Neurosci 12: 31. PubMed ID: 32116655
The search for new disease-modifying drugs for Parkinson's disease (PD) is a slow and highly expensive process, and the repurposing of drugs already approved for different medical indications is becoming a compelling alternative option for researchers. Genetic variables represent a predisposing factor to the disease and mutations in leucine-rich repeat kinase 2 (LRRK2) locus have been correlated to late-onset autosomal-dominant PD. The common fruit fly Drosophila melanogaster carrying the mutation LRRK2 loss-of-function in the WD40 domain (LRRK2(WD40)), is a simple in vivo model of PD and is a valid tool to first evaluate novel therapeutic approaches to the disease. Recent studies have suggested a neuroprotective activity of immunomodulatory agents in PD models. In this study the immunomodulatory drug Pomalidomide (POM), a Thalidomide derivative, was examined in the Drosophila LRRK2(WD40) genetic model of PD. Mutant and wild type flies received increasing POM doses (1, 0.5, 0.25 mM) through their diet from day 1 post eclosion, until postnatal day (PN) 7 or 14, when POM's actions were evaluated by quantifying changes in climbing behavior as a measure of motor performance, the number of brain dopaminergic neurons and T-bars, mitochondria integrity. LRRK2(WD40) flies displayed a spontaneous age-related impairment of climbing activity, and POM significantly and dose-dependently improved climbing performance both at PN 7 and PN 14. LRRK2(WD40) fly motor disability was underpinned by a progressive loss of dopaminergic neurons in posterior clusters of the protocerebrum, which are involved in the control of locomotion, by a low number of T-bars density in the presynaptic bouton active zones. POM treatment fully rescued the cell loss in all posterior clusters at PN 7 and PN 14 and significantly increased the T-bars density. Moreover, several damaged mitochondria with dilated cristae were observed in LRRK2(WD40) flies treated with vehicle but not following POM. This study demonstrates the neuroprotective activity of the immunomodulatory agent POM in a genetic model of PD. POM is an FDA-approved clinically available and well-tolerated drug used for the treatment of multiple myeloma. If further validated in mammalian models of PD, POM could rapidly be clinically tested in humans.
Yatsenko, A. S., Kucherenko, M. M., Xie, Y., Aweida, D., Urlaub, H., Scheibe, R. J., Cohen, S. and Shcherbata, H. R. (2020). Profiling of the muscle-specific dystroglycan interactome reveals the role of Hippo signaling in muscular dystrophy and age-dependent muscle atrophy. BMC Med 18(1): 8. PubMed ID: 31959160
Dystroglycanopathies are a group of inherited disorders characterized by vast clinical and genetic heterogeneity and caused by abnormal functioning of the ECM receptor dystroglycan (Dg). Remarkably, among many cases of diagnosed dystroglycanopathies, only a small fraction can be linked directly to mutations in Dg or its regulatory enzymes, implying the involvement of other, not-yet-characterized, Dg-regulating factors. To advance disease diagnostics and develop new treatment strategies, new approaches to find dystroglycanopathy-related factors should be considered. The Dg complex is highly evolutionarily conserved; therefore, model genetic organisms provide excellent systems to address this challenge. In particular, Drosophila is amenable to experiments not feasible in any other system, allowing original insights about the functional interactors of the Dg complex. To identify new players contributing to dystroglycanopathies, Drosophila was used as a genetic muscular dystrophy model. Using mass spectrometry, muscle-specific Dg interactors were sought. Next, in silico analyses allowed determination of their association with diseases and pathological conditions in humans. Using immunohistochemical, biochemical, and genetic interaction approaches followed by the detailed analysis of the muscle tissue architecture, Dg interaction with some of the discovered factors was verified. Analyses of mouse muscles and myocytes were used to test if interactions are conserved in vertebrates. The muscle-specific Dg complexome revealed novel components that influence the efficiency of Dg function in the muscles. The closest human homologs for Dg-interacting partners were identfied, their significant enrichment in disease-associations was determined, and some of the newly identified Dg interactions were verified.Dg associates with two components of the mechanosignaling Hippo pathway: the WW domain-containing proteins Kibra and Yorkie. Importantly, this conserved interaction manages adult muscle size and integrity. The results presented in this study provide a new list of muscle-specific Dg interactors, further analysis of which could aid not only in the diagnosis of muscular dystrophies, but also in the development of new therapeutics. To regulate muscle fitness during aging and disease, Dg associates with Kibra and Yorkie and acts as a transmembrane Hippo signaling receptor that transmits extracellular information to intracellular signaling cascades, regulating muscle gene expression.
Yu, L., Li, G., Deng, J., Jiang, X., Xue, J., Zhu, Y., Huang, W., Tang, B. and Duan, R. (2020). The UFM1 cascade times mitosis entry associated with microcephaly. Faseb j 34(1): 1319-1330. PubMed ID: 31914610
Posttranslational modifications enhance the functional diversity of the proteome by modifying the substrates. The UFM1 cascade is a novel ubiquitin-like modification system. The mutations in UFM1, its E1 (UBA5) and E2 (UFC1), have been identified in patients with microcephaly. However, its pathological mechanisms remain unclear. This study observed the disruption of the UFM1 cascade in Drosophila neuroblasts (NBs) decreased the number of NBs, leading to a smaller brain size. The lack of ufmylation in NBs resulted in an increased mitotic index and an extended G2/M phase, indicating a defect in mitotic progression. In addition, live imaging of the embryos revealed an impaired E3 ligase (Ufl1) function resulted in premature entry into mitosis and failed cellularization. Even worse, the embryonic lethality occurred as early as within the first few mitotic cycles following the depletion of Ufm1. Knockdown of ufmylation in the fixed embryos exhibited severe phenotypes, including detached centrosomes, defective microtubules, and DNA bridge. Furthermore, the UFM1 cascade could alter the level of phosphorylation on tyrosine-15 of CDK1 (pY15-CDK1), which is a negative regulator of the G2 to M transition. These findings yield unambiguous evidence suggesting that the UFM1 cascade is a microcephaly-causing factor that regulates the progression of the cell cycle at mitosis phase entry.
Ali, M. S., Suda, K., Kowada, R., Ueoka, I., Yoshida, H. and Yamaguchi, M. (2020). Neuron-specific knockdown of solute carrier protein SLC25A46a induces locomotive defects, an abnormal neuron terminal morphology, learning disability, and shortened lifespan. IBRO Rep 8: 65-75. PubMed ID: 32140609
Various mutations in the SLC25A46 gene have been reported in mitochondrial diseases that are sometimes classified as type 2 Charcot-Marie-Tooth disease, optic atrophy, and Leigh syndrome. Two Drosophila genes, dSLC25A46a and dSLC25A46b have been identified as candidate homologs of human SLC25A46. In the present study, pan-neuron-specific dSLC25A46a knockdown flies were developed and their phenotypes examined. Neuron-specific dSLC25A46a knockdown resulted in reduced mobility in larvae as well as adults. An aberrant morphology for neuromuscular junctions (NMJs), such as a reduced synaptic branch length and decreased number and size of boutons, was observed in dSLC25A46a knockdown flies. Learning ability was also reduced in the larvae of knockdown flies. In dSLC25A46a knockdown flies, mitochondrial hyperfusion was detected in NMJ synapses together with the accumulation of reactive oxygen species and reductions in ATP. These phenotypes were very similar to those of dSLC25A46b knockdown flies, suggesting that dSLC25A46a and dSLC25A46b do not have redundant roles in neurons. Collectively, these results show that the depletion of SLC25A46a leads to mitochondrial defects followed by an aberrant synaptic morphology, resulting in locomotive defects and learning disability. Thus, the dSLC25A46a knockdown fly summarizes most of the phenotypes in patients with mitochondrial diseases, offering a useful tool for studying these diseases.
Aleyakpo, B., Umukoro, O., Kavlie, R., Ranson, D. C., Thompsett, A., Corcoran, O. and Casalotti, S. O. (2019). G-protein alphaq gene expression plays a role in alcohol tolerance in Drosophila melanogaster. Brain Neurosci Adv 3: 2398212819883081. PubMed ID: 32166184
Ethanol is a psychoactive substance causing both short- and long-term behavioural changes in humans and animal models. This study used the fruit fly Drosophila melanogaster to investigate the effect of ethanol exposure on the expression of the Galphaq protein subunit. Repetitive exposure to ethanol causes a reduction in sensitivity (tolerance) to ethanol, which was measured as the time for 50% of a set of flies to become sedated after exposure to ethanol (ST50). It was demonstrated that the same treatment that induces an increase in ST50 over consecutive days (tolerance) also causes a decrease in Galphaq protein subunit expression at both the messenger RNA and protein level. To identify whether there may be a causal relationship between these two outcomes, strains of flies were developed in which Galphaq messenger RNA expression is suppressed in a time- and tissue-specific manner. In these flies, the sensitivity to ethanol and the development of tolerance are altered. This work further supports the value of Drosophila as a model to dissect the molecular mechanisms of the behavioural response to alcohol and identifies G proteins as potentially important regulatory targets for alcohol use disorders.
Feuillette, S., Charbonnier, C., Frebourg, T., Campion, D. and Lecourtois, M. (2020). A Connected Network of Interacting Proteins Is Involved in Human-Tau Toxicity in Drosophila. Front Neurosci 14: 68. PubMed ID: 32116515
Tauopathies are neurodegenerative diseases characterized by the presence of aggregates of abnormally phosphorylated Tau. Deciphering the pathophysiological mechanisms that lead from the alteration of Tau biology to neuronal death depends on the identification of Tau cellular partners. Combining genetic and transcriptomic analyses in Drosophila, this study identified 77 new modulators of human Tau-induced toxicity, bringing to 301 the number of Tau genetic interactors identified so far in flies. Network analysis showed that 229 of these genetic modulators constitute a connected network. The addition of 77 new genes strengthened the network structure, increased the intergenic connectivity and brought up key hubs with high connectivities, namely Src64B/FYN, Src42A/FRK, kuz/ADAM10, heph/PTBP1, scrib/SCRIB, and Cam/CALM3. Interestingly, this study established a genetic link between Tau-induced toxicity and ADAM10, a recognized Alzheimer Disease protective factor. In addition, these data support the importance of the presynaptic compartment in mediating Tau toxicity.

Tuesday, April 7th - Adult Development

Munoz-Nava, L. M., Alvarez, H. A., Flores-Flores, M., Chara, O. and Nahmad, M. (2020). A dynamic cell recruitment process drives growth of the Drosophila wing by overscaling the Vestigial expression pattern. Dev Biol. PubMed ID: 32197891
Organs mainly attain their size by cell growth and proliferation, but sometimes also grow through recruitment of undifferentiated cells. This study investigated the participation of cell recruitment in establishing the pattern of Vestigial (Vg), the product of the wing selector gene in Drosophila. The Vg pattern overscales along the dorsal-ventral (DV) axis of the wing imaginal disc, i.e., it expands faster than the DV length of the pouch. The overscaling of the Vg pattern cannot be explained by differential proliferation, apoptosis, or oriented-cell divisions, but can be recapitulated by a mathematical model that explicitly considers cell recruitment. When impairing cell recruitment genetically, it was found that the Vg pattern almost perfectly scales and adult wings are approximately 20% smaller. Conversely, impairing cell proliferation results in very small wings, suggesting that cell recruitment and cell proliferation additively contribute to organ growth in this system. Furthermore, using fluorescent reporter tools, direct evidence is provided that cell recruitment is initiated between early and mid third-instar larval development. Altogether, this work quantitatively shows when, how, and by how much cell recruitment shapes the Vg pattern and drives growth of the Drosophila wing.
Duncan, S., Delage, S., Chioran, A., Sirbu, O., Brown, T. J. and Ringuette, M. J. (2020) . The predicted collagen-binding domains of Drosophila SPARC are essential for survival and for collagen IV distribution and assembly into basement membranes. Dev Biol. PubMed ID: 32087195
The assembly of basement membranes (BMs) into tissue-specific morphoregulatory structures requires non-core BM components. Work in Drosophila indicates a principal role of collagen-binding matricellular glycoprotein SPARC (Secreted Protein, Acidic, Rich in Cysteine) in larval fat body BM assembly. This study reports that SPARC and collagen IV (Col(IV)) first colocalize in the trans-Golgi of hemocyte-like cell lines. Mutating the collagen-binding domains of Drosophila SPARC led to the loss of colocalization with Col(IV), a fibrotic-like BM, and 2(nd) instar larval lethality, indicating that SPARC binding to Col(IV) is essential for survival. Analysis of this mutant at 2(nd) instar reveals increased Col(IV) puncta within adipocytes, reflecting a disruption in the intracellular chaperone-like activity of SPARC. Both SPARC mutants exhibited altered fat body BM pore topography. Wing imaginal disc-derived SPARC did not localize within Col(IV)-rich matrices. This raises the possibility that SPARC interaction with Col(IV) requires initial intracellular interaction to colocalize at the BM or that wing-derived SPARC undergoes differential post-translational modifications that impacts its function. Collectively, these data provide evidence that the chaperone-like activity of SPARC on Col(IV) begins just prior to their co-secretion and demonstrate for the first time that the Col(IV) chaperone-like activity of SPARC is necessary for Drosophila development beyond the 2(nd) instar.
Barrio, L. and Milan, M. (2020). Regulation of Anisotropic Tissue Growth by Two Orthogonal Signaling Centers. Dev Cell 52(5): 659-672. PubMed ID: 32084357
The Drosophila wing has served as a paradigm to mechanistically characterize the role of morphogens in patterning and growth. Wingless (Wg) and Decapentaplegic (Dpp) are expressed in two orthogonal signaling centers, and their gradients organize patterning by regulating the expression of well-defined target genes. By contrast, graded activity of these morphogens is not an absolute requirement for wing growth. Despite their permissive role in regulating growth, this study shows that Wg and Dpp are utilized in a non-interchangeable manner by the two existing orthogonal signaling centers to promote preferential growth along the two different axes of the developing wing. The data indicate that these morphogens promote anisotropic growth by making use of distinct and non-interchangeable molecular mechanisms. Whereas Dpp drives growth along the anterior-posterior axis by maintaining Brinker levels below a growth-repressing threshold, Wg exerts its action along the proximal-distal axis through a double repression mechanism involving the T cell factor (TCF) Pangolin.
Dong, W., Gao, Y. H., Zhang, X. B., Moussian, B. and Zhang, J. Z. (2020). Chitinase10 controls chitin amounts and organization in the wing cuticle of Drosophila. Insect Sci. PubMed ID: 32129536
Wings are essential for insect fitness. A number of proteins and enzymes have been identified to be involved in wing terminal differentiation, which is characterized by the formation of the wing cuticle. This study addressed the question whether Chitinase 10 (Cht10) may play an important role in chitin organization in the wings of the fruit fly Drosophila melanogaster. Cht10 expression was found to coincide with the expression of the chitin synthase coding gene kkv. This suggests that the respective proteins may cooperate during wing differentiation. In tissue-specific RNA interference experiments, it was demonstrated that suppression of Cht10 causes an excess in chitin amounts in the wing cuticle. Chitin organization is severely disrupted in these wings. Based on these data, it is hypothesized that Cht10 restricts chitin amounts produced by Kkv in order to ensure normal chitin organization and wing cuticle formation. In addition, it was found by scanning electron microscopy that Cht10 suppression also affects the cuticle surface. In turn, cuticle inward permeability is enhanced in Cht10-less wings. Moreover, flies with reduced Cht10 function are unable to fly. In conclusion, Cht10 is essential for wing terminal differentiation and function.
Borne, F., Kovalev, A., Gorb, S. and Courtier-Orgogozo, V. (2020). The glue produced by Drosophila melanogaster for pupa adhesion is universal. J Exp Biol. PubMed ID: 32165432
Insects produce a variety of adhesives for diverse functions such as locomotion, mating, egg or pupal anchorage to substrates. Although they are important for the biology of organisms and potentially represent a great resource for developing new materials, insect adhesives have been little studied so far. This study examined the adhesive properties of the larval glue of Drosophila melanogaster. This glue is made of glycosylated proteins and allows the animal to adhere to a substrate during metamorphosis. This study designed an adhesion test to measure the pull-off force required to detach a pupa from a substrate and to evaluate the contact area covered by the glue. The pupa adheres with similar forces to a variety of substrates (with distinct roughness, hydrophilic and charge properties). An average pull-off force was obtained of 217 mN, corresponding to adhesion strength of 137-244 kPa. Surprisingly, the pull-off forces did not depend on the contact area. This study paves the way for a genetic dissection of the components of D. melanogaster glue that confer its particular adhesive properties.
Bataille, L., Colombie, N., Pelletier, A., Paululat, A., Lebreton, G., Carrier, Y., Frendo, J. L. and Vincent, A. (2020). Alary muscles and TARMs, a novel type of striated muscles maintaining internal organs positions. Development. PubMed ID: 32188630
Alary muscles (AMs) have been described as a component of the cardiac system in various arthropods. Lineage-related thoracic muscles (TARMs), linking the exoskeleton to specific gut regions, have recently been discovered in Drosophila Asymmetrical attachments of AMs and TARMs, to the exoskeleton on one side, and internal organs on the other, suggested an architectural function in moving larvae. This study analysed AMs and TARMs striated organisation, and imaged their atypical deformability in crawling larvae. Then AMs and TARMs were selectively eliminated by targeted apoptosis. Elimination of AMs revealed that AMs are required for suspending the heart in proper intra-hemocelic position and opening of the heart lumen, and constrain the curvature of the trachea, the respiratory system, during crawling; TARMs are required for proper positioning of visceral organs and efficient food transit. AM/TARM cardiac versus visceral attachment depends on Hox control, with visceral attachment being the ground state. TARMs and AMs are the first example of multinucleate striated muscles connecting the skeleton to the cardiac and visceral systems in bilaterians, with multiple physiological functions.

Monday April 8th - Monday Evolution

Kurbalija Novicic, Z., Sayadi, A., Jelic, M. and Arnqvist, G. (2020). Negative frequency dependent selection contributes to the maintenance of a global polymorphism in mitochondrial DNA. BMC Evol Biol 20(1): 20. PubMed ID: 32019493
Understanding the forces that maintain diversity across a range of scales is at the very heart of biology. Frequency-dependent processes are generally recognized as the most central process for the maintenance of ecological diversity. The same is, however, not generally true for genetic diversity. Negative frequency dependent selection, where rare genotypes have an advantage, is often regarded as a relatively weak force in maintaining genetic variation in life history traits because recombination disassociates alleles across many genes. Yet, many regions of the genome show low rates of recombination and genetic variation in such regions (i.e., supergenes) may in theory be upheld by frequency dependent selection. This group studied what is essentially a ubiquitous life history supergene (i.e., mitochondrial DNA) in the fruit fly Drosophila subobscura, showing sympatric polymorphism with two main mtDNA genotypes co-occurring in populations world-wide. Using an experimental evolution approach involving manipulations of genotype starting frequencies, this study shows that negative frequency dependent selection indeed acts to maintain genetic variation in this region. Moreover, the strength of selection was affected by food resource conditions. This work provides novel experimental support for the view that balancing selection through negative frequency dependency acts to maintain genetic variation in life history genes. It is suggested that the emergence of negative frequency dependent selection on mtDNA is symptomatic of the fundamental link between ecological processes related to resource use and the maintenance of genetic variation.
Vaught, R. C., Voigt, S., Dobler, R., Clancy, D. J., Reinhardt, K. and Dowling, D. K. (2020).. Interactions between cytoplasmic and nuclear genomes confer sex-specific effects on lifespan in Drosophila melanogaster. J Evol Biol. PubMed ID: 32053259
Genetic variation outside of the cell nucleus can affect the phenotype. The cytoplasm is home to the mitochondria, and in arthropods often hosts intracellular bacteria such as Wolbachia. While numerous studies have implicated epistatic interactions between cytoplasmic and nuclear genetic variation as mediators of phenotypic expression, two questions remain. Firstly, it remains unclear whether outcomes of cyto-nuclear interactions will manifest differently across the sexes, as might be predicted given that cytoplasmic genomes are screened by natural selection only through females as a consequence of their maternal inheritance. Secondly, the relative contribution of mitochondrial genetic variation to other cytoplasmic sources of variation, such as Wolbachia infection, in shaping phenotypic outcomes of cyto-nuclear interactions remains unknown. This study addresses these questions, creating a fully-crossed set of replicated cyto-nuclear populations derived from three geographically distinct populations of Drosophila melanogaster, measuring the lifespan of males and females from each population. It was observed that cyto-nuclear interactions shape lifespan, and that the outcomes of these interactions differ across the sexes. Yet, no evidence was found that placing the cytoplasms from one population alongside the nuclear background of others (generating putative cyto-nuclear mismatches) leads to decreased lifespan in either sex. Although it was difficult to partition mitochondrial from Wolbachia effects, the results suggest at least some of the cytoplasmic genotypic contribution to lifespan was directly mediated by an effect of sequence variation in the mtDNA. Future work should explore the degree to which cyto-nuclear interactions result in sex differences in expression of other components of organismal life-history.
Tracy, C. B., Nguyen, J., Abraham, R. and Shirangi, T. R. (2020). Evolution of sexual size dimorphism in the wing musculature of Drosophila. PeerJ 8: e8360. PubMed ID: 31988804
Male courtship songs in Drosophila are exceedingly diverse across species. While much of this variation is understood to have evolved from changes in the central nervous system, evolutionary transitions in the wing muscles that control the song may have also contributed to song diversity. Focusing on a group of four wing muscles that are known to influence courtship song in Drosophila melanogaster, this study investigated the evolutionary history of wing muscle anatomy of males and females from 19 Drosophila species. Three of the wing muscles have evolved sexual dimorphisms in size multiple independent times, whereas one has remained monomorphic in the phylogeny. These data suggest that evolutionary changes in wing muscle anatomy may have contributed to species variation in sexually dimorphic wing-based behaviors, such as courtship song. Moreover, wing muscles appear to differ in their propensity to evolve size dimorphisms, which may reflect variation in the functional constraints acting upon different wing muscles.
Sato, K., Tanaka, R., Ishikawa, Y. and Yamamoto, D. (2020). Behavioral Evolution of Drosophila: Unraveling the Circuit Basis. Genes (Basel) 11(2). PubMed ID: 32024133
Behavior is a readout of neural function. Therefore, any difference in behavior among different species is, in theory, an outcome of interspecies diversification in the structure and/or function of the nervous system. However, the neural diversity underlying the species-specificity in behavioral traits and its genetic basis have been poorly understood. This article discusses potential neural substrates for species differences in the courtship pulse song frequency and mating partner choice in the Drosophila melanogaster subgroup. Possible neurogenetic mechanisms are discussed whereby a novel behavioral repertoire emerges based on the study of nuptial gift transfer, a trait unique to D. subobscura in the genus Drosophila. The conserved central circuit composed primarily of fruitless-expressing neurons (the fru-circuit) serves for the execution of courtship behavior, whereas the sensory pathways impinging onto the fru-circuit or the motor pathways downstream of the fru-circuit are susceptible to changes associated with behavioral species differences.
Polovina, E. S., Parakatselaki, M. E. and Ladoukakis, E. D. (2020). Paternal leakage of mitochondrial DNA and maternal inheritance of heteroplasmy in Drosophila hybrids. Sci Rep 10(1): 2599. PubMed ID: 32054873
Mitochondrial DNA (mtDNA) is maternally transmitted in animals and therefore, individuals are expected to have a single mtDNA haplotype (homoplasmy). Yet, heteroplasmic individuals have been observed in a large number of animal species. Heteroplasmy may emerge as a result of somatic mtDNA mutations, paternal leakage during fertilization or be inherited from a heteroplasmic mother. Understanding the causes of heteroplasmy could shed light into the evolution of mtDNA inheritance. This study examined heteroplasmy in progeny from heterospecific crosses of Drosophila for two consecutive generations. The generation of heteroplasmy from paternal leakage and the maternal transmission of heteroplasmy were examined. The data reveal non-random patterns in the emergence and transmission of heteroplasmy and suggest that heteroplasmy depends on the family of origin.
Bracewell, R. and Bachtrog, D. (2020). Complex evolutionary history of the Y chromosome in flies of the Drosophila obscura species group. Genome Biol Evol. PubMed ID: 32176296
The Drosophila obscura species group shows dramatic variation in karyotype, including transitions among sex chromosomes. Members of the affinis and pseudoobscura subgroups contain a neo-X chromosome (a fusion of the X with an autosome), and it was shown that ancestral Y genes have become autosomal in species harboring the neo-X. Detailed analysis of species in the pseudoobscura subgroup revealed that ancestral Y genes became autosomal through a translocation to the small dot chromosome. This study shows that the Y-dot translocation is restricted to the pseudoobscura subgroup, and translocation of ancestral Y genes in the affinis subgroup likely followed a different route. Most ancestral Y genes appear to have translocated to unique autosomal or X-linked locations in different taxa of the affinis subgroup, and a dynamic model of sex chromosome formation and turnover in the obscura species group is proposed. The results suggest that Y genes can find unique paths to escape unfavorable genomic environments that form after sex chromosome-autosome fusions.

Friday, April 3rd - Stem Cells

Baral, S. S., Lieux, M. E. and DiMario, P. J. (2020). Nucleolar stress in Drosophila neuroblasts, a model for human ribosomopathies. Biol Open. PubMed ID: 32184230
Different stem cells or progenitor cells display variable threshold requirements for functional ribosomes. This is particularly true for several human ribosomopathies in which select embryonic neural crest cells or adult bone marrow stem cells, but not others, show lethality due to failures in ribosome biogenesis or function (now known as nucleolar stress). To determine if various Drosophila neuroblasts display differential sensitivities to nucleolar stress, CRISPR-Cas9 was used to disrupt the Nopp140 gene that encodes two splice variant ribosome biogenesis factors (RBFs). Disruption of Nopp140 induced nucleolar stress that arrested larvae in the second instar stage. While the majority of larval neuroblasts arrested development, the Mushroom Body (MB) neuroblasts continued to proliferate as shown by their maintenance of deadpan, a neuroblast-specific transcription factor, and by their continued EdU incorporation. MB neuroblasts in wild type larvae appeared to contain more fibrillarin and Nopp140 in their nucleoli as compared to other neuroblasts, indicating that MB neuroblasts stockpile RBFs as they proliferate in late embryogenesis while other neuroblasts normally enter quiescence. A greater abundance of Nopp140 encoded by maternal transcripts in Nopp140-/- MB neuroblasts of 1-2 day old larvae likely rendered these cells more resilient to nucleolar stress.
Khaminets, A., Ronnen-Oron, T., Baldauf, M., Meier, E. and Jasper, H. (2020). Cohesin controls intestinal stem cell identity by maintaining association of Escargot with target promoters. Elife 9. PubMed ID: 32022682
Intestinal stem cells (ISCs) maintain regenerative capacity of the intestinal epithelium. Their function and activity are regulated by transcriptional changes, yet how such changes are coordinated at the genomic level remains unclear. The Cohesin complex regulates transcription globally by generating topologically-associated DNA domains (TADs) that link promotor regions with distant enhancers. The Cohesin complex prevents premature differentiation of Drosophila ISCs into enterocytes (ECs). Depletion of the Cohesin subunit Rad21 and the loading factor Nipped-B triggers an ISC to EC differentiation program that is independent of Notch signaling, but can be rescued by over-expression of the ISC-specific escargot (esg) transcription factor. Using damID and transcriptomic analysis, this study that Cohesin regulates Esg binding to promoters of differentiation genes, including a group of Notch target genes involved in ISC differentiation. It is proposed that Cohesin ensures efficient Esg-dependent gene repression to maintain stemness and intestinal homeostasis.
Meng, F. W., Rojas Villa, S. E. and Biteau, B. (2020). Sox100B Regulates Progenitor-Specific Gene Expression and Cell Differentiation in the Adult Drosophila Intestine. Stem Cell Reports 14(2): 226-240. PubMed ID: 32032550
Robust production of terminally differentiated cells from self-renewing resident stem cells is essential to maintain proper tissue architecture and physiological functions, especially in high-turnover tissues. However, the transcriptional networks that precisely regulate cell transition and differentiation are poorly understood in most tissues. This study identified Sox100B, a Drosophila Sox E family transcription factor, as a critical regulator of adult intestinal stem cell differentiation. Sox100B is expressed in stem and progenitor cells and required for differentiation of enteroblast progenitors into absorptive enterocytes. Mechanistically, Sox100B regulates the expression of another critical stem cell differentiation factor, Sox21a. Supporting a direct control of Sox21a by Sox100B, a Sox21a intronic enhancer was identified that is active in all intestinal progenitors and directly regulated by Sox100B. Taken together, these results demonstrate that the activity and regulation of two Sox transcription factors are essential to coordinate stem cell differentiation and proliferation and maintain intestinal tissue homeostasis.
Li, Z., Guo, X., Huang, H., Wang, C., Yang, F., Zhang, Y., Wang, J., Han, L., Jin, Z., Cai, T. and Xi, R. (2020). A Switch in Tissue Stem Cell Identity Causes Neuroendocrine Tumors in Drosophila Gut. Cell Rep 30(6): 1724-1734. PubMed ID: 32049006
Intestinal stem cells (ISCs) are able to generate gut-specific enterocytes, as well as neural-like enteroendocrine cells. It is unclear how the tissue identity of the ISC lineage is regulated to confer cell-lineage fidelity. This study shows that, in adult Drosophila midgut, loss of the transcriptional repressor Tramtrack in ISCs causes a self-renewal program switch to neural stem cell (NSC)-like, and that switch drives neuroendocrine tumor development. In Tramtrack-depleted ISCs, the ectopically expressed Deadpan acts as a major self-renewal factor for cell propagation, and Sequoia acts as a differentiation factor for the neuroendocrine phenotype. In addition, the expression of Sequoia renders NSC-specific self-renewal genes responsive to Notch in ISCs, thus inverting the differentiation-promoting function of Notch into a self-renewal role as in normal NSCs. These results suggest an active maintenance mechanism for the gut identity of ISCs, whose disruption may lead to an improper acquisition of NSC-like traits and tumorigenesis.
Arnaoutov, A., Lee, H., Plevock Haase, K., Aksenova, V., Jarnik, M., Oliver, B., Serpe, M. and Dasso, M. (2020). IRBIT Directs Differentiation of Intestinal Stem Cell Progeny to Maintain Tissue Homeostasis. iScience 23(3): 100954. PubMed ID: 32179478
The maintenance of the intestinal epithelium is ensured by the controlled proliferation of intestinal stem cells (ISCs) and differentiation of their progeny into various cell types, including enterocytes (ECs) that both mediate nutrient absorption and provide a barrier against pathogens. The signals that regulate transition of proliferative ISCs into differentiated ECs are not fully understood. IRBIT (CG9977) is an evolutionarily conserved protein that regulates ribonucleotide reductase (RNR), an enzyme critical for the generation of DNA precursors. This study shows that IRBIT expression in ISC progeny within the Drosophila midgut epithelium cells regulates their differentiation via suppression of RNR activity. Disruption of this IRBIT-RNR regulatory circuit causes a premature loss of intestinal tissue integrity. Furthermore, age-related dysplasia can be reversed by suppression of RNR activity in ISC progeny. Collectively, these findings demonstrate a role of the IRBIT-RNR pathway in gut homeostasis.
Hakes, A. E. and Brand, A. H. (2020). Tailless/TLX reverts intermediate neural progenitors to stem cells driving tumourigenesis via repression of asense/ASCL1. Elife 9. PubMed ID: 32073402
Understanding the sequence of events leading to cancer relies in large part upon identifying the tumour cell of origin. Glioblastoma is the most malignant brain cancer but the early stages of disease progression remain elusive. Neural lineages have been implicated as cells of origin, as have glia. Interestingly, high levels of the neural stem cell regulator TLX correlate with poor patient prognosis. This study shows that high levels of the Drosophila TLX homologue, Tailless, initiate tumourigenesis by reverting intermediate neural progenitors to a stem cell state. Strikingly, tumour formation could be blocked completely by re-expressing Asense (homologue of human ASCL1), which is a direct target of Tailless. These results predict that expression of TLX and ASCL1 should be mutually exclusive in glioblastoma, which was verified in single-cell RNA-seq of human glioblastoma samples. Counteracting high TLX is a potential therapeutic strategy for suppressing tumours originating from intermediate progenitor cells.

Thursday, April 2nd - Behavior

Auer, T. O., Khallaf, M. A., Silbering, A. F., Zappia, G., Ellis, K., Alvarez-Ocana, R., Arguello, J. R., Hansson, B. S., Jefferis, G., Caron, S. J. C., Knaden, M. and Benton, R. (2020). Olfactory receptor and circuit evolution promote host specialization. Nature 579(7799): 402-408. PubMed ID: 32132713
The evolution of animal behaviour is poorly understood. Despite numerous correlations between interspecific divergence in behaviour and nervous system structure and function, demonstrations of the genetic basis of these behavioural differences remain rare. This study develop a neurogenetic model, Drosophila sechellia, a species that displays marked differences in behaviour compared to its close cousin Drosophila melanogaster that are linked to its extreme specialization on noni fruit (Morinda citrifolia). Using calcium imaging, this study identified olfactory pathways in D. sechellia that detect volatiles emitted by the noni host. This mutational analysis indicates roles for different olfactory receptors in long- and short-range attraction to noni, and cross-species allele-transfer experiments demonstrate that the tuning of one of these receptors is important for species-specific host-seeking. The molecular determinants of this functional change were identified, and their evolutionary origin and behavioural importance were characterized. Circuit tracing was performed in the D. sechellia brain, and receptor adaptations were found to be accompanied by increased sensory pooling onto interneurons as well as species-specific central projection patterns. This work reveals an accumulation of molecular, physiological and anatomical traits that are linked to behavioural divergence between species, and defines a model for investigating speciation and the evolution of the nervous system.
Abhilash, L. and Sharma, V. K. (2020). Mechanisms of photic entrainment of activity/rest rhythms in populations of Drosophila selected for divergent timing of eclosion. Chronobiol Int: 1-16. PubMed ID: 32079418
It is a common notion that phases-of-entrainment of circadian rhythms are adaptive, in that they enable organisms to time their behavior to specific times of the day to enhance their fitness. Previous studies have shown that selection for morning and evening phasing of adult emergence in Drosophila melanogaster populations leads to divergent coevolution of free-running periods of both adult emergence and activity/rest rhythms, such that early (morning) and late (evening) adult emergence chronotypes have shorter and longer circadian periods, respectively. However, there is little evidence to support the notion that phases-of-entrainment in these fly stocks is indeed driven by non-parametric mechanisms. Extending from a previous hypothesis based on anecdotal evidence for parametric mechanisms being in play, this study explored the extent of non-parametric and parametric effects of light on circadian clocks of early and late chronotypes. Predictions of the non-parametric model of entrainment were systematically tested, the Circadian Integrated Response Characteristic (CIRC) of the stocks , the effect of light pulses on amplitude of the behavior and the effect of duration of light pulse on phase-shifts of the clock were assessed were sketched. In addition to the differences in clock period, divergent CIRCs contribute to entrainment of the activity/rest rhythm. The differences in CIRC could be explained by differential transient amplitude responses and duration responses of the clock's phase between the early and late chronotypes. This study thus highlights the role of amplitude responses and phase-shifts due to long durations of light in entrainment of circadian rhythms of D. melanogaster.
Ishimoto, H. and Kamikouchi, A. (2020). A feedforward circuit regulates action selection of pre-mating courtship behavior in female Drosophila. Curr Biol 30(3): 396-407.e394. PubMed ID: 31902724
In the early phase of courtship, female fruit flies exhibit an acute rejection response to avoid unfavorable mating. This pre-mating rejection response is evolutionarily paralleled across species, but the molecular and neuronal basis of that behavior is unclear. This study shows that a putative incoherent feedforward circuit comprising ellipsoid body neurons, cholinergic R4d, and its repressor GABAergic R2/R4m neurons regulates the pre-mating rejection response in the virgin female Drosophila melanogaster. Both R4d and R2/R4m are positively regulated, via specific dopamine receptors, by a subset of neurons in the dopaminergic PPM3 cluster. Genetic deprivation of GABAergic signal via GABAA receptor RNA interference in this circuit induces a massive rejection response, whereas activation of GABAergic R2/R4m or suppression of cholinergic R4d increases receptivity. Moreover, glutamatergic signaling via N-methyl-d-aspartate receptors induces NO-mediated retrograde regulation potentially from R4d to R2/R4m, likely providing flexible control of the behavioral switching from rejection to acceptance. This study elucidates the molecular and neural mechanisms regulating the behavioral selection process of the pre-mating female.
Newell, N. R., Ray, S., Dalton, J. E., Fortier, J. C., Kao, J. Y., Chang, P. L., Nuzhdin, S. V. and Arbeitman, M. N. (2020). The Drosophila post-mating response: Gene expression and behavioral changes reveal perdurance and variation in cross-tissue interactions. G3 (Bethesda). PubMed ID: 31907222
Examining cross-tissue interactions is important for understanding physiology and homeostasis. In animals, the female gonad produces signaling molecules that act distally. This study examined gene expression in Drosophila melanogaster female head tissues in 1) virgins without a germline compared to virgins with a germline, 2) post-mated females with and without a germline compared to virgins, and 3) post-mated females mated to males with and without a germline compared to virgins. In virgins, the absence of a female germline results in expression changes in genes with known roles in nutrient homeostasis. At one- and three-days post-mating, genes that change expression are enriched with those that function in metabolic pathways, in all conditions. Female post-mating impacts were systematically examined on sleep, food preference and re-mating, in the strains and time points used for gene expression analyses and compare to published studies. Post-mating, gene expression changes vary by strain, prompting an examination of variation in female re-mating. A genome-wide association study was performed that identified several DNA polymorphisms, including four in/near Wnt signaling pathway genes. Together, these data reveal how gene expression and behavior in females are influenced by cross-tissue interactions, by examining the impact of mating, fertility, and genotype.
Jiang, L., Cheng, Y., Gao, S., Zhong, Y., Ma, C., Wang, T. and Zhu, Y. (2020). Emergence of social cluster by collective pairwise encounters in Drosophila. Elife 9. PubMed ID: 31959283
=Many animals exhibit an astonishing ability to form groups of large numbers of individuals. The dynamic properties of such groups have been the subject of intensive investigation. The actual grouping processes and underlying neural mechanisms, however, remain elusive. This study established a social clustering paradigm in Drosophila to investigate the principles governing social group formation. Fruit flies spontaneously assembled into a stable cluster mimicking a distributed network. Social clustering was exhibited as a highly dynamic process including all individuals, which participated in stochastic pair-wise encounters mediated by appendage touches. Depriving sensory inputs resulted in abnormal encounter responses and a high failure rate of cluster formation. Furthermore, the social distance of the emergent network was regulated by ppk-specific neurons, which were activated by contact-dependent social grouping. Taken together, these findings revealed the development of an orderly social structure from initially unorganised individuals via collective actions.
Asirim, E. Z., Humberg, T. H., Maier, G. L. and Sprecher, S. G. (2020). Circadian and Genetic Modulation of Visually-Guided Navigation in Drosophila Larvae. Sci Rep 10(1): 2752. PubMed ID: 32066794
Organisms possess an endogenous molecular clock which enables them to adapt to environmental rhythms and to synchronize their metabolism and behavior accordingly. Circadian rhythms govern daily oscillations in numerous physiological processes. Drosophila larvae have relatively simple nervous system compared to their adult counterparts, yet they both share a homologous molecular clock with mammals, governed by interlocking transcriptional feedback loops with highly conserved constituents. Larvae exhibit a robust light avoidance behavior, presumably enabling them to avoid predators and desiccation, and DNA-damage by exposure to ultraviolet light, hence are crucial for survival. Circadian rhythm has been shown to alter light-dark preference, however it remains unclear how distinct behavioral strategies are modulated by circadian time. To address this question, this study investigate the larval visual navigation at different time-points of the day employing a computer-based tracking system, which allows detailed evaluation of distinct navigation strategies. The results show that due to circadian modulation specific to light information processing, larvae avoid light most efficiently at dawn, and a functioning clock mechanism at both molecular and neuro-signaling level is necessary to conduct this modulation.

Wednesday April 1 - Adult Physiology

Wat, L. W., Chao, C., Bartlett, R., Buchanan, J. L., Millington, J. W., Chih, H. J., Chowdhury, Z. S., Biswas, P., Huang, V., Shin, L. J., Wang, L. C., Gauthier, M. L., Barone, M. C., Montooth, K. L., Welte, M. A. and Rideout, E. J. (2020). A role for triglyceride lipase brummer in the regulation of sex differences in Drosophila fat storage and breakdown. PLoS Biol 18(1): e3000595. PubMed ID: 31961851
Triglycerides are the major form of stored fat in all animals. One important determinant of whole-body fat storage is whether an animal is male or female. This study used Drosophila, an established model for studies on triglyceride metabolism, to gain insight into the genes and physiological mechanisms that contribute to sex differences in fat storage. This analysis of triglyceride storage and breakdown in both sexes identified a role for triglyceride lipase brummer (bmm) in the regulation of sex differences in triglyceride homeostasis. Normally, male flies have higher levels of bmm mRNA both under normal culture conditions and in response to starvation, a lipolytic stimulus. Loss of bmm largely eliminates the sex difference in triglyceride storage and abolishes the sex difference in triglyceride breakdown via strongly male-biased effects. Although this study shows that bmm function in the fat body affects whole-body triglyceride levels in both sexes, in males, an additional role for bmm function was identified in the somatic cells of the gonad and in neurons in the regulation of whole-body triglyceride homeostasis. Furthermore, it was demonstrated that lipid droplets are normally present in both the somatic cells of the male gonad and in neurons, revealing a previously unrecognized role for bmm function, and possibly lipid droplets, in these cell types in the regulation of whole-body triglyceride homeostasis. Taken together, these data reveal a role for bmm function in the somatic cells of the gonad and in neurons in the regulation of male-female differences in fat storage and breakdown and identify bmm as a link between the regulation of triglyceride homeostasis and biological sex.
Ng'oma, E., Williams-Simon, P. A., Rahman, A. and King, E. G. (2020). Diverse biological processes coordinate the transcriptional response to nutritional changes in a Drosophila melanogaster multiparent population. BMC Genomics 21(1): 84. PubMed ID: 31992183
Environmental variation in the amount of resources available to populations challenge individuals to optimize the allocation of those resources to key fitness functions. This coordination of resource allocation relative to resource availability is commonly attributed to key nutrient sensing gene pathways in laboratory model organisms, chiefly the insulin/TOR signaling pathway. However, the genetic basis of diet-induced variation in gene expression is less clear. To describe the natural genetic variation underlying nutrient-dependent differences, an outbred panel was used derived from a multiparental population, the Drosophila Synthetic Population Resource. RNA sequence data was analyzed from multiple female tissue samples dissected from flies reared in three nutritional conditions: high sugar (HS), dietary restriction (DR), and control (C) diets. A large proportion of genes in the experiment (19.6% or 2471 genes) were significantly differentially expressed for the effect of diet, and 7.8% (978 genes) for the effect of the interaction between diet and tissue type. Interestingly, similar patterns of gene expression were observed relative to the C diet, in the DR and HS treated flies, a response likely reflecting diet component ratios. Hierarchical clustering identified 21 robust gene modules showing intra-modularly similar patterns of expression across diets, all of which were highly significant for diet or diet-tissue interaction effects. Gene set enrichment analysis for different diet-tissue combinations revealed a diverse set of pathways and gene ontology (GO) terms. GO analysis on individual co-expressed modules likewise showed a large number of terms encompassing many cellular and nuclear processes. Although a handful of genes in the IIS/TOR pathway including Ilp5, Rheb, and Sirt2 showed significant elevation in expression, many key genes such as InR, chico, most insulin peptide genes, and the nutrient-sensing pathways were not observed. These results suggest that a more diverse network of pathways and gene networks mediate the diet response in these population. These results have important implications for future studies focusing on diet responses in natural populations.
Beebe, K., Robins, M. M., Hernandez, E. J., Lam, G., Horner, M. A. and Thummel, C. S. (2020). Drosophila estrogen-related receptor directs a transcriptional switch that supports adult glycolysis and lipogenesis. Genes Dev. PubMed ID: 32165409
Metabolism and development must be closely coupled to meet the changing physiological needs of each stage in the life cycle. The molecular mechanisms that link these pathways, however, remain poorly understood. This study shows that the Drosophila estrogen-related receptor (ERR) directs a transcriptional switch in mid-pupae that promotes glucose oxidation and lipogenesis in young adults. ERR mutant adults are viable but display reduced locomotor activity, susceptibility to starvation, elevated glucose, and an almost complete lack of stored triglycerides. Molecular profiling by RNA-seq, ChIP-seq, and metabolomics revealed that glycolytic and pentose phosphate pathway genes are induced by ERR, and their reduced expression in mutants is accompanied by elevated glycolytic intermediates, reduced TCA cycle intermediates, and reduced levels of long chain fatty acids. Unexpectedly, it was found that the central pathways of energy metabolism, including glycolysis, the tricarboxylic acid cycle, and electron transport chain, are coordinately induced at the transcriptional level in mid-pupae and maintained into adulthood, and this response is partially dependent on ERR, leading to the metabolic defects observed in mutants. These data support the model that ERR contributes to a transcriptional switch during pupal development that establishes the metabolic state of the adult fly.
Wang, Y., Norum, M., Oehl, K., Yang, Y., Zuber, R., Yang, J., Farine, J. P., Gehring, N., Flotenmeyer, M., Ferveur, J. F. and Moussian, B. (2020). Dysfunction of Oskyddad causes Harlequin-type ichthyosis-like defects in Drosophila melanogaster. PLoS Genet 16(1): e1008363. PubMed ID: 31929524
Prevention of desiccation is a constant challenge for terrestrial organisms. Land insects have an extracellular coat, the cuticle, that plays a major role in protection against exaggerated water loss. This study reports that the ABC transporter Oskyddad (Osy)-a human ABCA12 paralog-contributes to the waterproof barrier function of the cuticle in the fruit fly Drosophila melanogaster. The reduction or elimination of Osy function provokes rapid desiccation. Osy is also involved in defining the inward barrier against xenobiotics penetration. Consistently, the amounts of cuticular hydrocarbons that are involved in cuticle impermeability decrease markedly when Osy activity is reduced. GFP-tagged Osy localises to membrane nano-protrusions within the cuticle, likely pore canals. This suggests that Osy is mediating the transport of cuticular hydrocarbons (CHC) through the pore canals to the cuticle surface. The envelope, which is the outermost cuticle layer constituting the main barrier, is unaffected in osy mutant larvae. This contrasts with the function of Snu, another ABC transporter needed for the construction of the cuticular inward and outward barriers, that nevertheless is implicated in CHC deposition. Hence, Osy and Snu have overlapping and independent roles to establish cuticular resistance against transpiration and xenobiotic penetration. The osy deficient phenotype parallels the phenotype of Harlequin ichthyosis caused by mutations in the human abca12 gene. Thus, it seems that the cellular and molecular mechanisms of lipid barrier assembly in the skin are conserved during evolution.
Arce-Molina, R., Cortes-Molina, F., Sandoval, P. Y., Galaz, A., Alegria, K., Schirmeier, S., Barros, L. F. and San Martin, A. (2020). A highly responsive pyruvate sensor reveals pathway-regulatory role of the mitochondrial pyruvate carrier MPC. Elife 9. PubMed ID: 32142409
Mitochondria generate ATP and building blocks for cell growth and regeneration, using pyruvate as the main substrate. This study introduced PyronicSF, a user-friendly GFP-based sensor of improved dynamic range that enables real-time subcellular quantitation of mitochondrial pyruvate transport, concentration and flux. Cultured mouse astrocytes were shown to maintain mitochondrial pyruvate in the low micromolar range, below cytosolic pyruvate, which means that the mitochondrial pyruvate carrier MPC is poised to exert ultrasensitive control on the balance between respiration and anaplerosis/gluconeogenesis. The functionality of the sensor in living tissue is demonstrated in the brain of Drosophila melanogaster larvae. Mitochondrial subpopulations are known to coexist within a given cell, which differ in their morphology, mobility, membrane potential, and vicinity to other organelles. The present tool can be used to investigate how mitochondrial diversity relates to metabolism, to study the role of MPC in disease, and to screen for small-molecule MPC modulators.
Wu, Q., Yu, G., Park, S. J., Gao, Y., Ja, W. W. and Yang, M. (2020). Excreta Quantification (EX-Q) for Longitudinal Measurements of Food Intake in Drosophila. iScience 23(1): 100776. PubMed ID: 31901635
Longitudinal measurements of food intake remain a challenge in Drosophila studies of nutrition and behavior. Here, this study reports an improved method for measuring fly food intake using dye-labeled food and excreta quantification (EX-Q). Reducing the surface area of the medium maximized excreta recovery and the accuracy in estimating total consumption. The EX-Q method is compatible with agar-based medium and makes it possible to measure consumption over an extended period and at multiple time points without sacrificing flies. Using EX-Q, nutrient- and age-specific features of Drosophila feeding behavior was revealed. Daily consumption of a chemically defined diet was relatively consistent over the first 25 days of adulthood. Omitting amino acids or vitamins from the diet reduced consumption in both sexes, whereas omitting sugars or cholesterol primarily affected female food intake. These results demonstrate EX-Q as a simple, reliable, and nondestructive method for longitudinal studies of solid food intake in Drosophila.
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