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Friday, January 31, 2020 - Enhancers and Transcriptional Regulation

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Volkova, E. I., Andreyenkova, N. G., Andreyenkov, O. V., Sidorenko, D. S., Zhimulev, I. F. and Demakov, S. A. (2019). Structural and Functional Dissection of the 5' Region of the Notch Gene in Drosophila melanogaster. Genes (Basel) 10(12). PubMed ID: 31842424
Notch is a key factor of a signaling cascade which regulates cell differentiation in all multicellular organisms. Numerous investigations have been directed mainly at studying the mechanism of Notch protein action; however, very little is known about the regulation of activity of the gene itself. This study provides the results of targeted 5'-end editing of the Drosophila Notch gene in its native environment and genetic and cytological effects of these changes. Using the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9 (CRISPR/Cas9) system in combination with homologous recombination, a founder fly stock was obtained in which a 4-kb fragment, including the 5' nontranscribed region, the first exon, and a part of the first intron of Notch, was replaced by an attachment Phage (attP) site. Then, fly lines carrying a set of six deletions within the 5'untranscribed region of the gene were obtained by PhiC31-mediated integration of transgenic constructs. Part of these deletions does not affect gene activity, but their combinations with transgenic construct in the first intron of the gene cause defects in the Notch target tissues. At the polytene chromosome level a DNA segment (~250 bp) in the Notch 5'-nontranscribed region was defined that when deleted leads to disappearance of the 3C6/C7 interband and elimination of CTC-Factor (CTCF) and Chromator (CHRIZ) insulator proteins in this region.
Mahmud, A., Yang, D., Stenberg, P., Ioshikhes, I. and Nandi, S. (2019). Exploring a Drosophila transcription factor interaction network to identify cis-regulatory modules. J Comput Biol. PubMed ID: 31855461
Multiple transcription factors (TFs) bind to specific sites in the genome and interact among themselves to form the cis-regulatory modules (CRMs). They are essential in modulating the expression of genes, and it is important to study this interplay to understand gene regulation. This study integrated experimentally identified TF binding sites collected from published studies with computationally predicted TF binding sites to identify Drosophila CRMs. Along with the detection of the previously known CRMs, this approach identified novel protein combinations. High-occupancy target sites were detected, where a large number of TFs bind. Investigating these sites revealed that Giant, Dichaete, and Knirp are highly enriched in these locations. A common TAG team motif was observed at these sites, which might play a role in recruiting other TFs. While comparing the binding sites at distal and proximal promoters, it was found that certain regulatory TFs, such as Zelda, were highly enriched in enhancers. This study has shown that, from the information available concerning the TF binding sites, the real CRMs could be predicted accurately and efficiently. Although it is only possible to claim co-occurrence of these proteins in this study, it may actually point to their interaction (as known interaction proteins typically co-occur together). Such an integrative approach can, therefore, help to provide a better understanding of the interplay among the factors, even though further experimental verification is required.
Lammers, N. C., Galstyan, V., Reimer, A., Medin, S. A., Wiggins, C. H. and Garcia, H. G. (2019). Multimodal transcriptional control of pattern formation in embryonic development. Proc Natl Acad Sci U S A. PubMed ID: 31882445
Predicting how interactions between transcription factors and regulatory DNA sequence dictate rates of transcription and, ultimately, drive developmental outcomes remains an open challenge in physical biology. Using stripe 2 of the even-skipped gene in Drosophila embryos as a case study, this study dissected the regulatory forces underpinning a key step along the developmental decision-making cascade: the generation of cytoplasmic mRNA patterns via the control of transcription in individual cells. Using live imaging and computational approaches, it was found that the transcriptional burst frequency is modulated across the stripe to control the mRNA production rate. However, it was discovered that bursting alone cannot quantitatively recapitulate the formation of the stripe and that control of the window of time over which each nucleus transcribes even-skipped plays a critical role in stripe formation. Theoretical modeling revealed that these regulatory strategies (bursting and the time window) respond in different ways to input transcription factor concentrations, suggesting that the stripe is shaped by the interplay of 2 distinct underlying molecular processes.
Pal, K., Forcato, M., Jost, D., Sexton, T., Vaillant, C., Salviato, E., Mazza, E. M. C., Lugli, E., Cavalli, G. and Ferrari, F. (2019). Global chromatin conformation differences in the Drosophila dosage compensated chromosome X. Nat Commun 10(1): 5355. PubMed ID: 31767860
In Drosophila melanogaster the single male chromosome X undergoes an average twofold transcriptional upregulation for balancing the transcriptional output between sexes. Previous literature hypothesised that a global change in chromosome structure may accompany this process. However, recent studies based on Hi-C failed to detect these differences. This study showed that global conformational differences are specifically present in the male chromosome X and detectable using Hi-C data on sex-sorted embryos, as well as male and female cell lines, by leveraging custom data analysis solutions. The male chromosome X has more mid-/long-range interactions. Differences were identified at structural domain boundaries containing BEAF-32 in conjunction with CP190 or Chromator. Weakening of these domain boundaries in male chromosome X co-localizes with the binding of the dosage compensation complex and its co-factor CLAMP, reported to enhance chromatin accessibility. Together, these data strongly indicate that chromosome X dosage compensation affects global chromosome structure.
Rieder, L. E., Jordan, W. T., 3rd and Larschan, E. N. (2019). Targeting of the dosage-compensated male X-chromosome during early Drosophila development. Cell Rep 29(13): 4268-4275. PubMed ID: 31875538
Dosage compensation, which corrects for the imbalance in X-linked gene expression between XX females and XY males, represents a model for how genes are targeted for coordinated regulation. However, the mechanism by which dosage compensation complexes identify the X chromosome during early development remains unknown because of the difficulty of sexing embryos before zygotic transcription using X- or Y-linked reporter transgenes. This study used meiotic drive to sex Drosophila embryos before zygotic transcription and ChIP-seq to measure the dynamics of dosage compensation factor targeting. The Drosophila male-specific lethal dosage compensation complex (MSLc) requires the ubiquitous zinc-finger protein chromatin-linked adaptor for MSL proteins (CLAMP) to identify the X chromosome. A multi-stage process was observed in which MSLc first identifies CLAMP binding sites throughout the genome, followed by concentration at the strongest X-linked MSLc sites. Insight is provided into the dynamics of binding site recognition by a large transcription complex during early development.
Rahimi, N., Carmon, S., Averbukh, I., Khajouei, F., Sinha, S., Schejter, E. D., Barkai, N. and Shilo, B. Z. (2020). Global shape of Toll activation is determined by wntD enhancer properties. Proc Natl Acad Sci U S A. PubMed ID: 31900360
Buffering variability in morphogen distribution is essential for reproducible patterning. A theoretically proposed class of mechanisms, termed "distal pinning," achieves robustness by combining local sensing of morphogen levels with global modulation of gradient spread. This study demonstrates a critical role for morphogen sensing by a gene enhancer, which ultimately determines the final global distribution of the morphogen and enables reproducible patterning. Specifically, this study shows that, while the pattern of Toll activation in the early Drosophila embryo is robust to gene dosage of its locally produced regulator, WntD, it is sensitive to a single-nucleotide change in the wntD enhancer. Thus, enhancer properties of locally produced WntD directly impinge on the global morphogen profile.

Thursday, December 30th - Adult Physiology

Sah Pamboro, E. L., Brown, E. B. and Keene, A. C. (2019). Dietary fatty acids promote sleep through a taste-independent mechanism. Genes Brain Behav: e12629. PubMed ID: 31845509
Consumption of foods that are high in fat contribute to obesity and metabolism-related disorders. Dietary lipids are comprised of triglycerides and fatty acids, and the highly palatable taste of dietary fatty acids promotes food consumption, activates reward centers in mammals, and underlies hedonic feeding. Despite the central role of dietary fats in the regulation of food intake and the etiology of metabolic diseases, little is known about how fat consumption regulates sleep. The fruit fly, Drosophila melanogaster, provides a powerful model system for the study of sleep and metabolic traits, and flies potently regulate sleep in accordance with food availability. To investigate the effects of dietary fats on sleep regulation, fatty acids were supplemented into the diet of Drosophila and their effects on sleep and activity were measured. Flies fed a diet of hexanoic acid, a medium-chain fatty acid that is a by-product of yeast fermentation, slept more than flies starved on an agar diet. To assess whether dietary fatty acids regulate sleep through the taste system, sleep was assessed in flies with a mutation in the hexanoic acid receptor Ionotropic receptor 56D, which is required for fatty acid taste perception. These flies also sleep more than agar-fed flies when fed a hexanoic acid diet, suggesting the sleep promoting effect of hexanoic acid is not dependent on sensory perception. Taken together, these findings provide a platform to investigate the molecular and neural basis for fatty acid-dependent modulation of sleep.
Singh, S., Giesecke, A., Damulewicz, M., Fexova, S., Mazzotta, G. M., Stanewsky, R. and Dolezel, D. (2019). New Drosophila circadian clock mutants affecting temperature compensation induced by targeted mutagenesis of timeless. Front Physiol 10: 1442. PubMed ID: 31849700
Drosophila has served as an excellent genetic model to decipher the molecular basis of the circadian clock. Two key proteins, Period (Per) and Timeless (Tim), are particularly well explored and a number of various arrhythmic, slow, and fast clock mutants have been identified in classical genetic screens. Interestingly, the free running period (tau, τ) is influenced by temperature in some of these mutants, whereas τ is temperature-independent in other mutant lines as in wild-type flies. This, so-called "temperature compensation" ability is compromised in the mutant timeless allele "ritsu" (timrit), and also in the timblind allele. A collection of new mutants was generated, and functional protein domains involved in the regulation of τ and in general clock function were mapped. Twenty new timeless mutant alleles exhibited various impairments of temperature compensation. Molecular characterization revealed that the mutations included short in-frame insertions, deletions, or substitutions of a few amino acids resulting from the non-homologous end joining repair process. Several mutations with a strong temperature compensation defect map to one specific region of Tim. In silico analysis suggests they affect a putative nuclear export signal (NES) and phosphorylation sites of Tim. Immunostaining for Per was performed on two Tim mutants that display longer τ at 25 degrees C and complete arrhythmicity at 28 degrees C. Consistently with the behavioral phenotype, Per immunoreactivity was reduced in circadian clock neurons of flies exposed to elevated temperatures.
Krupp, J. J., Nayal, K., Wong, A., Millar, J. G. and Levine, J. D. (2019). Desiccation resistance is an adaptive life-history trait dependent upon cuticular hydrocarbons, and influenced by mating status and temperature in D. melanogaster. J Insect Physiol: 103990. PubMed ID: 31830467
Terrestrial insects are susceptible to desiccation and conserve internal water stores by preventing the loss of water due to transpiration across the cuticle. The epicuticle, a thin waxy layer on the outer surface of the insect cuticle is comprised primarily of a complex blend of cuticular hydrocarbons (CHCs) and is integral to preventing cuticular water loss. How the composition of epicuticular lipids (quantity and quality of the specific hydrocarbons) relates to desiccation resistance, however, has been difficult to determine. This study establish a model system to test the capacity of CHCs to protect against desiccation in the vinegar fly, Drosophila melanogaster. Using this system, it was demonstrated that the oenocytes and CHCs produced by these cells are critically important for desiccation resistance, as measured by survival under desiccative conditions. Additionally, it was shown that both mating status and developmental temperature influence desiccation resistance. Prior mating increased desiccation survival through the direct transfer of CHCs between sexual partners, as well as through a female-specific response to a male-derived factor transferred during copulation. Together, these results demonstrate that desiccation resistance is an adaptive life-history trait dependent upon CHCs and influenced by prior social interactions and environmental conditions.
Leon, K. E., Fruin, A. M., Nowotarski, S. L. and DiAngelo, J. R. (2019). The regulation of triglyceride storage by ornithine decarboxylase (Odc1) in Drosophila. Biochem Biophys Res Commun. PubMed ID: 31870547
Polyamines are low molecular weight, organic cations that play a critical role in many major cellular processes including cell cycle regulation and apoptosis, cellular division, tissue proliferation, and cellular differentiation; however, the functions of polyamines in regulating the storage of metabolic fuels such as triglycerides and glycogen is poorly understood. To address this question, focus was placed on the Drosophila homolog of ornithine decarboxylase (Odc1), the first rate-limiting enzyme in the synthesis of polyamines. Mutants in Odc1 are lethal, but heterozygotes were viable to adulthood. Odc1 heterozygotes appeared larger than their genetic background control flies and consistent with this observation, weighed more than the controls. However, the increased weight was not due to increased food consumption as heterozygotes ate less than the controls. Interestingly, Odc1 heterozygous flies had augmented triglyceride storage, and this lipid phenotype was due to increased triglyceride storage per cell and an increase in the number of fat cells produced. Odc1 heterozygous flies also displayed increased expression of the lipid synthesis genes fatty acid synthase (FASN) and acetyl-CoA carboxylase (ACC), suggesting increased lipid synthesis was the cause of the augmented triglyceride phenotype. These results provide a link between the expression of Odc1 and triglyceride storage suggesting that the polyamine pathway plays a role in regulating lipid metabolism.
Gandara, L., Durrieu, L., Behrensen, C. and Wappner, P. (2019). A genetic toolkit for the analysis of metabolic changes in Drosophila provides new insights into metabolic responses to stress and malignant transformation. Sci Rep 9(1): 19945. PubMed ID: 31882718
Regulation of the energetic metabolism occurs fundamentally at the cellular level, so analytical strategies must aim to attain single cell resolution to fully embrace its inherent complexity. This study has developed methods to utilize a toolset of metabolic FRET sensors for assessing lactate, pyruvate and 2-oxoglutarate levels of Drosophila tissues in vivo by imaging techniques. The study shows how the energetic metabolism is altered by hypoxia: While some larval tissues respond to low oxygen levels by executing a metabolic switch towards lactic fermentation, the fat body and salivary glands do not alter their energetic metabolism. Analysis of tumor metabolism revealed that depending on the genetic background, some tumors undergo a lactogenic switch typical of the Warburg effect, while other tumors do not. This toolset allows for developmental and physiologic studies in genetically manipulated Drosophila individuals in vivo.
Wang, X., Zheng, H., Jia, Z., Lei, Z., Li, M., Zhuang, Q., Zhou, H., Qiu, Y., Fu, Y., Yang, X., Xi, Y. and Yan, Q. (2019). Drosophila Prominin-like, a homolog of CD133, interacts with ND20 to maintain mitochondrial function. Cell Biosci 9: 101. PubMed ID: 31890150
Drosophila Prominin-like is a homolog of mammalian CD133, which is recognized as a biomarker for stem cells. This study found that Drosophila Prominin-like interacts with ND-20, a subunit of mitochondrial respiratory complex I. Prominin-like is a six-transmembrane glycoprotein which localizes on cellular membranes. Prominin-like localizes in the mitochondria. The knockdown of prominin-like in S2 cells resulted in transient mitochondrial dysfunctions as evidenced by reduced ATP production, elevated ROS generation and an accompanied reduction in mitochondrial proteins. Mitochondrial dysfunctions were detected in aged prominin-like mutant flies. The data indicates that Prominin-like acts to maintain mitochondrial function through its interaction with ND-20 which, itself, is active in the mitochondrial electron transport chain.

Wednesday, January 29th - Signaling

Morotz, G. M., Glennon, E. B., Greig, J., Lau, D. H. W., Bhembre, N., Mattedi, F., Muschalik, N., Noble, W., Vagnoni, A. and Miller, C. C. J. (2019). Kinesin light chain-1 serine-460 phosphorylation is altered in Alzheimer's disease and regulates axonal transport and processing of the amyloid precursor protein. Acta Neuropathol Commun 7(1): 200. PubMed ID: 31806024
Damage to axonal transport is an early pathogenic event in Alzheimer's disease. The amyloid precursor protein (APP) is a key axonal transport cargo since disruption to APP transport promotes amyloidogenic processing of APP. Moreover, altered APP processing itself disrupts axonal transport. The mechanisms that regulate axonal transport of APP are therefore directly relevant to Alzheimer's disease pathogenesis. APP is transported anterogradely through axons on kinesin-1 motors and one route for this transport involves calsyntenin-1, a type-1 membrane spanning protein that acts as a direct ligand for kinesin-1 light chains (KLCs). Thus, loss of calsyntenin-1 disrupts APP axonal transport and promotes amyloidogenic processing of APP. Phosphorylation of KLC1 on serine-460 has been shown to reduce anterograde axonal transport of calsyntenin-1 by inhibiting the KLC1-calsyntenin-1 interaction. This study demonstrates that in Alzheimer's disease frontal cortex, KLC1 levels are reduced and the relative levels of KLC1 serine-460 phosphorylation are increased; these changes occur relatively early in the disease process. It was also shown that a KLC1 serine-460 phosphomimetic mutant inhibits axonal transport of APP in both mammalian neurons in culture and in Drosophila neurons in vivo. Finally, it was demonstrated that expression of the KLC1 serine-460 phosphomimetic mutant promotes amyloidogenic processing of APP. Together, these results suggest that increased KLC1 serine-460 phosphorylation contributes to Alzheimer's disease.
Scarpelli, E. M., Trinh, V. Y., Tashnim, Z., Krans, J. L., Keller, L. C. and Colodner, K. J. (2019). Developmental expression of human tau in Drosophila melanogaster glial cells induces motor deficits and disrupts maintenance of PNS axonal integrity, without affecting synapse formation. PLoS One 14(12): e0226380. PubMed ID: 31821364
Tauopathies are a class of neurodegenerative diseases characterized by the abnormal phosphorylation and accumulation of the microtubule-associated protein, tau (see Drosophila Tau), in both neuronal and glial cells. Though tau pathology in glial cells is a prominent feature of many of these disorders, the pathological contribution of these lesions to tauopathy pathogenesis remains largely unknown. Moreover, while tau pathology is predominantly found in the central nervous system, a role for tau in the cells of the peripheral nervous system has been described, though not well characterized. To investigate the effects of glial tau expression on the development and maintenance of the peripheral nervous system, a Drosophila melanogaster model of tauopathy was used that expresses human wild-type tau in glial cells during development. Glial tau expression during development was found to result in larval locomotor deficits and organismal lethality at the pupal stage, without affecting larval neuromuscular junction synapse development or post-synaptic amplitude. There was, however, a significant decrease in the decay time of synaptic potentials upon repeated stimulation of the motoneuron. Behavioral abnormalities were accompanied by glial cell death, disrupted maintenance of glial-axonal integrity, and the abnormal accumulation of the presynaptic protein, Bruchpilot, in peripheral nerve axons. Together, these data demonstrate that human tau expression in Drosophila glial cells does not affect neuromuscular junction synapse formation during development, but is deleterious to the maintenance of glial-axonal interactions in the peripheral nervous system.
Portela, M., Venkataramani, V., Fahey-Lozano, N., Seco, E., Losada-Perez, M., Winkler, F. and Casas-Tinto, S. (2019). Glioblastoma cells vampirize WNT from neurons and trigger a JNK/MMP signaling loop that enhances glioblastoma progression and neurodegeneration. PLoS Biol 17(12): e3000545. PubMed ID: 31846454
Glioblastoma (GB) is the most lethal brain tumor, and Wingless (Wg)-related integration site (WNT) pathway activation in these tumors is associated with a poor prognosis. Clinically, the disease is characterized by progressive neurological deficits. However, whether these symptoms result from direct or indirect damage to neurons is still unresolved. Using Drosophila and primary xenografts as models of human GB, this study describes a mechanism that leads to activation of WNT signaling (Wg in Drosophila) in tumor cells. GB cells display a network of tumor microtubes (TMs) that enwrap neurons, accumulate Wg receptor Frizzled1 (Fz1), and, thereby, deplete Wg from neurons, causing neurodegeneration. This process has been defined process as "vampirization." Furthermore, GB cells establish a positive feedback loop to promote their expansion, in which the Wg pathway activates cJun N-terminal kinase (JNK) in GB cells, and, in turn, JNK signaling leads to the post-transcriptional up-regulation and accumulation of matrix metalloproteinases (MMPs), which facilitate TMs' infiltration throughout the brain, TMs' network expansion, and further Wg depletion from neurons. Consequently, GB cells proliferate because of the activation of the Wg signaling target, beta-catenin, and neurons degenerate because of Wg signaling extinction. These findings reveal a molecular mechanism for TM production, infiltration, and maintenance that can explain both neuron-dependent tumor progression and also the neural decay associated with GB.
Rizzo, M. J. and Johnson, E. C. (2019). Homodimerization of Drosophila Class A neuropeptide GPCRs: Evidence for conservation of GPCR dimerization throughout metazoan evolution. Biochem Biophys Res Commun. PubMed ID: 31864711
While many instances of GPCR dimerization have been reported for vertebrate receptors, invertebrate GPCR dimerization remains poorly investigated, with few invertebrate GPCRs having been shown to assemble as dimers. To date, no Drosophila GPCRs have been shown to assemble as dimers. To explore the evolutionary conservation of GPCR dimerization, an acceptor-photobleaching FRET methodology was used to evaluate whether multiple subclasses of Drosophila GPCRs assembled as homodimers when heterologously expressed in HEK-293 T cells. Multiple Drosophila neuropeptide GPCRs that exhibited structural homology with a vertebrate GPCR family member previously shown to assemble as a dimer were C-terminally tagged with CFP and YFP fluorophores, and these receptors were visualized through confocal microscopy. FRET responses were determined based on the increase in CFP emission intensity following YFP photobleaching for each receptor pair tested. A significant FRET response was observed for each receptor expressed as a homodimer pair, while non-significant FRET responses were displayed by both cytosolic CFP and YFP expressed alone, and a heterodimeric pair of receptors from unrelated families. These findings suggest that receptors exhibiting positive FRET responses assemble as homodimers at the plasma membrane and are the first to suggest that Drosophila GPCRs assemble as homodimeric complexes. It is proposed that GPCR dimerization arose early in metazoan evolution and likely plays an important and underappreciated role in the cellular signaling of all animals.
Robles-Murguia, M., Rao, D., Finkelstein, D., Xu, B., Fan, Y. and Demontis, F. (2020). Muscle-derived Dpp regulates feeding initiation via endocrine modulation of brain dopamine biosynthesis. Genes Dev 34(1-2): 37-52. PubMed ID: 31831628
In animals, the brain regulates feeding behavior in response to local energy demands of peripheral tissues, which secrete orexigenic and anorexigenic hormones. Although skeletal muscle is a key peripheral tissue, it remains unknown whether muscle-secreted hormones regulate feeding. In Drosophila, this study found that decapentaplegic (dpp), the homolog of human bone morphogenetic proteins BMP2 and BMP4, is a muscle-secreted factor (a myokine) that is induced by nutrient sensing and that circulates and signals to the brain. Muscle-restricted dpp RNAi promotes foraging and feeding initiation, whereas dpp overexpression reduces it. This regulation of feeding by muscle-derived Dpp stems from modulation of brain tyrosine hydroxylase (TH) expression and dopamine biosynthesis. Consistently, Dpp receptor signaling in dopaminergic neurons regulates TH expression and feeding initiation via the downstream transcriptional repressor Schnurri. Moreover, pharmacologic modulation of TH activity rescues the changes in feeding initiation due to modulation of dpp expression in muscle. These findings indicate that muscle-to-brain endocrine signaling mediated by the myokine Dpp regulates feeding behavior.
Senos Demarco, R. and Jones, D. L. (2019). Mitochondrial fission regulates germ cell differentiation by suppressing ROS-mediated activation of Epidermal Growth Factor signaling in the Drosophila larval testis. Sci Rep 9(1): 19695. PubMed ID: 31873089
Mitochondria are essential organelles that have recently emerged as hubs for several metabolic and signaling pathways in the cell. Mitochondrial morphology is regulated by constant fusion and fission events to maintain a functional mitochondrial network and to remodel the mitochondrial network in response to external stimuli. Although the role of mitochondria in later stages of spermatogenesis has been investigated in depth, the role of mitochondrial dynamics in regulating early germ cell behavior is relatively less-well understood. Previously it was demonstrated that mitochondrial fusion is required for germline stem cell (GSC) maintenance in the Drosophila testis. This study shows that mitochondrial fission is also important for regulating the maintenance of early germ cells in larval testes. Inhibition of Drp1 in early germ cells resulted in the loss of GSCs and spermatogonia due to the accumulation of reactive oxygen species (ROS) and activation of the EGFR pathway in adjacent somatic cyst cells. EGFR activation contributed to premature germ cell differentiation. These data provide insights into how mitochondrial dynamics can impact germ cell maintenance and differentiation via distinct mechanisms throughout development.

Tuesday, January 28th - Disease models

McDaniel, S. L., Hollatz, A. J., Branstad, A. M., Gaskill, M. M., Fox, C. A. and Harrison, M. M. (2019). Tissue-specific DNA replication defects in Drosophila melanogaster caused by a Meier-Gorlin syndrome mutation in Orc4. Genetics. PubMed ID: 31818869
Meier-Gorlin syndrome is a rare recessive disorder characterized by a number of distinct tissue-specific developmental defects. Genes encoding members of the origin recognition complex (ORC) and additional proteins essential for DNA replication (CDC6, CDT1, GMNN, CDC45, MCM5, and DONSON) are mutated in individuals diagnosed with MGS. The essential role of ORC is to license origins during the G1 phase of the cell cycle, but ORC has also been implicated in several non-replicative functions. Because of its essential role in DNA replication, ORC is required for every cell division during development. Thus, it is unclear how the Meier-Gorlin syndrome mutations in genes encoding ORC lead to the tissue-specific defects associated with the disease. To begin to address these issues, Cas9-mediated genome engineering was used to generate a Drosophila melanogaster model of individuals carrying a specific Meier-Gorlin syndrome mutation in ORC4 along with control strains. Together these strains provide the first metazoan model for an MGS mutation in which the mutation was engineered at the endogenous locus along with precisely defined control strains. Flies homozygous for the engineered MGS allele reach adulthood, but with several tissue-specific defects. Genetic analysis revealed that this Orc4 allele was a hypomorph. Mutant females were sterile, and phenotypic analyses suggested that defects in DNA replication was an underlying cause. By leveraging the well-studied Drosophila system, this study provides evidence that a disease-causing mutation in Orc4 disrupts DNA replication, and it is proposed that in individuals with MGS defects arise preferentially in tissues with a high-replication demand.
Moron-Oset, J., Super, T., Esser, J., Isaacs, A. M., Gronke, S. and Partridge, L. (2019). Glycine-alanine dipeptide repeats spread rapidly in a repeat length- and age-dependent manner in the fly brain. Acta Neuropathol Commun 7(1): 209. PubMed ID: 31843021
Hexanucleotide repeat expansions of variable size in C9orf72 are the most prevalent genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. Sense and antisense transcripts of the expansions are translated by repeat-associated non-AUG translation into five dipeptide repeat proteins (DPRs). Of these, the polyGR, polyPR and, to a lesser extent, polyGA DPRs are neurotoxic, with polyGA the most abundantly detected DPR in patient tissue. Trans-cellular transmission of protein aggregates has recently emerged as a major driver of toxicity in various neurodegenerative diseases. In vitro evidence suggests that the C9 DPRs can spread. However, whether this phenomenon occurs under more complex in vivo conditions remains unexplored. This study used the adult fly brain to investigate whether the C9 DPRs can spread in vivo upon expression in a subset of neurons. Only polyGA was found to progressively spread throughout the brain, accumulating in the shape of aggregate-like puncta inside recipient cells. Interestingly, GA transmission occurred as early as 3 days after expression induction. By comparing the spread of 36, 100 and 200 polyGA repeats, it was found that polyGA spread is enhanced upon expression of longer GA DPRs. Transmission of polyGA is greater in older flies, indicating that age-associated factors exacerbate the spread. These data highlight a unique propensity of polyGA to spread throughout the brain, which could contribute to the greater abundance of polyGA in patient tissue. In addition, a model of early GA transmission is presented that is suitable for genetic screens to identify mechanisms of spread and its consequences in vivo.
Jain, P. B., Guerreiro, P. S., Canato, S. and Janody, F. (2019). The spectraplakin Dystonin antagonizes YAP activity and suppresses tumourigenesis. Sci Rep 9(1): 19843. PubMed ID: 31882643
Aberrant expression of the Spectraplakin Dystonin (DST) has been observed in various cancers, including those of the breast. However, little is known about its role in carcinogenesis. This report demonstrates that Dystonin is a candidate tumour suppressor in breast cancer and provides an underlying molecular mechanism. In MCF10A cells, Dystonin is necessary to restrain cell growth, anchorage-independent growth, self-renewal properties and resistance to doxorubicin. Strikingly, while Dystonin maintains focal adhesion integrity, promotes cell spreading and cell-substratum adhesion, it prevents Zyxin accumulation, stabilizes LATS and restricts YAP activation. Moreover, treating DST-depleted MCF10A cells with the YAP inhibitor Verteporfin prevents their growth. In vivo, the Drosophila Dystonin Short stop also restricts tissue growth by limiting Yorkie activity. As the two Dystonin isoforms BPAG1eA and BPAG1e are necessary to inhibit the acquisition of transformed features and are both downregulated in breast tumour samples and in MCF10A cells with conditional induction of the Src proto-oncogene, they could function as the predominant Dystonin tumour suppressor variants in breast epithelial cells. Thus, their loss could deem as promising prognostic biomarkers for breast cancer.
Yedlapudi, D., Xu, L., Luo, D., Marsh, G. B., Todi, S. V. and Dutta, A. K. (2019). Targeting alpha synuclein and amyloid beta by a multifunctional, brain-penetrant dopamine D2/D3 agonist D-520: Potential therapeutic application in Parkinson's disease with dementia. Sci Rep 9(1): 19648. PubMed ID: 31873106
A significant number of people with Parkinson's disease (PD) develop dementia in addition to cognitive dysfunction and are diagnosed as PD with dementia (PDD). This is characterized by cortical and limbic alpha synuclein (alpha-syn) accumulation, and high levels of diffuse amyloid beta (Abeta) plaques in the striatum and neocortical areas. In this regard, this study evaluated the effect of a brain-penetrant, novel multifunctional dopamine D2/D3 agonist, D-520 on the inhibition of Abeta aggregation and disintegration of alpha-syn and Abeta aggregates in vitro using purified proteins and in a cell culture model that produces intracellular Abeta-induced toxicity. The effect of D-520 was evaluated in a Drosophila model of Abeta1-42 toxicity. It is reported that D-520 inhibits the formation of Abeta aggregates in vitro and promotes the disaggregation of both alpha-syn and Abeta aggregates. Finally, in an in vivo Drosophila model of Abeta1-42 dependent toxicity, D-520 exhibited efficacy by rescuing fly eyes from retinal degeneration caused by Abeta toxicity. These data indicate the potential therapeutic applicability of D-520 in addressing motor dysfunction and neuroprotection in PD and PDD, as well as attenuating dementia in people with PDD.
Marchesin, V., Perez-Marti, A., Le Meur, G., Pichler, R., Grand, K., Klootwijk, E. D., Kesselheim, A., Kleta, R., Lienkamp, S. and Simons, M. (2019). Molecular basis for autosomal-dominant renal Fanconi Syndrome caused by HNF4A. Cell Rep 29(13): 4407-4421. PubMed ID: 31875549
HNF4A is a nuclear hormone receptor that binds DNA as an obligate homodimer. While all known human heterozygous mutations are associated with the autosomal-dominant diabetes form MODY1, one particular mutation (p.R85W) in the DNA-binding domain (DBD) causes additional renal Fanconi syndrome (FRTS). This study finds that expression of the conserved fly ortholog HNF4 harboring the FRTS mutation in Drosophila nephrocytes caused nuclear depletion and cytosolic aggregation of a wild-type HNF4 reporter protein. While the nuclear depletion led to mitochondrial defects and lipid droplet accumulation, the cytosolic aggregates triggered the expansion of the endoplasmic reticulum (ER), autophagy, and eventually cell death. The latter effects could be fully rescued by preventing nuclear export through interfering with serine phosphorylation in the DBD. These data describe a genomic and a non-genomic mechanism for FRTS in HNF4A-associated MODY1 with important implications for the renal proximal tubule and the regulation of other nuclear hormone receptors.
Ahlers, L. R. H., Trammell, C. E., Carrell, G. F., Mackinnon, S., Torrevillas, B. K., Chow, C. Y., Luckhart, S. and Goodman, A. G. (2019). Insulin Potentiates JAK/STAT Signaling to Broadly Inhibit Flavivirus Replication in Insect Vectors. Cell Rep 29(7): 1946-1960.e1945. PubMed ID: 31722209
The World Health Organization estimates that more than half of the world's population is at risk for vector-borne diseases, including arboviruses. Because many arboviruses are mosquito borne, investigation of the insect immune response will help identify targets to reduce the spread of arboviruses. This study used a genetic screening approach to identify an insulin-like receptor as a component of the immune response to arboviral infection. It was determined that vertebrate insulin reduces West Nile virus (WNV) replication in Drosophila melanogaster as well as WNV, Zika, and dengue virus titers in mosquito cells. Mechanistically, this study shows that insulin signaling activates the JAK/STAT, but not RNAi, pathway via ERK to control infection in Drosophila cells and Culex mosquitoes through an integrated immune response. Finally, insulin priming of adult female Culex mosquitoes through a blood meal reduces WNV infection, demonstrating an essential role for insulin signaling in insect antiviral responses to human pathogens.

Monday, January 27th - RNA

Tsai, Y. W., Sung, H. H., Li, J. C., Yeh, C. Y., Chen, P. Y., Cheng, Y. J., Chen, C. H., Tsai, Y. C. and Chien, C. T. (2019). Glia-derived exosomal miR-274 targets Sprouty in trachea and synaptic boutons to modulate growth and responses to hypoxia. Proc Natl Acad Sci U S A. PubMed ID: 31666321
Secreted exosomal microRNAs (miRNAs) mediate interorgan/tissue communications by modulating target gene expression, thereby regulating developmental and physiological functions. However, the source, route, and function in target cells have not been formally established for specific miRNAs. This study shows that glial miR-274 non-cell-autonomouslymiR-274 modulates the growth of synaptic boutons and tracheal branches. Whereas the precursor form of miR-274 is expressed in glia, the mature form of miR-274 distributes broadly, including in synaptic boutons, muscle cells, and tracheal cells. Mature miR-274 is secreted from glia to the circulating hemolymph as an exosomal cargo, a process requiring ESCRT components in exosome biogenesis and Rab11 and Syx1A in exosome release. This study further shows that miR-274 can function in the neurons or tracheal cells to modulate the growth of synaptic boutons and tracheal branches, respectively. Also, miR-274 uptake into the target cells by AP-2-dependent mechanisms modulates target cell growth. In the target cells, miR-274 down-regulates Sprouty (Sty) through a targeting sequence at the sty 3' untranslated region, thereby enhancing MAPK signaling and promoting cell growth. miR-274 expressed in glia of an mir-274 null mutant is released as an exosomal cargo in the circulating hemolymph, and such glial-specific expression resets normal levels of Sty and MAPK signaling and modulates target cell growth. mir-274 mutant larvae are hypersensitive to hypoxia, which is suppressed by miR-274 expression in glia or by increasing tracheal branches. Thus, glia-derived miR-274 coordinates growth of synaptic boutons and tracheal branches to modulate larval hypoxia responses.
Ma, X., Ibrahim, F., Kim, E. J., Shaver, S., Becker, J., Razvi, F., Cerny, R. L. and Cerutti, H. (2019). An ortholog of the Vasa intronic gene is required for small RNA-mediated translation repression in Chlamydomonas reinhardtii. Proc Natl Acad Sci U S A. PubMed ID: 31871206
Small RNAs (sRNAs) associate with Argonaute (AGO) proteins in effector complexes, termed RNA-induced silencing complexes (RISCs), which regulate complementary transcripts by translation inhibition and/or RNA degradation. In the unicellular alga Chlamydomonas, several metazoans, and land plants, emerging evidence indicates that polyribosome-associated transcripts can be translationally repressed by RISCs without substantial messenger RNA (mRNA) destabilization. However, the mechanism of translation inhibition in a polyribosomal context is not understood. This study shows that Chlamydomonas VIG1, an ortholog of the Drosophila melanogaster Vasa intronic gene (VIG), is required for this process. VIG1 localizes predominantly in the cytosol and comigrates with monoribosomes and polyribosomes by sucrose density gradient sedimentation. A VIG1-deleted mutant shows hypersensitivity to the translation elongation inhibitor cycloheximide, suggesting that VIG1 may have a nonessential role in ribosome function/structure. Additionally, FLAG-tagged VIG1 copurifies with AGO3 and Dicer-like 3 (DCL3), consistent with it also being a component of the RISC. Indeed, VIG1 is necessary for the repression of sRNA-targeted transcripts at the translational level but is dispensable for cleavage-mediated RNA interference and for the association of the AGO3 effector with polyribosomes or target transcripts. These results suggest that VIG1 is an ancillary ribosomal component and plays a role in sRNA-mediated translation repression of polyribosomal transcripts.
Arvola, R. M., Chang, C. T., Buytendorp, J. P., Levdansky, Y., Valkov, E., Freddolino, P. L. and Goldstrohm, A. C. (2019). Unique repression domains of Pumilio utilize deadenylation and decapping factors to accelerate destruction of target mRNAs. Nucleic Acids Res. PubMed ID: 31863588
Pumilio is an RNA-binding protein that represses a network of mRNAs to control embryogenesis, stem cell fate, fertility and neurological functions in Drosophila. This study sought to identify the mechanism of Pumilio-mediated repression and found that it accelerates degradation of target mRNAs, mediated by three N-terminal Repression Domains (RDs), which are unique to Pumilio orthologs. The repressive activities of the Pumilio RDs depend on specific subunits of the Ccr4-Not (CNOT) deadenylase complex. Depletion of Pop2, Not1, Not2, or Not3 subunits alleviates Pumilio RD-mediated repression of protein expression and mRNA decay, whereas depletion of other CNOT components had little or no effect. Moreover, the catalytic activity of Pop2 deadenylase is important for Pumilio RD activity. Further, this study shows that the Pumilio RDs directly bind to the CNOT complex. Also, the decapping enzyme, Dcp2, participates in repression by the N-terminus of Pumilio. These results support a model wherein Pumilio utilizes CNOT deadenylase and decapping complexes to accelerate destruction of target mRNAs. Because the N-terminal RDs are conserved in mammalian Pumilio orthologs, the results of this work broadly enhance understanding of Pumilio function and roles in diseases including cancer, neurodegeneration and epilepsy.
Agbu, P., Cassidy, J. J., Braverman, J., Jacobson, A. and Carthew, R. W. (2019). MicroRNA miR-7 regulates secretion of insulin-like peptides. Endocrinology. PubMed ID: 31875904
The insulin/IGF pathway is essential for linking nutritional status to growth and metabolism. MicroRNAs (miRNAs) are short RNAs that are players in the regulation of this process. The miRNA miR-7 shows highly conserved expression in insulin-producing cells across the animal kingdom. However, its conserved functions in regulation of insulin-like peptides (ILPs) remain unknown. Using Drosophila as a model, this study demonstrates that miR-7 limits ILP availability by inhibiting its production and secretion. Increasing miR-7 alters body growth and metabolism in an ILP-dependent manner, elevating circulating sugars and total body triglycerides, while decreasing animal growth. These effects are not due to direct targeting of ILP mRNA, but instead arise through alternate targets that affect the function of ILP-producing cells. The Drosophila F-actin capping protein alpha, CPA, is a direct target of miR-7, and knockdown of CPA in IPCs phenocopies the effects of miR-7 on ILP secretion. This regulation of CPA is conserved in mammals, with the mouse ortholog Capza1 also targeted by miR-7 in beta-islet cells. Taken together, these results support a role for miR-7 regulation of an actin capping protein in insulin regulation, and highlight a conserved mechanism of action for an evolutionarily ancient microRNA.
Tay, M. L. and Pek, J. W. (2019). SON protects nascent transcripts from unproductive degradation by counteracting DIP1. PLoS Genet 15(11): e1008498. PubMed ID: 31730657
Gene expression involves the transcription and splicing of nascent transcripts through the removal of introns. In Drosophila, a double-stranded RNA binding protein Disco-interacting protein 1 (DIP1) targets INE-1 stable intronic sequence RNAs (sisRNAs) for degradation after splicing. How nascent transcripts that also contain INE-1 sequences escape degradation remains unknown. This study observes that these nascent transcripts can also be bound by DIP1 but the Drosophila homolog of SON (Dsn) protects them from unproductive degradation in ovaries. Dsn localizes to the satellite body where active decay of INE-1 sisRNAs by DIP1 occurs. Dsn is a repressor of DIP1 posttranslational modifications (primarily sumoylation) that are assumed to be required for efficient DIP1 activity. Moreover, the pre-mRNA destabilization caused by Dsn depletion is rescued in DIP1 or Sumo heterozygous mutants, suggesting that Dsn is a negative regulator of DIP1. These results reveal that under normal circumstances nascent transcripts are susceptible to DIP1-mediated degradation, however intronic sequences are protected by Dsn until intron excision has taken place.
Samuels, T. J., Jarvelin, A. I., Ish-Horowicz, D. and Davis, I. (2020). Imp/IGF2BP levels modulate individual neural stem cell growth and division through myc mRNA stability. Elife 9. PubMed ID: 31934860
The numerous neurons and glia that form the brain originate from tightly controlled growth and division of neural stem cells, regulated systemically by important known stem cell-extrinsic signals. However, the cell-intrinsic mechanisms that control the distinctive proliferation rates of individual neural stem cells are unknown. This study shows that the size and division rates of Drosophila neural stem cells (neuroblasts) are controlled by the highly conserved RNA binding protein Imp (IGF2BP), via one of its top binding targets in the brain, myc mRNA. Imp stabilises myc mRNA leading to increased Myc protein levels, larger neuroblasts, and faster division rates. Declining Imp levels throughout development limit myc mRNA stability to restrain neuroblast growth and division, and heterogeneous Imp expression correlates with myc mRNA stability between individual neuroblasts in the brain. It is proposed that Imp-dependent regulation of myc mRNA stability fine-tunes individual neural stem cell proliferation rates.

Friday, January 24th - Adult Development

Rojas Villa, S. E., Meng, F. W. and Biteau, B. (2019). zfh2 controls progenitor cell activation and differentiation in the adult Drosophila intestinal absorptive lineage. PLoS Genet 15(12): e1008553. PubMed ID: 31841513
Many tissues rely on resident stem cell population to maintain homeostasis. The balance between cell proliferation and differentiation is critical to permit tissue regeneration and prevent dysplasia, particularly following tissue damage. Thus, understanding the cellular processes and genetic programs that coordinate these processes is essential. This study reports that the conserved transcription factor zfh2 is specifically expressed in Drosophila adult intestinal stem cell and progenitors and is a critical regulator of cell differentiation in this lineage. zfh2 expression is shown to be required and sufficient to drive the activation of enteroblasts, the non-proliferative progenitors of absorptive cells. This transition is characterized by the transient formation of thin membrane protrusions, morphological changes characteristic of migratory cells and compensatory stem cell proliferation. zfh2 acts in parallel to insulin signaling and upstream of the TOR growth-promoting pathway during early differentiation. Finally, maintaining zfh2 expression in late enteroblasts blocks terminal differentiation and leads to the formation of highly dysplastic lesions, defining a new late cell differentiation transition. Together, this study greatly improves understanding of the cascade of cellular changes and regulatory steps that control differentiation in the adult fly midgut and identifies zfh2 as a major player in these processes.
Maier, D., Nagel, A. C. and Preiss, A. (2019). Genetic interactions between Protein Kinase D and Lobe mutants during eye development of Drosophila melanogaster. Hereditas 156: 37. PubMed ID: 31889943
In Drosophila, the development of the fly eye involves the activity of several, interconnected pathways that first define the presumptive eye field within the eye anlagen, followed by establishment of the dorso-ventral boundary, and the regulation of growth and apoptosis. In Lobe (L) mutant flies, parts of the eye or even the complete eye are absent because the eye field has not been properly defined. This study has identified Protein Kinase D (PKD) as a strong modifier of the L mutant phenotype. PKD belongs to the PKC/CAMK class of Ser/Thr kinases that have been involved in diverse cellular processes including stress resistance and growth. Despite the many roles of PKD, Drosophila PKD null mutants are without apparent phenotype apart from sensitivity to oxidative stress. This study reports an involvement of PKD in eye development in the sensitized genetic background of Lobe. Absence of PKD strongly enhanced the dominant eye defects of heterozygous L (2) flies, and decreased their viability. Moreover, eye-specific overexpression of an activated isoform of PKD considerably ameliorated the dominant L (2) phenotype. This genetic interaction was not allele specific but similarly seen with three additional, weaker L alleles, demonstrating its specificity. It is proposed that PKD-mediated phosphorylation is involved in underlying processes causing the L phenotype, i.e. in the regulation of growth, the epidermal transformation of eye tissue and apoptosis, respectively.
Jiang, Y. F., Lin, H. L., Wang, L. J., Hsu, T. and Fu, C. Y. (2019). Coordinated organization of mitochondrial lamellar cristae and gain of COX function during mitochondrial maturation in Drosophila. Mol Biol Cell: mbcE19080450. PubMed ID: 31746672
Mitochondrial cristae contain electron transport chain (ETC) complexes and are distinct from the inner boundary membrane (IBM). While many details regarding the regulation of mitochondrial structure are known, the relationship between cristae structure and function during organelle development is not fully described. This study used serial-section tomography to characterize the formation of lamellar cristae in immature mitochondria during a period of dramatic mitochondrial development that occurs after Drosophila emergence as an adult. The formation of lamellar cristae was found to be associated with the gain of COX function, and the COX subunit, COX4, was localized predominantly to organized lamellar cristae. Interestingly, 3D tomography showed some COX-positive lamellar cristae were not connected to IBM. It is hypothesized that some lamellar cristae may be organized by a vesicle germination process in the matrix, in addition to invagination of IBM. OXA1 protein, which mediates membrane insertion of COX proteins, was also localized to cristae and reticular structures isolated in the matrix additional to the IBM, suggesting that it may participate in the formation of vesicle germination-derived cristae. Overall, this study elaborates on how cristae morphogenesis and functional maturation are intricately associated. The data support the vesicle germination and membrane invagination models of cristae formation.
Klipa, O. and Hamaratoglu, F. (2019). Cell elimination strategies upon identity switch via modulation of apterous in Drosophila wing disc. PLoS Genet 15(12): e1008573. PubMed ID: 31877129
The ability to establish spatial organization is an essential feature of any developing tissue and is achieved through well-defined rules of cell-cell communication. Maintenance of this organization requires elimination of cells with inappropriate positional identity, a poorly understood phenomenon. Mechanisms regulating cell elimination were studied in the context of a growing tissue, the Drosophila wing disc and its dorsal determinant Apterous. Systematic analysis of apterous mutant clones along with their twin spots shows that they are eliminated from the dorsal compartment via three different mechanisms: relocation to the ventral compartment, basal extrusion, and death, depending on the position of the clone in the wing disc. Basal extrusion is the main elimination mechanism in the hinge, whereas apoptosis dominates in the pouch and in the notum. In the absence of apoptosis, extrusion takes over to ensure clearance in all regions. Notably, clones in the hinge grow larger than those in the pouch, emphasizing spatial differences. Mechanistically, it was found that limiting cell division within the clones does not prevent their extrusion. Indeed, even clones of one or two cells can be extruded basally, demonstrating that the clone size is not the main determinant of the elimination mechanism to be used. Overall, this study revealed three elimination mechanisms and their spatial biases for preserving pattern in a growing organ.
Li, Y., Zhang, F., Jiang, N., Liu, T., Shen, J. and Zhang, J. (2019). Decapentaplegic signaling regulates Wingless ligand production and target activation during Drosophila wing development. FEBS Lett. PubMed ID: 31814119
The Decapentaplegic (Dpp) and Wingless (Wg) signaling pathways are essential for animal development. However, how these two signals are integrated in distinct tissues is not fully understood. This study describes a novel mode of Dpp-Wg crosstalk during Drosophila wing development. The canonical Dpp signaling is shown to be required for Wg target gene activation. In addition, Dpp signaling inhibits the transcription of wg through the schnurri (shn) repressor complex. A Dpp-responsive shn/pMad/Med silencer element (SSE) is identified in the genomic loci of the wg gene. ChIP analysis suggests that Shn interacts with this element in vivo. These findings support a model in which Dpp signaling plays a dual role in transcriptional regulation of both the wg gene and downstream targets.
Kumar, A., Rizvi, M. S., Athilingam, T., Parihar, S. S. and Sinha, P. (2019). Heterophilic cell-cell adhesion of atypical cadherins fat and dachsous regulate epithelial cell size dynamics during Drosophila thorax morphogenesis. Mol Biol Cell: mbcE19080468. PubMed ID: 31877063
Spatio-temporal changes in epithelial cell sizes-or epithelial cell size dynamics (ECD)-during morphogenesis entail interplays between two opposing forces: cell contraction via acto-myosin cytoskeleton and cell expansion via cell-cell adhesion. Cell-cell adhesion-based ECD, however, has not been clearly demonstrated yet. For instance, changing levels of homophilic E-cadherin-based cell-cell adhesion induce cell-sorting, but not ECD. This study shows that cell expansive forces of heterophilic cell-cell adhesion regulate ECD: higher cell-cell adhesion results in cell size enlargement. Thus, ECD during morphogenesis in the heminotal epithelia of Drosophila pupa leading to thorax closure corresponds with spatio-temporal gradients of two heterophilic atypical cadherins-Fat (Ft) and Dachsous (Ds)-and the levels of Ft-Ds heterodimers formed concomitantly. Mathematical modeling and genetic tests validate this mechanism of dynamic heterophilic cell-cell adhesion-based regulation of ECD. Conservation of these atypical cadherins suggests a wider prevalence of heterophilic cell-cell adhesion-based ECD regulation during animal morphogenesis.

Thursday, January 23rd - Signaling

Chang, K., Kang, P., Liu, Y., Huang, K., Miao, T., Sagona, A. P., Nezis, I. P., Bodmer, R., Ocorr, K. and Bai, H. (2019). TGFB-INHB/activin signaling regulates age-dependent autophagy and cardiac health through inhibition of MTORC2. Autophagy: 1-16. PubMed ID: 31884871
Age-related impairment of macroautophagy/autophagy and loss of cardiac tissue homeostasis contribute significantly to cardiovascular diseases later in life. MTOR (mechanistic target of rapamycin kinase) signaling is the most well-known regulator of autophagy, cellular homeostasis, and longevity. The MTOR signaling consists of two structurally and functionally distinct multiprotein complexes, MTORC1 and MTORC2. This study has identified TGFB-INHB/activin signaling as a novel upstream regulator of MTORC2 to control autophagy and cardiac health during aging. Cardiac-specific knockdown of TGFB-INHB/activin-like protein daw induces autophagy and alleviates age-related heart dysfunction, including cardiac arrhythmias and bradycardia. Interestingly, the downregulation of daw activates TORC2 signaling to regulate cardiac autophagy. Activation of TORC2 alone through overexpressing its subunit protein rictor promotes autophagic flux and preserves cardiac function with aging. In contrast, activation of TORC1 does not block autophagy induction in daw knockdown flies. Lastly, either daw knockdown or rictor overexpression in fly hearts prolongs lifespan, suggesting that manipulation of these pathways in the heart has systemic effects on longevity control. Thus, these studies discover the TGFB-INHB/activin-mediated inhibition of TORC2 as a novel mechanism for age-dependent decreases in autophagic activity and cardiac health.
Koca, Y., Housden, B. E., Gault, W. J., Bray, S. J. and Mlodzik, M. (2019). Notch signaling coordinates ommatidial rotation in the Drosophila eye via transcriptional regulation of the EGF-Receptor ligand Argos. Sci Rep 9(1): 18628. PubMed ID: 31819141
In all metazoans, a small number of evolutionarily conserved signaling pathways are reiteratively used during development to orchestrate critical patterning and morphogenetic processes. Among these, Notch (N) signaling is essential for most aspects of tissue patterning where it mediates the communication between adjacent cells to control cell fate specification. In Drosophila, Notch signaling is required for several features of eye development, including the R3/R4 cell fate choice and R7 specification. This study shows that hypomorphic alleles of Notch, belonging to the N(facet) class, reveal a novel phenotype: while photoreceptor specification in the mutant ommatidia is largely normal, defects are observed in ommatidial rotation (OR), a planar cell polarity (PCP)-mediated cell motility process. During OR Notch signaling is specifically required in the R4 photoreceptor to upregulate the transcription of argos (aos), an inhibitory ligand to the epidermal growth factor receptor (EGFR), to fine-tune the activity of EGFR signaling. Consistently, the loss-of-function defects of N(facet) alleles and EGFR-signaling pathway mutants are largely indistinguishable. A Notch-regulated aos enhancer confers R4 specific expression arguing that aos is directly regulated by Notch signaling in this context via Su(H)-Mam-dependent transcription.
Beati, H., Langlands, A., Ten Have, S. and Muller, H. J. (2019). SILAC-based quantitative proteomic analysis of Drosophila gastrula stage embryos mutant for fibroblast growth factor signalling. Fly (Austin): 1-19. PubMed ID: 31873056
Quantitative proteomic analyses in combination with genetics provide powerful tools in developmental cell signalling research. Drosophila melanogaster is one of the most widely used genetic models for studying development and disease. This study combined quantitative proteomics with genetic selection to determine changes in the proteome upon depletion of Heartless (Htl) Fibroblast-Growth Factor (FGF) receptor signalling in Drosophila embryos at the gastrula stage. A robust, single generation SILAC (stable isotope labelling with amino acids in cell culture) protocol is presented for labelling proteins in early embryos. An independent genetic marker was developed for the selection of homozygously mutant embryos at the pre-gastrula stage. These analyses detected quantitative changes in the global proteome of htl mutant embryos during gastrulation. Distinct classes of downregulated and upregulated proteins were identified, and network analyses indicated functionally related groups of proteins in each class. In addition, changes were identified in the abundance of phosphopeptides. In summary, this quantitative proteomic analysis reveals global changes in metabolic, nucleoplasmic, cytoskeletal and transport proteins in htl mutant embryos.
Neuert, H., Deing, P., Krukkert, K., Naffin, E., Steffes, G., Risse, B., Silies, M. and Klambt, C. (2019). The Drosophila NCAM homolog Fas2 signals independent of adhesion. Development. PubMed ID: 31862845
The development of tissues and organs requires close interaction of cells. To do so, cells express adhesion proteins such as the neural cell adhesion molecule (NCAM) or its Drosophila orthologue Fasciclin 2 (Fas2). Both are members of the Ig-domain superfamily of proteins that mediate homophilic adhesion. These proteins are expressed as different isoforms differing in their membrane anchorage and their cytoplasmic domains. To study the function of single isoforms a comprehensive genetic analysis of fas2 was performed. The expression pattern was revealed of all major Fas2 isoforms, two of which are GPI-anchored. The remaining five isoforms carry transmembrane domains with variable cytoplasmic tails. fas2 mutants were generated expressing only single isoforms. In contrast to the null mutation which causes embryonic lethality, these mutants are viable, indicating redundancy among the different isoforms. Cell type specific rescue experiments showed that glial secreted Fas2 can rescue the fas2 mutant phenotype to viability. This demonstrates cytoplasmic Fas2 domains have no apparent essential functions and indicate that Fas2 has function(s) other than homophilic adhesion. In conclusion, these data propose novel mechanistic aspects of a long studied adhesion protein.
Iida, C., Ohsawa, S., Taniguchi, K., Yamamoto, M., Morata, G. and Igaki, T. (2019). JNK-mediated Slit-Robo signaling facilitates epithelial wound repair by extruding dying cells. Sci Rep 9(1): 19549. PubMed ID: 31863086
Multicellular organisms repair injured epithelium by evolutionarily conserved biological processes including activation of c-Jun N-terminal kinase (JNK) signaling. This study showed in Drosophila imaginal epithelium that physical injury leads to the emergence of dying cells, which are extruded from the wounded tissue by JNK-induced Slit-Roundabout2 (Robo2) repulsive signaling. Reducing Slit-Robo2 signaling in the wounded tissue suppresses extrusion of dying cells and generates aberrant cells with highly upregulated growth factors Wingless (Wg) and Decapentaplegic (Dpp). The inappropriately elevated Wg and Dpp impairs wound repair, as halving one of these growth factor genes cancelled wound healing defects caused by Slit-Robo2 downregulation. These data suggest that JNK-mediated Slit-Robo2 signaling contributes to epithelial wound repair by promoting extrusion of dying cells from the wounded tissue, which facilitates transient and appropriate induction of growth factors for proper wound healing.
Ji, Z., Kiparaki, M., Folgado, V., Kumar, A., Blanco, J., Rimesso, G., Chuen, J., Liu, Y., Zheng, D. and Baker, N. E. (2019). Drosophila RpS12 controls translation, growth, and cell competition through Xrp1. PLoS Genet 15(12): e1008513. PubMed ID: 31841522
Whereas complete loss of Rp function is generally lethal, most heterozygous Rp mutants grow more slowly and are subject to competitive loss from mosaics tissues that also contain wild type cells. The rpS12 gene has a special role in the cell competition of other Ribosomal Protein (Rp) mutant cells in Drosophila. Elimination by cell competition is promoted by higher RpS12 levels and prevented by a specific rpS12 mis-sense mutation, identifying RpS12 as a key effector of cell competition due to mutations in other Rp genes. This study showed that RpS12 is also required for other aspects of Rp mutant phenotypes, including hundreds of gene expression changes that occur in 'Minute' Rp heterozygous wing imaginal discs, overall translation rate, and the overall rate of organismal development, all through the bZip protein Xrp1 that is one of the RpS12-regulated genes. These findings outline the regulatory response to mutations affecting essential Rp genes that controls overall translation, growth, and cell competition, and which may contribute to cancer and other diseases.

Wednesday, January 22nd - Behavior

Sethi, S., Lin, H. H., Shepherd, A. K., Volkan, P. C., Su, C. Y. and Wang, J. W. (2019). Social context enhances hormonal modulation of pheromone detection in Drosophila. Curr Biol 29(22): 3887-3898. PubMed ID: 31679932
Critical to evolutionary fitness, animals regulate social behaviors by integrating signals from both their external environments and internal states. This study found that population density modulates the courtship behavior of male Drosophila melanogaster in an age-dependent manner. In a competitive mating assay, males reared in a social environment have a marked advantage in courting females when pitted against males reared in isolation. Group housing promotes courtship in mature (7-day) but not immature (2-day) males; this behavioral plasticity requires the Or47b pheromone receptor. Using single-sensillum recordings, this study found that group housing increases the response of Or47b olfactory receptor neurons (ORNs) only in mature males. The effect of group housing on olfactory response and behavior can be mimicked by chronically exposing single-housed males to an Or47b ligand. At the molecular level, group housing elevates Ca(2+) levels in Or47b ORNs, likely leading to CaMKI-mediated activation of the histone-acetyl transferase CBP. This signaling event in turn enhances the efficacy of juvenile hormone, an age-related regulator of reproductive maturation in flies. Furthermore, the male-specific Fruitless isoform (Fru(M)) is required for the sensory plasticity, suggesting that Fru(M) functions as a downstream genomic coincidence detector in Or47b ORNs-integrating reproductive maturity, signaled by juvenile hormone, and population density, signaled by CBP. In all, this study has identify a neural substrate and activity-dependent mechanism by which social context can directly influence pheromone sensitivity, thereby modulating social behavior according to animals' life-history stage.
Toshima, N., Kantar Weigelt, M., Weiglein, A., Boetzl, F. A. and Gerber, B. (2019). An amino-acid mixture can be both rewarding and punishing to larval Drosophila. J Exp Biol. PubMed ID: 31672727
Amino acids are important nutrients for animals because they are necessary for protein synthesis in particular during growth, as well as for neurotransmission. However, little is known about how animals use past experience to guide their search for amino-acid-rich food. It was reasoned that the larvae of Drosophila melanogaster are suitable for investigating this topic because they are the feeding and growth stages in the life cycle of these holometabolous insects. Specifically, whether experiencing an odour with a 20-amino-acid mixture as a semi-natural tastant during training establishes odour-tastant associative memories was investigated. Across a broad concentration range (0.01-20 mM), such an amino-acid mixture was found to have a rewarding effect, establishing appetitive memory for the odour. Surprisingly, however, manipulation of the test conditions revealed that relatively high concentrations of the amino-acid mixture (3.3 mM and higher) in addition establish aversive memory for the odour. Both these oppositely-valenced memories were then characterized in terms of their dependency on the number of training trials, their temporal stability, their modulation through starvation, and the specific changes in locomotion underlying them. Collectively, and in the light of what is known about the neuronal organization of odour-food memory in larval Drosophila, the data suggest that these memories are established in parallel. The similarity of these results to what has been reported for sodium chloride is discussed along with the possible neurogenetic bases for concentration-dependent changes in valence when these tastants are used as reinforcers.
Shahandeh, M. P., Pischedda, A., Rodriguez, J. M. and Turner, T. L. (2019). The genetics of male pheromone preference difference between Drosophila melanogaster and Drosophila simulans. G3 (Bethesda). PubMed ID: 31748379
Species of flies in the genus Drosophila differ dramatically in their preferences for mates, but little is known about the genetic or neurological underpinnings of this evolution. Recent advances have been made to understanding of one case: pheromone preference evolution between the species D. melanogaster and D. simulans. Males of both species are very sensitive to the pheromone 7,11-HD that is present only on the cuticle of female D. melanogaster. In one species this cue activates courtship, and in the other it represses it. This change in valence was recently shown to result from the modification of central processing neurons, rather than changes in peripherally expressed receptors, but nothing is known about the genetic changes that are responsible. This study shows that a 1.35 Mb locus on the X chromosome has a major effect on male 7,11-HD preference. Unfortunately, when this locus is divided, the effect is largely lost. Instead this study attempted to filter the 159 genes within this region using newfound understanding of the neuronal underpinnings of this phenotype to identify and test candidate genes. The results of these tests are presented, and the difficulty of identifying the genetic architecture of behavioral traits and the potential of connecting these genetic changes to the neuronal modifications that elicit different behaviors is discussed.
Verschut, T. A., Carlsson, M. A. and Hamback, P. A. (2019). Scaling the interactive effects of attractive and repellent odours for insect search behaviour. Sci Rep 9(1): 15309. PubMed ID: 31653955
Insects searching for resources are exposed to a complexity of mixed odours, often involving both attractant and repellent substances. Understanding how insects respond to this complexity of cues is crucial for understanding consumer-resource interactions, but also to develop novel tools to control harmful pests. To advance understanding of insect responses to combinations of attractive and repellent odours, this study formulated three qualitative hypotheses; the response-ratio hypothesis, the repellent-threshold hypothesis and the odour-modulation hypothesis. The hypotheses were tested by exposing Drosophila melanogaster in a wind tunnel to combinations of vinegar as attractant and four known repellents; benzaldehyde, 1-octen-3-ol, geosmin and phenol. The responses to benzaldehyde, 1-octen-3-ol and geosmin provided support for the response-ratio hypothesis, which assumes that the behavioural response depends on the ratio between attractants and repellents. The response to phenol, rather supported the repellent-threshold hypothesis, where aversion only occurs above a threshold concentration of the repellent due to overshadowing of the attractant. It is hypothesized that the different responses may be connected to the localization of receptors, as receptors detecting phenol are located on the maxillary palps whereas receptors detecting the other odorants are located on the antennae.
Agrawal, P., Kao, D., Chung, P. and Looger, L. L. (2020). The neuropeptide Drosulfakinin regulates social isolation-induced aggression in Drosophila. J Exp Biol. PubMed ID: 31900346
Social isolation strongly modulates behavior across the animal kingdom. This study utilized the fruit fly Drosophila melanogaster to study social isolation-driven changes in animal behavior and gene expression in the brain. RNA-seq identified several head-expressed genes strongly responding to social isolation or enrichment. Of particular interest, social isolation downregulated expression of the gene encoding the neuropeptide Drosulfakinin (Dsk), the homologue of vertebrate cholecystokinin (CCK), which is critical for many mammalian social behaviors. Dsk knockdown significantly increased social isolation-induced aggression. Genetic activation or silencing of Dsk neurons each similarly increased isolation-driven aggression. The results suggest a U-shaped dependence of social isolation-induced aggressive behavior on Dsk signaling, similar to the actions of many neuromodulators in other contexts.
Melo, N., Wolff, G. H., Costa-da-Silva, A. L., Arribas, R., Triana, M. F., Gugger, M., Riffell, J. A., DeGennaro, M. and Stensmyr, M. C. (2019). Geosmin attracts Aedes aegypti mosquitoes to oviposition sites. Curr Biol. PubMed ID: 31839454
Geosmin is one of the most recognizable microbial smells. Some insects, like mosquitoes, require microbial-rich environments for their progeny, whereas for other insects such microbes may prove dangerous. In Drosophila, geosmin is decoded in a precise fashion and induces aversion. This study investigated the effect of geosmin on the behavior of the yellow fever mosquito Aedes aegypti. In contrast to flies, geosmin is not aversive but mediates egg-laying site selection. Female mosquitoes likely associate geosmin with microbes, including cyanobacteria consumed by larvae, who also find geosmin-as well as geosmin-producing cyanobacteria-attractive. Using in vivo multiphoton calcium imaging from transgenic PUb-GCaMP6s mosquitoes, this study shows that Ae. aegypti code geosmin in a qualitatively similar fashion to flies, i.e., through a single olfactory channel with a high degree of sensitivity for this volatile. It was further demonstrated that geosmin can be used as bait under field conditions, and geosmin, which is both expensive and difficult to obtain, can be substituted by beetroot peel extract, providing a cheap and viable potential means for mosquito control and surveillance in developing countries.

Tuesday, January 21st - Adult neural development and function

Gur, B., Sporar, K., Lopez-Behling, A. and Silies, M. (2019). Distinct expression of potassium channels regulates visual response properties of lamina neurons in Drosophila melanogaster. J Comp Physiol A Neuroethol Sens Neural Behav Physiol. PubMed ID: 31823004
The computational organization of sensory systems depends on the diversification of individual cell types with distinct signal-processing capabilities. The Drosophila visual system, for instance, splits information into channels with different temporal properties directly downstream of photoreceptors in the first-order interneurons of the OFF pathway, L2 and L3. However, the biophysical mechanisms that determine this specialization are largely unknown. This study shows that the voltage-gated Ka channels Shaker and Shal contribute to the response properties of the major OFF pathway input L2. L3 calcium response kinetics postsynaptic to photoreceptors resemble the sustained calcium signals of photoreceptors, whereas L2 neurons decay transiently. Based on a cell-type-specific RNA-seq data set and endogenous protein tagging, this study identified Shaker and Shal as the primary candidates to shape L2 responses. Using in vivo two-photon imaging of L2 calcium signals in combination with pharmacological and genetic perturbations of these Ka channels, it was shown that the wild-type Shaker and Shal function is to enhance L2 responses and cell-autonomously sharpen L2 kinetics. These results reveal a role for Ka channels in determining the signal-processing characteristics of a specific cell type in the visual system.
Li, R., Liang, Y., Zheng, S., He, Q. and Yang, L. (2019). The atypical cadherin flamingo determines the competence of neurons for activity-dependent fine-scale topography. Mol Brain 12(1): 109. PubMed ID: 31823796
The topographic projection of afferent terminals into two-dimensional maps is essential for sensory systems to encode the locations of sensory stimuli. In vertebrates, guidance cues are critical for establishing a coarse topographic map, while neuronal activity directs fine-scale topography between adjacent afferent terminals. However, the molecular mechanism underlying activity-dependent fine-scale topography is not well known. Studies in the Drosophila visual system have demonstrated that cell-adhesion molecules direct fine-scale topography, but whether or not these molecules are involved in activity-dependent fine-scale topography remains to be determined. Previously it was reported that the nociceptors in Drosophila larvae form an activity-dependent fine-scale topographic system. The establishment of this system is instructed by the level of neuronal activity in individual nociceptors. This study shows that the atypical cadherin Flamingo (Fmi) is required for establishing the nociceptor topographic map. The topographic defect caused by loss of fmi was found to be epistatic to the inhibition of neuronal activity and the overexpression of the activity-regulated gene Trim9. These results suggest that Fmi and neuronal activity interact to regulate fine-scale topography. This study provides a link between neuronal activity and the cell-adhesion molecule in the establishment of fine-scale topography.
Gonzalez-Gutierrez, A., Ibacache, A., Esparza, A., Barros, L. F. and Sierralta, J. (2019). Neuronal lactate levels depend on glia-derived lactate during high brain activity in Drosophila. Glia. PubMed ID: 31876077
Lactate/pyruvate transport between glial cells and neurons is thought to play an important role in how brain cells sustain the high-energy demand that neuronal activity requires. However, the in vivo mechanisms and characteristics that underlie the transport of monocarboxylates are poorly described. This study used Drosophila expressing genetically encoded FRET sensors to provide an ex vivo characterization of the transport of monocarboxylates in motor neurons and glial cells from the larval ventral nerve cord. Lactate/pyruvate transport in glial cells was shown to be coupled to protons and is more efficient than in neurons. Glial cells maintain higher levels of intracellular lactate generating a positive gradient toward neurons. Interestingly, during high neuronal activity, raised lactate in motor neurons is dependent on transfer from glial cells mediated in part by the previously described monocarboxylate transporter Chaski, providing support for in vivo glia-to-neuron lactate shuttling during neuronal activity.
Grabe, V., Schubert, M., Strube-Bloss, M., Reinert, A., Trautheim, S., Lavista-Llanos, S., Fiala, A., Hansson, B. S. and Sachse, S. (2019). Odor-induced multi-level inhibitory maps in Drosophila. eNeuro. PubMed ID: 31888962
Optical imaging of intracellular Ca(2+) influx as a correlate of neuronal excitation represents a standard technique for visualizing spatiotemporal activity of neuronal networks. However, the information-processing properties of single neurons and neuronal circuits likewise involve inhibition of neuronal membrane potential. This study reports spatially resolved optical imaging of odor-evoked inhibitory patterns in the olfactory circuitry of Drosophila using a genetically encoded fluorescent Cl(-) sensor. In combination with the excitatory component reflected by intracellular Ca(2+) dynamics, a comprehensive functional map is presented of both odor-evoked neuronal activation and inhibition at different levels of olfactory processing. Odor-evoked inhibition carried by Cl(-) influx is present both in sensory neurons and second-order projection neurons, and is characterized by stereotypic, odor-specific patterns. Cl(-) mediated inhibition features distinct dynamics in different neuronal populations. The data support a dual role of inhibitory neurons in the olfactory system: global gain control across the neuronal circuitry and glomerulus-specific inhibition to enhance neuronal information processing.
Gruntman, E., Romani, S. and Reiser, M. B. (2019). The computation of directional selectivity in the Drosophila OFF motion pathway. Elife 8. PubMed ID: 31825313
In flies, the direction of moving ON and OFF features is computed separately. T4 (ON) and T5 (OFF) are the first neurons in their respective pathways to extract a directionally selective response from their non-selective inputs. A recent study of T4 found that the integration of offset depolarizing and hyperpolarizing inputs is critical for the generation of directional selectivity. However, T5s lack small-field inhibitory inputs, suggesting they may use a different mechanism. This study used whole-cell recordings of T5 neurons and found a similar receptive field structure: fast depolarization and persistent, spatially offset hyperpolarization. By assaying pairwise interactions of local stimulation across the receptive field, no amplifying responses, only suppressive responses were found to the non-preferred motion direction. Passive, biophysical models were evaluated, and ut was found that a model using direct inhibition, but not the removal of excitation, can accurately predict T5 responses to a range of moving stimuli.
Jung, Y., Kennedy, A., Chiu, H., Mohammad, F., Claridge-Chang, A. and Anderson, D. J. (2019). Neurons that function within an integrator to promote a persistent behavioral state in Drosophila. Neuron. PubMed ID: 31810837
Innate behaviors involve both reflexive motor programs and enduring internal states, but how these responses are coordinated by the brain is not clear. In Drosophila, male-specific P1 interneurons promote courtship song, as well as a persistent internal state that prolongs courtship and enhances aggressiveness. However, P1 neurons themselves are not persistently active. This study identified pCd neurons as persistently active, indirect P1 targets that are required for P1-evoked persistent courtship and aggression. Acute activation of pCd neurons alone is inefficacious but enhances and prolongs courtship or aggression promoted by female cues. Brief female exposure induces a persistent increase in male aggressiveness, an effect abrogated by interruption of pCd activity. pCd activity is not sufficient but necessary for persistent physiological activity, implying an essential role in a persistence network. Thus, P1 neurons coordinate both command-like control of courtship song and a persistent internal state of social arousal mediated by pCd neurons.

Monday, January 20th - Gonadogenesis

Rathje, C. C., Randle, S. J., Al Rawi, S., Skinner, B. M., Nelson, D. E., Majumdar, A., Johnson, E. E. P., Bacon, J., Vlazaki, M., Affara, N. A., Ellis, P. J. and Laman, H. (2019). A conserved requirement for Fbxo7 during male germ cell cytoplasmic memodeling. Front Physiol 10: 1278. PubMed ID: 31649556
Fbxo7 is the substrate-recognition subunit of an SCF-type ubiquitin E3 ligase complex. It has physiologically important functions in regulating mitophagy, proteasome activity and the cell cycle in multiple cell types, like neurons, lymphocytes and erythrocytes. This study shows that in addition to the previously known Parkinsonian and hematopoietic phenotypes, male mice with reduced Fbxo7 expression are sterile. In these males, despite successful meiosis, nuclear elongation and eviction of histones from chromatin, the developing spermatids are phagocytosed by Sertoli cells during late spermiogenesis, as the spermatids undergo cytoplasmic remodeling. Surprisingly, despite the loss of all germ cells, there was no evidence of the symplast formation and cell sloughing that is typically associated with spermatid death in other mouse sterility models, suggesting that novel cell death and/or cell disposal mechanisms may be engaged in Fbxo7 mutant males. Mutation of the Drosophila Fbxo7 ortholog, nutcracker (ntc) also leads to sterility with germ cell death during cytoplasmic remodeling, indicating that the requirement for Fbxo7 at this stage is conserved. The ntc phenotype was attributed to decreased levels of the proteasome regulator, DmPI31 and reduced proteasome activity. Consistent with the fly model, a reduction in PI31 levels was observed in mutant mice; however, there is no alteration in proteasome activity in whole mouse testes. These results are consistent with findings that Fbxo7 regulates PI31 protein levels, and indicates that a defect at the late stages of spermiogenesis, possibly due to faulty spatial dynamics of proteasomes during cytoplasmic remodeling, may underlie the fertility phenotype in mice.
Weiss, I. and Bohrmann, J. (2019). Electrochemical gradients are involved in regulating cytoskeletal patterns during epithelial morphogenesis in the Drosophila ovary. BMC Dev Biol 19(1): 22. PubMed ID: 31718540
During Drosophila oogenesis, the follicular epithelium differentiates into several morphologically distinct follicle-cell populations. Characteristic bioelectrical properties make this tissue a suitable model system for studying connections between electrochemical signals and the organisation of the cytoskeleton. This study analysed the patterns of basal microfilaments (bMF) and microtubules (MT) in relation to electrochemical signals. The bMF- and MT-patterns in developmental stages 8 to 12 were visualised using labelled phalloidin and an antibody against acetylated alpha-tubulin as well as follicle-cell specific expression of GFP-actin and GFP-alpha-tubulin. In order to test whether cytoskeletal modifications depend directly on bioelectrical changes, inhibitors of ion-transport mechanisms were used that have previously been shown to modify pHi and Vmem as well as the respective gradients. While relative alkalisation and/or hyperpolarisation stabilised the parallel transversal alignment of bMF, acidification led to increasing disorder and to condensations of bMF. On the other hand, relative acidification as well as hyperpolarisation stabilised the longitudinal orientation of MT, whereas alkalisation led to loss of this arrangement and to partial disintegration of MT. It is conclude that the pHi- and Vmem-changes induced by inhibitors of ion-transport mechanisms simulate bioelectrical changes occurring naturally and leading to the cytoskeletal changes observed during differentiation of the follicle-cell epithelium. Therefore, gradual modifications of electrochemical signals can serve as physiological means to regulate cell and tissue architecture by modifying cytoskeletal patterns.
Tourmente, M., Archer, C. R. and Hosken, D. J. (2019). Complex interactions between sperm viability and female fertility. Sci Rep 9(1): 15366. PubMed ID: 31653962
Sperm viability is a major male fitness component, with higher sperm viability associated with enhanced sperm competitiveness. While many studies have focussed on sperm viability from the male fitness standpoint, its impact on female fitness is less clear. This study used a panel of 32 isogenic Drosophila simulans lines to test for genetic variation in sperm viability (percentage of viable cells). Then, whether sperm viability affected female fitness was tested by mating females to males from low or high sperm viability genotypes. Significant variation was found in sperm viability among genotypes, and consistent with this, sperm viability was highly repeatable within genotypes. Additionally, females mated to high sperm viability males laid more eggs in the first seven hours after mating, and produced more offspring in total. However, the early increase in oviposition did not result in more offspring in the 8 hours following mating, suggesting that mating with high sperm-viability genotypes leads to egg wastage for females shortly after copulation. Although mating with high sperm-viability males resulted in higher female fitness in the long term, high quality ejaculates would result in a short-term female fitness penalty, or at least lower realised fitness, potentially generating sexual conflict over optimal sperm viability.
Lobo-Pecellin, M., Marin-Menguiano, M. and Gonzalez-Reyes, A. (2019). mastermind regulates niche ageing independently of the Notch pathway in the Drosophila ovary. Open Biol 9(11): 190127. PubMed ID: 31744422
Proper stem cell activity in tissues ensures the correct balance between proliferation and differentiation, thus allowing tissue homeostasis and repair. The Drosophila ovary develops well-defined niches that contain on average 2-4 germline stem cells (GSCs), whose maintenance depends on systemic signals and local factors. A known player in the decline of tissue homeostasis is ageing, which correlates with the waning of resident stem cell populations. In Drosophila, ovaries from old females contain fewer GSCs than those from young flies. Niche cells of aged ovaries were isolated, a transcriptomic analysis was performed and mastermind (mam) was identified as a factor for Drosophila ovarian niche functionality during ageing. mam is upregulated in aged niche cells, and premature GSC loss can be induced by overexpressing mam in otherwise young niche cells. High mam levels in niche cells induce reduced Hedgehog amounts, a decrease in cadherin levels and a likely increase in reactive oxygen species, three scenarios known to provoke GSC loss. Mam is a canonical co-activator of the Notch pathway in many Drosophila tissues. However, this study presents evidence to support a Notch-independent role for mam in the ovarian germline niche.
Lapart, J. A., Gottardo, M., Cortier, E., Duteyrat, J. L., Augiere, C., Mange, A., Jerber, J., Solassol, J., Gopalakrishnan, J., Thomas, J. and Durand, B. (2019). Dzip1 and Fam92 form a ciliary transition zone complex with cell type specific roles in Drosophila. Elife 8. PubMed ID: 31821146
Cilia and flagella are conserved eukaryotic organelles essential for cellular signaling and motility. Cilia dysfunctions cause life-threatening ciliopathies, many of which are due to defects in the transition zone (TZ), a complex structure of the ciliary base. Therefore, understanding TZ assembly, which relies on ordered interactions of multiprotein modules, is of critical importance. This study shows that Drosophila Dzip1 and Fam92 form a functional module which constrains the conserved core TZ protein, Cep290, to the ciliary base. Cell type specific roles of this functional module were identified in two different tissues. While it is required for TZ assembly in all Drosophila ciliated cells, it also regulates basal-body growth and docking to the plasma membrane during spermatogenesis. This study therefore demonstrate a novel regulatory role for Dzip1 and Fam92 in mediating membrane/basal-body interactions and show that these interactions exhibit cell type specific functions in basal-body maturation and TZ organization.
Fadiga, J. and Nystul, T. G. (2019). The follicle epithelium in the Drosophila ovary is maintained by a small number of stem cells. Elife 8. PubMed ID: 31850843
The follicle stem cells (FSCs) in the Drosophila ovary are an important experimental model for the study of epithelial stem cell biology. Although decades of research support the conclusion that there are two FSCs per ovariole, a recent study used a novel clonal marking system to conclude that there are 15-16 FSCs per ovariole. This study performed clonal analysis using both this novel clonal marking system and standard clonal marking systems, and several problems were identified that may have contributed to the overestimate of FSC number. In addition, new methods were developed for accurately measuring clone size, and FSC clones were found to produce, on average, half of the follicle cells in each ovariole. These findings provide strong independent support for the conclusion that there are typically two active FSCs per ovariole, though they are consistent with up to four FSCs per germarium.

Friday, October 17th - Evolution

Hoedjes, K. M., van den Heuvel, J., Kapun, M., Keller, L., Flatt, T. and Zwaan, B. J. (2019). Distinct genomic signals of lifespan and life history evolution in response to postponed reproduction and larval diet in Drosophila. Evol Lett 3(6): 598-609. PubMed ID: 31867121
Reproduction and diet are two major factors controlling the physiology of aging and life history, but how they interact to affect the evolution of longevity is unknown. Moreover, although studies of large-effect mutants suggest an important role of nutrient sensing pathways in regulating aging, the genetic basis of evolutionary changes in lifespan remains poorly understood. To address these questions, the genomes of experimentally evolved Drosophila melanogaster populations were subjected to a factorial combination of two selection regimes: reproductive age (early versus postponed), and diet during the larval stage ("low," "control," "high"), resulting in six treatment combinations with four replicate populations each. Selection on reproductive age consistently affected lifespan, with flies from the postponed reproduction regime having evolved a longer lifespan. In contrast, larval diet affected lifespan only in early-reproducing populations: flies adapted to the "low" diet lived longer than those adapted to control diet. This study found genomic evidence for strong independent evolutionary responses to either selection regime, as well as loci that diverged in response to both regimes, thus representing genomic interactions between the two. Overall, the genomic basis of longevity was found to be largely independent of dietary adaptation. Differentiated loci were not enriched for "canonical" longevity genes, suggesting that naturally occurring genic targets of selection for longevity differ qualitatively from variants found in mutant screens. Comparing the candidate loci to those from other "evolve and resequence" studies of longevity demonstrated significant overlap among independent experiments. This suggests that the evolution of longevity, despite its presumed complex and polygenic nature, might be to some extent convergent and predictable.
Tambones, I. L., Haudry, A., Simao, M. C. and Carareto, C. M. A. (2019). High frequency of horizontal transfer in Jockey families (LINE order) of drosophilids. Mob DNA 10: 43. PubMed ID: 31709017
The use of large-scale genomic analyses has resulted in an improvement of transposable element sampling and a significant increase in the number of reported HTT (horizontal transfer of transposable elements) events by expanding the sampling of transposable element sequences in general and of specific families of these elements in particular, which were previously poorly sampled. This study investigated the occurrence of HTT events in a group of elements that, until recently, were uncommon among the HTT records in Drosophila - the Jockey elements, members of the LINE (long interspersed nuclear element) order of non-LTR (long terminal repeat) retrotransposons. The sequences of 111 Jockey families deposited in Repbase that met the criteria of the analysis were used to identify Jockey sequences in 48 genomes of Drosophilidae (genus Drosophila, subgenus Sophophora: melanogaster, obscura and willistoni groups; subgenus Drosophila: immigrans, melanica, repleta, robusta, virilis and grimshawi groups; subgenus Dorsilopha: busckii group; genus/subgenus Zaprionus and genus Scaptodrosophila). Phylogenetic analyses revealed 72 Jockey families in 41 genomes. Combined analyses revealed 15 potential HTT events between species belonging to different genera and species groups of Drosophilidae, providing evidence for the flow of genetic material favoured by the spatio-temporal sharing of these species present in the Palaeartic or Afrotropical region. These results provide phylogenetic, biogeographic and temporal evidence of horizontal transfers of the Jockey elements, increase the number of rare records of HTT in specific families of LINE elements, increase the number of known occurrences of these events, and enable a broad understanding of the evolutionary dynamics of these elements and the host species.
Sprengelmeyer, Q. D., Mansourian, S., Lange, J. D., Matute, D. R., Cooper, B. S., Jirle, E. V., Stensmyr, M. C. and Pool, J. E. (2019). Recurrent collection of Drosophila melanogaster from Wild african environments and genomic insights into species history. Mol Biol Evol. PubMed ID: 31730190
Drosophila melanogaster is thought to originate from sub-Saharan Africa. This study documents the collection of 288 D. melanogaster individuals from multiple African wilderness areas in Zambia, Zimbabwe, and Namibia. The presence of D. melanogaster in these remote woodland environments is consistent with an ancestral range in southern-central Africa, as opposed to equatorial regions. After sequencing the genomes of 17 wilderness-collected flies collected from Kafue National Park in Zambia, reduced genetic diversity relative to town populations, elevated chromosomal inversion frequencies, and strong differences at specific genes including known insecticide targets were found. Combining these genomes with existing data, this study probed the history of this species' geographic expansion. Demographic estimates indicated that expansion from southern-central Africa began approximately 10,000 years ago, with a Saharan crossing soon after, but expansion from the Middle East into Europe did not begin until roughly 1,400 years ago. This improved model of demographic history will provide an important resource for future evolutionary and genomic studies of this key model organism. These findings add context to the history of D. melanogaster, while opening the door for future studies on the biological basis of adaptation to human environments.
Dyer, K. A. and Hall, D. W. (2019). Fitness consequences of a non-recombining sex-ratio drive chromosome can explain its prevalence in the wild. Proc Biol Sci 286(1917): 20192529. PubMed ID: 31847762
Understanding the pleiotropic consequences of gene drive systems on host fitness is essential to predict their spread through a host population. This paper reports a study sex-ratio (SR) X-chromosome drive in the fly Drosophila recens, where SR causes the death of Y-bearing sperm in male carriers. SR males only sire daughters, which all carry SR, thus giving the chromosome a transmission advantage. The prevalence of the SR chromosome appears stable, suggesting pleiotropic costs. It was previously shown that females homozygous for SR are sterile, and this study tests for additional fitness costs of SR. Females heterozygous for SR were found to have reduced fecundity, and male SR carriers were found to have reduced fertility in conditions of sperm competition. Fitness estimates were used to parametrize theoretical models of SR drive, and sthe decrease in fecundity and sperm competition performance were shown to account for the observed prevalence of SR in natural populations. In addition, it was found that the expected equilibrium frequency of the SR chromosome is particularly sensitive to the degree of multiple mating and performance in sperm competition. Together, these data suggest that the mating system of the organism should be carefully considered during the development of gene drive systems.
Zurovcova, M., Benes, V., Zurovec, M. and Kucerova, L. (2019). Expansion of imaginal disc growth factor gene family in diptera reflects the evolution of novel functions. Insects 10(10). PubMed ID: 31635152
Imaginal disc growth factors (IDGFs) are a small protein family found in insects. They are related to chitinases and implicated in multiple functions, including cell growth stimulation, antimicrobial activity, insect hemolymph clotting, and maintenance of the extracellular matrix. A number of new IDGFs have been found in several insect species and their detailed phylogenetic analysis provides a good basis for further functional studies. To achieve this goal, Idgf cDNAs were sequenced from several lepidopteran and trichopteran species and the data was supplemented with sequences retrieved from public databases. A comparison of Idgf genes in different species showed that Diptera typically contain several Idgf paralogs with a simple exon-intron structure (2-3 exons), whereas lepidopteran Idgfs appear as a single copy per genome and contain a higher number of exons (around 9). These results show that, while lepidopteran Idgfs, having single orthologs, are characterized by low divergence and stronger purifying selection over most of the molecule, the duplicated Idgf genes in Diptera, Idgf1 and Idgf4, exhibit signs of positive selection. This characterization of IDGF evolution provides the first information on the changes that formed these important molecules.
Kawecki, T. J. (2019). Sexual selection reveals a cost of pathogen resistance undetected in life-history assays. Evolution. PubMed ID: 31814118
Mechanisms of resistance to pathogens and parasites are thought to be costly and thus to lead to evolutionary trade-offs between resistance and life-history traits expressed in the absence of the infective agents. On the other hand, sexually selected traits are often proposed to indicate "good genes" for resistance, which implies a positive genetic correlation between resistance and success in sexual selection. This study shows that experimental evolution of improved resistance to the intestinal pathogen Pseudomonas entomophila in Drosophila melanogaster was associated with a reduction in male sexual success. Males from four resistant populations achieved lower paternity than males from four susceptible control populations in competition with males from a competitor strain, indicating an evolutionary cost of resistance in terms of mating success and/or sperm competition. In contrast, no costs were found in larval viability, larval competitive ability and population productivity assayed under nutritional limitation; together with earlier studies this suggests that the costs of P. entomophila resistance for nonsexual fitness components are negligible. Thus, rather than indicating heritable pathogen resistance, sexually selected traits expressed in the absence of pathogens may be sensitive to costs of resistance, even if no such costs are detected in other fitness traits.

Thursday, January 17th - Synapse and Vesicles

Martin-Pena, A. and Ferrus, A. (2019). CCB is involved in actin-based axonal transport of selected synaptic proteins. J Neurosci. PubMed ID: 31754011
Synapse formation, maturation, and turnover require a finely regulated transport system that delivers selected cargos to specific synapses. However, the supporting mechanisms of this process are not fully understood. The present study unravels a new molecular system for vesicle-based axonal transport of proteins in male and female flies (Drosophila melanogaster). This study identifies the gene CG14579 as the transcription unit corresponding to the regulatory mutations known as central complex broad, ccb. These mutations were previously isolated for their morphological phenotype in R-neurons of the ellipsoid body, a component of the central complex. Mutant axons from R-neurons fail to cross the midline, which is indicative of an aberrant composition of the growth cone. However, the molecular mechanism remained to be deciphered. This study shows that CCB is involved in axonal trafficking of FasII and Synaptobrevin, but not Syntaxin. These results suggest that axonal transport of certain proteins is required for the correct pathfinding of R-neurons. The molecular network supporting the CCB system was further investigated, and CCB was found to co-localize and co-immunoprecipitate with Rab11. Epistasis studies indicated that Rab11 is positioned downstream of CCB within this axonal transport system. Interestingly, ccb also interacts with Actin and the Actin nucleator Spire. The data revealed that this interaction plays a key role in the development of axonal connections within the ellipsoid body. It is proposed that the CCB/Rab11/SPIRE system regulates axonal trafficking of synaptic proteins required for proper connectivity and synaptic function.
Reynolds, H. M., Zhang, L., Tran, D. T. and Ten Hagen, K. G. (2019). Tango1 coordinates the formation of ER/Golgi docking sites to mediate secretory granule formation. J Biol Chem. PubMed ID: 31690624
Regulated secretion is a conserved process occurring across diverse cells and tissues. Current models suggest that the conserved cargo receptor Tango1 mediates the packaging of collagen into large coat protein complex II (COPII) vesicles that move from the endoplasmic reticulum (ER) to the Golgi apparatus. However, how Tango1 regulates the formation of COPII carriers and influences the secretion of other cargo remains unknown. Through high-resolution imaging of Tango1, COPII, Golgi and secretory cargo (mucins) in Drosophila larval salivary glands, this study found that Tango1 forms ring-like structures that mediate the formation of COPII rings, rather than vesicles. These COPII rings act as docking sites for the cis-Golgi. Moreover, nascent secretory mucins were observed emerging from the Golgi side of these Tango1/COPII/Golgi complexes, suggesting that these structures represent functional docking sites/fusion points between the ER exit sites and the Golgi. Loss of Tango1 disrupted the formation of COPII rings, the association of COPII with the cis-Golgi, mucin O-glycosylation and secretory granule biosynthesis. Additionally, this study identified a Tango1 self-association domain that is essential for formation of this structure. These results provide evidence that Tango1 organizes an interaction site where secretory cargo is efficiently transferred from the ER to Golgi and then to secretory vesicles. These findings may explain how the loss of Tango1 can influence Golgi/ER morphology and affect the secretion of diverse proteins across many tissues.
Ueoka, I., Takai, A., Yamaguchi, M., Chiyonobu, T., Yoshida, H. and Yamaguchi, M. (2019). Novel genetic link between the ATP-binding cassette subfamily A gene and hippo gene in Drosophila. Exp Cell Res: 111733. PubMed ID: 31751555
The pan-neuron-specific knockdown of dABCA, a Drosophila homologue of the human ATP binding cassette subfamily A member 13 gene, increases social space without affecting climbing ability and induces the early onset of evening activity in adult flies followed by relatively high activity throughout the day. Satellite bouton numbers in the presynaptic terminals of motor neurons are increased in dABCA knockdown flies. This study further characterized pan-neuron-specific dABCA knockdown flies and found that active zones in the presynaptic terminals of motor neurons increased, whereas learning abilities decreased in larvae. Genetic crossing experiments revealed that the hippo mutation enhanced the hyperactivity phenotype of adults, but suppressed the increased satellite bouton phenotype induced by the dABCA knockdown. Drosophila ABCA is predicted to transport lipid molecules and impair the asymmetric distribution of phospholipids across the plasma membrane, and these local changes are considered to be important for various cellular functions. The disruption of lipid homeostasis in central and peripheral nervous systems by the dABCA knockdown may affect the Hippo-related signaling pathway in order to induce the observed phenotypes.
Hoover, K. M., Gratz, S. J., Qi, N., Herrmann, K. A., Liu, Y., Perry-Richardson, J. J., Vanderzalm, P. J., O'Connor-Giles, K. M. and Broihier, H. T. (2019). The calcium channel subunit alpha2delta-3 organizes synapses via an activity-dependent and autocrine BMP signaling pathway. Nat Commun 10(1): 5575. PubMed ID: 31811118
Synapses are highly specialized for neurotransmitter signaling, yet activity-dependent growth factor release also plays critical roles at synapses. While efficient neurotransmitter signaling relies on precise apposition of release sites and neurotransmitter receptors, molecular mechanisms enabling high-fidelity growth factor signaling within the synaptic microenvironment remain obscure. This study shows that the auxiliary calcium channel subunit alpha2delta-3 promotes the function of an activity-dependent autocrine Bone Morphogenetic Protein (BMP) signaling pathway at the Drosophila neuromuscular junction (NMJ). alpha2delta proteins have conserved synaptogenic activity, although how they execute this function has remained elusive. This study finds that alpha2delta-3 provides an extracellular scaffold for an autocrine BMP signal, suggesting a mechanistic framework for understanding alpha2delta's conserved role in synapse organization. A transcriptional requirement was established for activity-dependent, autocrine BMP signaling in determining synapse density, structure, and function. It is proposed that activity-dependent, autocrine signals provide neurons with continuous feedback on their activity state for modulating both synapse structure and function.
Chou, V. T., Johnson, S., Long, J., Vounatsos, M. and Van Vactor, D. (2019). dTACC restricts bouton addition and regulates microtubule organization at the Drosophila neuromuscular junction. Cytoskeleton (Hoboken). PubMed ID: 31702858
Regulation of the synaptic cytoskeleton is essential to proper neuronal development and wiring. Perturbations in neuronal microtubules (MTs) are associated with numerous pathologies, yet it remains unclear how changes in MTs may be coupled to synapse morphogenesis. Studies have identified many MT regulators that promote synapse growth. However, less is known about the factors that restrict growth, despite the potential links of synaptic overgrowth to severe neurological conditions. This study reports that dTACC, which is implicated in MT assembly and stability, prevents synapse overgrowth at the Drosophila neuromuscular junction by restricting addition of new boutons throughout larval development. dTACC localizes to the axonal MT lattice and is required to maintain tubulin levels and the integrity of higher-order MT structures in motor axon terminals. While previous reports have demonstrated the roles of MT-stabilizing proteins in promoting synapse growth, these findings suggest that in certain contexts, MT stabilization may correlate with restricted growth.
Kraut, R. S. and Knust, E. (2019). Changes in endolysosomal organization define a pre-degenerative state in the crumbs mutant Drosophila retina. PLoS One 14(12): e0220220. PubMed ID: 31834921
Mutations in the epithelial polarity gene crumbs (crb) lead to retinal degeneration in Drosophila and in humans. The overall morphology of the retina and its deterioration in Drosophila crb mutants has been well-characterized, but the cell biological origin of the degeneration is not well understood. Degenerative conditions in the retina and elsewhere in the nervous system often involve defects in degradative intracellular trafficking pathways. So far, however, effects of crb on the endolysosomal system, or on the spatial organization of these compartments in photoreceptor cells have not been described. This study therefore asked whether photoreceptors in crb mutants exhibit alterations in endolysosomal compartments under pre-degenerative conditions, where the retina is still morphologically intact. Data presented in this study show that, already well before the onset of degeneration, Arl8, Rab7, and Atg8-carrying endolysosomal and autophagosomal compartments undergo changes in morphology and positioning with respect to each other in crb mutant retinas. It is proposed that these changes may be early signs of the degeneration-prone condition in crb retinas.

Wednesday, January 15 - Apoptosis and Autophagy

Katsube, H., Hinami, Y., Yamazoe, T. and Inoue, Y. H. (2019). Endoplasmic reticulum stress-induced cellular dysfunction and cell death in insulin-producing cells results in diabetes-like phenotypes in Drosophila. Biol Open 8(12). PubMed ID: 31822470
The destruction of pancreatic beta cells leads to reduced insulin secretion and eventually causes diabetes. Various types of cellular stress are thought to be involved in destruction and/or malfunction of these cells. This study shows that endoplasmic reticulum (ER) stress accumulation in insulin-producing cells (IPCs) generated diabetes-like phenotypes in Drosophila. To promote the accumulation of extra ER stress, a dominant-negative form of a Drosophila ER chaperone protein (Hsc70-3(DN)) was induced; it was demonstrated to cause the unfolded-protein response (UPR) in various tissues. The numbers of IPCs decreased owing to apoptosis induction mediated by caspases. The apoptosis was driven by activation of Dronc, and subsequently by Drice and Dcp-1. Accordingly, the relative mRNA-expression levels of Drosophila insulin-like peptides significantly decreased. Consistent with these results, it was demonstrated that glucose levels in larval haemolymph were significantly higher than those of controls. Accumulation of ER stress induced by continuous Hsc70-3(DN) expression in IPCs resulted in the production of undersized flies. Ectopic expression of Hsc70-3(DN) can induce more efficient ER stress responses and more severe phenotypes. It is proposed that ER stress is responsible for IPC loss and dysfunction, which results in diabetes-related pathogenesis in this Drosophila diabetes model. Moreover, inhibiting apoptosis partially prevents the ER stress-induced diabetes-like phenotypes.
Wang, Z., Lee, G., Vuong, R. and Park, J. H. (2019). Two-factor specification of apoptosis: TGF-beta signaling acts cooperatively with ecdysone signaling to induce cell- and stage-specific apoptosis of larval neurons during metamorphosis in Drosophila melanogaster. Apoptosis 24(11-12): 972-989. PubMed ID: 31641960
Developmentally regulated programmed cell death (PCD) is one of the key cellular events for precise controlling of neuronal population during postembryonic development of the central nervous system. Previous work has shown that a group of corazonin-producing peptidergic neurons (vCrz) undergo apoptosis in response to ecdysone signaling via ecdysone receptor (EcR)-B isoforms and Ultraspiracle during early phase of metamorphosis. Further utilizing genetic, transgenic, and mosaic analyses, it was found that TGF-beta signaling mediated by a glia-produced ligand, Myoglianin, type-I receptor Baboon (particularly Babo-A isoform) and dSmad2, is also required autonomously for PCD of the vCrz neurons. These studies show that TGF-beta signaling is not acting epistatically to EcR or vice versa. It was also shown that ectopic expression of a constitutively active phosphomimetic form of dSmad2 (dSmad2(PM)) is capable of inducing premature death of vCrz neurons in larva but not other larval neurons. Intriguingly, the dSmad2(PM)-mediated killing is completely suppressed by coexpression of a dominant-negative form of EcR (EcR(DN)), suggesting that EcR function is required for the proapoptotic dSmad2(PM) function. Based on these data, it is suggested that TGF-beta and ecdysone signaling pathways act cooperatively to induce vCrz neuronal PCD. It is proposed that this type of two-factor authentication is a key developmental strategy to ensure the timely PCD of specific larval neurons during metamorphosis.
Nishikawa, S. and Takamatsu, A. (2019). Effects of cell death-induced proliferation on a cell competition system. Math Biosci 316: 108241. PubMed ID: 31449892
Cell death-induced proliferation (CDIP) is a phenomenon in which cell death activates neighboring cells and promotes their proliferation. It was first reported as "compensatory proliferation" in injured tissues, which functions to maintain normal tissues. On the other hand, this phenomenon also affects potentially tumorigenic mutant cells and promotes tumorigenesis. This discrepancy may complicate the understanding of a phenomenon called "cell competition" observed in a system consisting of wild-type (WT) cells and mutant cells in a single-layer tissue. In this system, the WT cells induce cell death in the adjacent mutant cells to eliminate them. Therefore, it is believed that CDIP serves WT cells by compensating the space previously occupied by mutant cells. On the other hand, CDIP may contribute to the expansion of a potentially tumorigenic mutant clone because this clone activates itself. With the aim to investigate the role of CDIP in cell competition, a mathematical model was constructed in this study by introducing a CDIP effect into the population-based cell competition model that was proposed in previous work. In contrast to the above-mentioned first expectation, the model suggests that the CDIP of WT cells that is derived from cell competition does not affect the fate whether it follows formation of normal tissue or overgrowth of a mutant clone after cell competition. It should be noted, however, that CDIP accelerates the speed of normal tissue formation; only this point is in agreement with expectations. In contrast, the CDIP of mutant cells that is derived from either autonomous cell death or cell competition helps mutant cells to survive.
Wang, Y., Huang, Y., Liu, J., Zhang, J., Xu, M., You, Z., Peng, C., Gong, Z. and Liu, W. (2019). Acetyltransferase GCN5 regulates autophagy and lysosome biogenesis by targeting TFEB. EMBO Rep: e48335. PubMed ID: 31750630
Accumulating evidence highlights the role of histone acetyltransferase GCN5 in the regulation of cell metabolism in metazoans. This study reports that GCN5 is a negative regulator of autophagy, a lysosome-dependent catabolic mechanism. In animal cells and Drosophila, GCN5 inhibits the biogenesis of autophagosomes and lysosomes by targeting TFEB, the master transcription factor for autophagy- and lysosome-related gene expression. GCN5 is a specific TFEB acetyltransferase, and acetylation by GCN5 results in the decrease in TFEB transcriptional activity. Induction of autophagy inactivates GCN5, accompanied by reduced TFEB acetylation and increased lysosome formation. It was further demonstrated that acetylation at K274 and K279 disrupts the dimerization of TFEB and the binding of TFEB to its target gene promoters. In a Tau-based neurodegenerative Drosophila model, deletion of dGcn5 improves the clearance of Tau protein aggregates and ameliorates the neurodegenerative phenotypes. Together, these results reveal GCN5 as a novel conserved TFEB regulator, and the regulatory mechanisms may be involved in autophagy- and lysosome-related physiological and pathological processes.
Nagata, R., Nakamura, M., Sanaki, Y. and Igaki, T. (2019). Cell competition is driven by autophagy. Dev Cell 51(1): 99-112. PubMed ID: 31543447
Cell competition is a quality control process that selectively eliminates unfit cells from the growing tissue via cell-cell interaction. Despite extensive mechanistic studies, the mechanism by which cell elimination is triggered has been elusive. Here, through a genetic screen in Drosophila, this study discovered that V-ATPase, an essential factor for autophagy, is required for triggering cell competition. Strikingly, autophagy is specifically elevated in prospective "loser" cells nearby wild-type "winner" cells, and blocking autophagy in loser cells abolishes their elimination. Mechanistically, elevated autophagy upregulates a proapoptotic gene hid through NFkappaB, and the elevated hid cooperates with JNK signaling to effectively induce loser's death. Crucially, this mechanism generally applies to cell competition caused by differences in protein synthesis between cells. These findings establish a common mechanism of cell competition whereby cells with higher protein synthesis induce autophagy in their neighboring cells, leading to elimination of unfit cells.
Reiff, T., Antonello, Z. A., Ballesta-Illan, E., Mira, L., Sala, S., Navarro, M., Martinez, L. M. and Dominguez, M. (2019). Notch and EGFR regulate apoptosis in progenitor cells to ensure gut homeostasis in Drosophila. EMBO J: e101346. PubMed ID: 31566767 The regenerative activity of adult stem cells carries a risk of cancer, particularly in highly renewable tissues. Members of the family of inhibitor of apoptosis proteins (IAPs) inhibit caspases and cell death, and are often deregulated in adult cancers; however, their roles in normal adult tissue homeostasis are unclear. This study show that regulation of the number of enterocyte-committed progenitor (enteroblast) cells in the adult Drosophila involves a caspase-mediated physiological apoptosis, which adaptively eliminates excess enteroblast cells produced by intestinal stem cells (ISCs) and, when blocked, can also lead to tumorigenesis. Importantly, it was found that Diap1 is expressed by enteroblast cells and that loss and gain of Diap1 led to changes in enteroblast numbers. Antagonistic interplay between Notch and EGFR signalling was found to govern enteroblast life/death decisions via the Klumpfuss/WT1 and Lozenge/RUNX transcription regulators, which also regulate enteroblast differentiation and cell fate plasticity. These data provide new insights into how caspases drive adult tissue renewal and protect against the formation of tumours.

Tuesday, January 14th - Enhancers and gene regulation

Dunipace, L., Akos, Z. and Stathopoulos, A. (2019). Coacting enhancers can have complementary functions within gene regulatory networks and promote canalization. PLoS Genet 15(12): e1008525. PubMed ID: 31830033
Developmental genes are often regulated by multiple enhancers exhibiting similar spatiotemporal outputs, which are generally considered redundantly acting though few have been studied functionally. Using CRISPR-Cas9, deletions of two enhancers, brk5' and brk3', were created that drive similar but not identical expression of the gene brinker (brk) in early Drosophila embryos. Utilizing both in situ hybridization and quantitative mRNA analysis, the changes were investigated in the gene network state caused by the removal of one or both of the early acting enhancers. brk5' deletion generally phenocopied the gene mutant, including expansion of the BMP ligand decapentaplegic (dpp) as well as inducing variability in amnioserosa tissue cell number suggesting a loss of canalization. In contrast, brk3' deletion presented unique phenotypes including dorsal expansion of several ventrally expressed genes and a decrease in amnioserosa cell number. Similarly, deletions were made for two enhancers associated with the gene short-gastrulation (sog), sog.int and sog.dist, demonstrating that they also exhibit distinct patterning phenotypes and affect canalization. In summary, this study shows that similar gene expression driven by coacting enhancers can support distinct, and sometimes complementary, functions within gene regulatory networks and, moreover, that phenotypes associated with individual enhancer deletion mutants can provide insight into new gene functions.
Mathiyalagan, N.,et al. (2019). Meta-analysis of Grainyhead-like dependent transcriptional networks: A roadmap for identifying novel conserved genetic pathways. Genes (Basel) 10(11). PubMed ID: 31683705
Mutations affecting Drosophila grainyhead (grh) and vertebrate Grainyhead-like (Grhl) transcription factors lead to a developmental and adult onset epithelial disease, such as aberrant skin barrier formation, facial/palatal clefting, impaired neural tube closure, age-related hearing loss, ectodermal dysplasia, and importantly, cancers of epithelial origin. Recently, mutations in the family member GRHL3 have been shown to lead to both syndromic and non-syndromic facial and palatal clefting in humans. Large-scale datasets have been generated to explore the grh/Grhl-dependent transcriptome, following ablation or mis-regulation of grh/Grhl-function. A meta-analysis was performed of all 41 currently published grh and Grhl RNA-SEQ, and microarray datasets, in order to identify and characterise the transcriptional networks controlled by grh/Grhl genes across disparate biological contexts. Moreover, this study has also cross-referenced the results with published ChIP and ChIP-SEQ datasets, in order to determine which of the critical effector genes are likely to be direct grh/Grhl targets, based on genomic occupancy by grh/Grhl genes. Lastly, to interrogate the predictive strength of this approach, the expression of the top 10 candidate grhl target genes in epithelial development were experimentally validated, in a zebrafish model lacking grhl3, and found that orthologues of seven of these (cldn23, ppl, prom2, ocln, slc6a19, aldh1a3, and sod3) were significantly down-regulated at 48 hours post-fertilisation. Therefore, this study provides a strong predictive resource for the identification of putative grh/grhl effector target genes.
Bell, K., Skier, K., Chen, K. H. and Gergen, J. P. (2019). Two pair-rule responsive enhancers regulate wingless transcription in the Drosophila blastoderm embryo. Dev Dyn. PubMed ID: 31837063
While many developmentally relevant enhancers act in a modular fashion, there is growing evidence for nonadditive interactions between distinct cis-regulatory enhancers. This study investigated if nonautonomous enhancer interactions underlie transcription regulation of the Drosophila segment polarity gene, wingless. Two wg enhancers active at the blastoderm stage were identified: wg 3613u, located from -3.6 to -1.3 kb upstream of the wg transcription start site (TSS) and 3046d, located in intron two of the wg gene, from 3.0 to 4.6 kb downstream of the TSS. Genetic experiments confirm that Even Skipped (Eve), Fushi-tarazu (Ftz), Runt, Odd-paired (Opa), Odd-skipped (Odd), and Paired (Prd) contribute to spatially regulated wg expression. Interestingly, there are enhancer specific differences in response to the gain or loss of function of pair-rule gene activity. Although each element recapitulates aspects of wg expression, a composite reporter containing both enhancers more faithfully recapitulates wg regulation than would be predicted from the sum of their individual responses. These results suggest that the regulation of wg by pair-rule genes involves nonadditive interactions between distinct cis-regulatory enhancers.
Guo, X., Yin, C., Yang, F., Zhang, Y., Huang, H., Wang, J., Deng, B., Cai, T., Rao, Y. and Xi, R. (2019). The cellular diversity and transcription factor code of Drosophila enteroendocrine cells. Cell Rep 29(12): 4172-4185. PubMed ID: 31851941
Enteroendocrine cells (EEs) in the intestinal epithelium have important endocrine functions, yet this cell lineage exhibits great local and regional variations that have hampered detailed characterization of EE subtypes. Through single-cell RNA-sequencing analysis, combined with a collection of peptide hormone and receptor knockin strains, this study provides a comprehensive analysis of cellular diversity, spatial distribution, and transcription factor (TF) code of EEs in adult Drosophila midgut. Ten major EE subtypes were identified that totally produced approximately 14 different classes of hormone peptides. Each EE on average co-produces approximately 2-5 different classes of hormone peptides. Functional screen with subtype-enriched TFs suggests a combinatorial TF code that controls EE cell diversity; class-specific TFs Mirr and Ptx1 respectively define two major classes of EEs, and regional TFs such as Esg, Drm, Exex, and Fer1 further define regional EE identity. These single-cell data should greatly facilitate Drosophila modeling of EE differentiation and function.
Gisselbrecht, S. S., Palagi, A., Kurland, J. V., Rogers, J. M., Ozadam, H., Zhan, Y., Dekker, J. and Bulyk, M. L. (2019). Transcriptional silencers in Drosophila serve a dual role as transcriptional enhancers in alternate cellular contexts. Mol Cell. PubMed ID: 31704182
A major challenge in biology is to understand how complex gene expression patterns are encoded in the genome. While transcriptional enhancers have been studied extensively, few transcriptional silencers have been identified, and they remain poorly understood. This study used a novel strategy to screen hundreds of sequences for tissue-specific silencer activity in whole Drosophila embryos. Almost all of the transcriptional silencers that were identified were also active enhancers in other cellular contexts. These elements are bound by more transcription factors than non-silencers. A subset of these silencers forms long-range contacts with promoters. Deletion of a silencer caused derepression of its target gene. These results challenge the common practice of treating enhancers and silencers as separate classes of regulatory elements and suggest the possibility that thousands or more bifunctional CRMs remain to be discovered in Drosophila and in humans.
Topfer, U., Bischoff, M. C., Bartkuhn, M. and Holz, A. (2019). Serpent/dGATAb regulates Laminin B1 and Laminin B2 expression during Drosophila embryogenesis. Sci Rep 9(1): 15910. PubMed ID: 31685844
Transcriptional regulation of Laminin expression during embryogenesis is a key step required for proper ECM assembly. This study shows that in Drosophila the Laminin B1 and Laminin B2 genes share expression patterns in mesodermal cells as well as in endodermal and ectodermal gut primordia, yolk and amnioserosa. In the absence of the GATA transcription factor Serpent, the spatial extend of Laminin reporter gene expression was strongly limited, indicating that Laminin expression in many tissues depends on Serpent activity. a direct binding of Serpent to the intronic enhancers of Laminin B1 and Laminin B2 was demonstrated. In addition, ectopically expressed Serpent activated enhancer elements of Laminin B1 and Laminin B2. These results reveal Serpent as an important regulator of Laminin expression across tissues.

Monday, January 13th - Disease models

Cacciottolo, R., Ciantar, J., Lanfranco, M., Borg, R. M., Vassallo, N., Bordonne, R. and Cauchi, R. J. (2019). SMN complex member Gemin3 self-interacts and has a functional relationship with ALS-linked proteins TDP-43, FUS and Sod1. Sci Rep 9(1): 18666. PubMed ID: 31822699
The predominant motor neuron disease in infants and adults is spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS), respectively. SMA is caused by insufficient levels of the Survival Motor Neuron (SMN) protein, which operates as part of the multiprotein SMN complex that includes the DEAD-box RNA helicase Gemin3/DDX20/DP103. C9orf72, SOD1, TDP-43 and FUS are ranked as the four major genes causing familial ALS. Accumulating evidence has revealed a surprising molecular overlap between SMA and ALS. This study asked the question of whether Drosophila can also be exploited to study shared pathogenic pathways. Focusing on motor behaviour, muscle mass and survival, disruption of either TBPH/TDP-43 or Caz/FUS was found to enhance defects associated with Gemin3 loss-of-function. Gemin3-associated neuromuscular junction overgrowth was however suppressed. Sod1 depletion had a modifying effect in late adulthood. This study also showed that Gemin3 self-interacts and Gem3(DeltaN), a helicase domain deletion mutant, retains the ability to interact with its wild-type counterpart. Importantly, mutant:wild-type dimers are favoured more than wild-type:wild-type dimers. In addition to reinforcing the link between SMA and ALS, further exploration of mechanistic overlaps is now possible in a genetically tractable model organism. Notably, Gemin3 can be elevated to a candidate for modifying motor neuron degeneration.
Bergkvist, L., Du, Z., Elovsson, G., Appelqvist, H., Itzhaki, L. S., Kumita, J. R., Kagedal, K. and Brorsson, A. C. (2019). Mapping pathogenic processes contributing to neurodegeneration in Drosophila models of Alzheimer's disease. FEBS Open Bio. PubMed ID: 31823504
Drosophila models of Alzheimer's disease (AD) include the Abeta fly model and the AbetaPP-BACE1 fly model. In the Abeta fly model, the Abeta peptide is fused to a secretion sequence and directly over-expressed. In the AbetaPP-BACE1 model, human AbetaPP and human BACE1 are expressed in the fly, resulting in in vivo production of Abeta peptides and other AbetaPP cleavage products. Although these two models have been used for almost two decades, the underlying mechanisms resulting in neurodegeneration are not yet clearly understood. This study has characterised toxic mechanisms in these two AD fly models. Neuronal cell death and increased protein carbonylation (indicative of oxidative stress) were detected in both AD fly models. In the Abeta fly model, this correlates with high Abeta1-42 levels and down-regulation of the levels of mRNA encoding lysosomal associated membrane protein 1, lamp1 (a lysosomal marker), while in the AbetaPP-BACE1 fly model, neuronal cell death correlates with low Abeta1-42 levels, up-regulation of lamp1 mRNA levels and increased levels of C-terminal fragments (CTFs). In addition, a significant amount of AbetaPP/Abeta antibody (4G8) positive species, located close to the endosomal marker rab5, were detected in the AbetaPP-BACE1 model. Taken together, this study highlights the similarities and differences in the toxic mechanisms which result in neuronal death in two different AD fly models. Such information is important to consider when utilising these models to study AD pathogenesis or screening for potential treatments.
Chung, H. J., Islam, M. S., Rahman, M. M. and Hong, S. T. (2019). Neuroprotective function of Omi to alpha-synuclein-induced neurotoxicity. Neurobiol Dis: 104706. PubMed ID: 31837423
The main pathological hallmark of Parkinson's disease (PD) is the presence of Lewy bodies, which mainly consist of aggregated alpha-synuclein. Based on the neurotoxicity of oligomeric alpha-synuclein and its significance in the aetiology of PD, there has been decades of effort to elucidate an enzyme specifically degrading oligomeric alpha-synuclein. This study reports an enzyme, Omi, which specifically recognizes and precisely degrades oligomeric alpha-synuclein but not monomeric alpha-synuclein. After enzymatic and functional analyses of Omi in in vitro, an in vivo assay system of dual gene interaction was developed in Drosophila to investigate further the etiological role of Omi in PD. Pan-neuronal expression of Omi rescued Parkinsonism in a Drosophila model of PD, while Knockout of Omi exacerbated Parkinsonism. Expression of Omi counteracted the alpha-synuclein-induced retinal degeneration, providing additional evidence for Omi's protective role oligomeric alpha-synuclein. This work reports identification of the catabolic pathway of oligomeric alpha-synuclein as well as showing how Omi functions as the key molecule in the recognition and degradation of toxic oligomeric alpha-synuclein, a possible cause of neurodegeneration in PD, without affecting monomeric alpha-synuclein which is a native essential molecule for the normal function of neurons.
Garrido-Maraver, J., Loh, S. H. Y. and Martins, L. M. (2019). Forcing contacts between mitochondria and the endoplasmic reticulum extends lifespan in a Drosophila model of Alzheimer's disease. Biol Open. PubMed ID: 31822473
Eukaryotic cells are complex systems containing internal compartments with specialised functions. Among these compartments, the endoplasmic reticulum (ER) plays a major role in processing proteins for modification and delivery to other organelles, whereas mitochondria generate energy in the form of ATP. Mitochondria and the ER form physical interactions, defined as mitochondria-ER contact sites (MERCs) to exchange metabolites such as calcium ions (Ca(2+)) and lipids. Sites of contact between mitochondria and the ER can regulate biological processes such as ATP generation and mitochondrial division. The interactions between mitochondria and the ER are dynamic and respond to the metabolic state of cells. Changes in MERCs have been linked to metabolic pathologies such as diabetes, neurodegenerative diseases and sleep disruption. This study explored the consequences of increasing contacts between mitochondria and the ER in flies using a synthetic linker. Enhancing MERCs was shown to increase locomotion and extend lifespan. In a Drosophila model of Alzheimer's disease linked to toxic amyloid beta (Abeta), linker expression can suppress motor impairment and extend lifespan. It is concluded that strategies for increasing contacts between mitochondria and the ER may improve symptoms of diseases associated with mitochondria dysfunction.
Casci, I., Krishnamurthy, K., Kour, S., Tripathy, V., Ramesh, N., Anderson, E. N., Marrone, L., Grant, R. A., Oliver, S., Gochenaur, L., Patel, K., Sterneckert, J., Gleixner, A. M., Donnelly, C. J., Ruepp, M. D., Sini, A. M., Zuccaro, E., Pennuto, M., Pasinelli, P. and Bhan Pandey, U. (2019). Muscleblind acts as a modifier of FUS toxicity by modulating stress granule dynamics and SMN localization. Nat Commun 10(1): 5583. PubMed ID: 31811140
Mutations in fused in sarcoma (FUS; see Drosophila Cabeza) lead to amyotrophic lateral sclerosis (ALS) with varying ages of onset, progression and severity. This suggests that unknown genetic factors contribute to disease pathogenesis. This study shows the identification of muscleblind as a novel modifier of FUS-mediated neurodegeneration in vivo. Muscleblind regulates cytoplasmic mislocalization of mutant FUS and subsequent accumulation in stress granules, dendritic morphology and toxicity in mammalian neuronal and human iPSC-derived neurons. Interestingly, genetic modulation of endogenous muscleblind was sufficient to restore survival motor neuron (SMN) protein localization in neurons expressing pathogenic mutations in FUS, suggesting a potential mode of suppression of FUS toxicity. Upregulation of SMN suppressed FUS toxicity in Drosophila and primary cortical neurons, indicating a link between FUS and SMN. These data provide in vivo evidence that muscleblind is a dominant modifier of FUS-mediated neurodegeneration by regulating FUS-mediated ALS pathogenesis.
Creed, T. M., Baldeosingh, R., Eberly, C. L., Schlee, C. S., Kim, M., Cutler, J. A., Pandey, A., Civin, C. I., Fossett, N. G. and Kingsbury, T. J. (2020). The PAX-SIX-EYA-DACH network modulates GATA-FOG function in fly hematopoiesis and human erythropoiesis. Development 147(1). PubMed ID: 31806659
The GATA (see Drosophila Serpent) and PAX-SIX-EYA-DACH transcriptional networks (PSEDNs) are essential for proper development across taxa. This study demonstrates novel PSEDN roles in vivo in Drosophila hematopoiesis and in human erythropoiesis in vitro. Using Drosophila genetics, PSEDN members were shown to function with GATA to block lamellocyte differentiation and maintain the prohemocyte pool. Overexpression of human SIX1 (see Drosophila Sine oculis) stimulated erythroid differentiation of human erythroleukemia TF1 cells and primary hematopoietic stem-progenitor cells. Conversely, SIX1 knockout impaired erythropoiesis in both cell types. SIX1 stimulation of erythropoiesis required GATA1, as SIX1 overexpression failed to drive erythroid phenotypes and gene expression patterns in GATA1 knockout cells. SIX1 can associate with GATA1 and stimulate GATA1-mediated gene transcription, suggesting that SIX1-GATA1 physical interactions contribute to the observed functional interactions. In addition, both fly and human SIX proteins regulated GATA protein levels. Collectively, this findings demonstrate that SIX proteins enhance GATA function at multiple levels, and reveal evolutionarily conserved cooperation between the GATA and PSEDN networks that may regulate developmental processes beyond hematopoiesis.

Friday, January 10th - Adult Neural Function

Xue, Y., Chiu, J. C. and Zhang, Y. (2019). SUR-8 interacts with PP1-87B to stabilize PERIOD and regulate circadian rhythms in Drosophila. PLoS Genet 15(11): e1008475. PubMed ID: 31710605
Circadian rhythms are generated by endogenous pacemakers that rely on transcriptional-translational feedback mechanisms conserved among species. In Drosophila, the stability of a key pacemaker protein PERIOD (PER) is tightly controlled by changes in phosphorylation status. A number of molecular players have been implicated in PER destabilization by promoting PER progressive phosphorylation. On the other hand, there have been few reports describing mechanisms that stabilize PER by delaying PER hyperphosphorylation. This study reports that the protein Suppressor of Ras (SUR-8) regulates circadian locomotor rhythms by stabilizing PER. Depletion of SUR-8 from circadian neurons lengthened the circadian period by about 2 hours and decreased PER abundance, whereas its overexpression led to arrhythmia and an increase in PER. Specifically SUR-8 promotes the stability of PER through phosphorylation regulation. Interestingly, downregulation of the protein phosphatase 1 catalytic subunit PP1-87B recapitulated the phenotypes of SUR-8 depletion. SUR-8 facilitates interactions between PP1-87B and PER. Depletion of SUR-8 decreased the interaction of PER and PP1-87B, which supports the role of SUR-8 as a scaffold protein. Interestingly, the interaction between SUR-8 and PER is temporally regulated: SUR-8 has more binding to PER at night than morning. Thus, these results indicate that SUR-8 interacts with PP1-87B to control PER stability to regulate circadian rhythms.
Akiba, M., Sugimoto, K., Aoki, R., Murakami, R., Miyashita, T., Hashimoto, R., Hiranuma, A., Yamauchi, J., Ueno, T. and Morimoto, T. (2019). Dopamine modulates the optomotor response to unreliable visual stimuli in Drosophila melanogaster. Eur J Neurosci. PubMed ID: 31834948
The precise neural mechanism underlying dopaminergic modulation of behaviour induced by sensory stimuli remains poorly understood. This study used Drosophila melanogaster to show that dopamine can modulate the optomotor response to moving visual stimuli including noise. The optomotor response is the head-turning response to moving objects, which is observed in most sight-reliant animals including mammals and insects. First, the effects of the dopamine system on the optomotor response were investigated in mutant flies deficient in dopamine receptors D1R1 (DopR) or D1R2, which are involved in the modulation of sleep-arousal in flies. This study examined the optomotor response in D1R1 knockout (D1R1 KO) and D1R2 knockout (D1R2 KO) flies and found that it was not affected in D1R1 KO flies; however, it was significantly reduced in D1R2 KO flies compared with the wild type. Using cell-type-specific expression of an RNA interference construct of D1R2, the fan-shaped body, a part of the central complex, was identified as responsible for dopamine-mediated modulation of the optomotor response. In particular, pontine cells in the fan-shaped body seemed important in the modulation of the optomotor response, and their neural activity was required for the optomotor response. These results suggest a novel role of the central complex in the modulation of a behaviour based on the processing of sensory stimulations.
Wu, S., Guo, C., Zhao, H., Sun, M., Chen, J., Han, C., Peng, Q., Qiao, H., Peng, P., Liu, Y., Luo, S. D. and Pan, Y. (2019). Drosulfakinin signaling in fruitless circuitry antagonizes P1 neurons to regulate sexual arousal in Drosophila. Nat Commun 10(1): 4770. PubMed ID: 31628317
Animals perform or terminate particular behaviors by integrating external cues and internal states through neural circuits. Identifying neural substrates and their molecular modulators promoting or inhibiting animal behaviors are key steps to understand how neural circuits control behaviors. This study identified the Cholecystokinin-like peptide Drosulfakinin (DSK) that functions at single-neuron resolution to suppress male sexual behavior in Drosophila. Dsk neurons physiologically interact with male-specific P1 neurons, part of a command center for male sexual behaviors, and function oppositely to regulate multiple arousal-related behaviors including sex, sleep and spontaneous walking. It was further found that the DSK-2 peptide functions through its receptor CCKLR-17D3 to suppress sexual behaviors in flies. Such a neuropeptide circuit largely overlaps with the fruitless-expressing neural circuit that governs most aspects of male sexual behaviors. Thus DSK/CCKLR signaling in the sex circuitry functions antagonistically with P1 neurons to balance arousal levels and modulate sexual behaviors.
Arimoto, E., Kawashima, Y., Choi, T., Unagami, M., Akiyama, S., Tomizawa, M., Yano, H., Suzuki, E. and Sone, M. (2019). Analysis of a cellular structure observed in the compound eyes of Drosophila white; yata mutants and white mutants. Biol Open. PubMed ID: 31862863
Previous work has identified the Drosophila yata mutant, which showed phenotypes including progressive vacuolization of the white-colored compound eye, progressive shrinkage of the brain and a shortened lifespan. The yata gene was shown to be involved in controlling intracellular trafficking of the APPL protein, which is an orthologue of APP that is a causative molecule of Alzheimer's disease. This study examined the phenotype of the compound eye of the yata mutant using electron microscopy and confocal microscopy. Abnormal cellular structures that seemed to originate from bleb-like structures and contained vesicles and organelles, such as multivesicular bodies and autophagosomes, were observed in aged white; yata mutants and aged white mutants. These structures were not observed in newly eclosed flies, and the presence of the structures was suppressed in flies grown under constant dark conditions after eclosion. The structures were not observed in newly eclosed red-eyed yata mutants or wild-type flies but were observed in very aged red-eyed wild-type flies. Thus, these data suggest that the observed structures are formed as a result of changes associated with exposure to light after eclosion in white mutants, white; yata mutants and aged flies.
Xiao, S., Sun, J. S. and Carlson, J. R. (2019). Robust olfactory responses in the absence of odorant binding proteins. Elife 8. PubMed ID: 31651397
Odorant binding proteins (Obps) are expressed at extremely high levels in the antennae of insects, and have long been believed essential for carrying hydrophobic odorants to odor receptors. Previously work found that when one functional type of olfactory sensillum in Drosophila was depleted of its sole abundant Obp, it retained a robust olfactory response. This study deleted all the Obp genes that are abundantly expressed in the antennal basiconic sensilla. All of six tested sensillum types responded robustly to odors of widely diverse chemical or temporal structure. One mutant gave a greater physiological and behavioral response to an odorant that affects oviposition. These results support a model in which many sensilla can respond to odorants in the absence of Obps, and many Obps are not essential for olfactory response, but that some Obps can modulate olfactory physiology and the behavior that it drives.
Dweck, H. K. M. and Carlson, J. R. (2019). Molecular Logic and Evolution of Bitter Taste in Drosophila. Curr Biol. PubMed ID: 31839451
Taste systems detect a vast diversity of toxins, which are perceived as bitter. When a species adapts to a new environment, its taste system must adapt to detect new death threats. This study deleted each of six commonly expressed bitter gustatory receptors (Grs) from Drosophila melanogaster. Systematic analysis revealed that requirements for these Grs differed for the same tastant in different neurons and for different tastants in the same neuron. Responses to some tastants in some neurons required four Grs, including Gr39a. Deletions also produced increased or novel responses, supporting a model of Gr-Gr inhibitory interactions. Coexpression of four Grs conferred several bitter responses to a sugar neuron. Bitter coding was examined in three other Drosophila species. Major evolutionary shifts were found. One shift depended on the concerted activity of seven Grs. This work shows how the complex logic of bitter coding provides the capacity to detect innumerable hazards and the flexibility to adapt to new ones.

Thursday, January 9th - Chromatin and Chromosomes

Bosso, G., Cipressa, F., Moroni, M. L., Pennisi, R., Albanesi, J., Brandi, V., Cugusi, S., Renda, F., Ciapponi, L., Polticelli, F., Antoccia, A., di Masi, A. and Cenci, G. (2019). NBS1 interacts with HP1 to ensure genome integrity. Cell Death Dis 10(12): 951. PubMed ID: 31836699
Heterochromatin Protein 1 (HP1) and the Mre11-Rad50-Nbs1 (MRN) complex are conserved factors that play crucial role in genome stability and integrity. Despite their involvement in overlapping cellular functions, ranging from chromatin organization, telomere maintenance to DNA replication and repair, a tight functional relationship between HP1 and the MRN complex has never been elucidated. This study shows that the Drosophila HP1a protein binds to the MRN complex through its chromoshadow domain (CSD). In addition, loss of any of the MRN members reduces HP1a levels indicating that the MRN complex acts as regulator of HP1a stability. Moreover, overexpression of HP1a in nbs (but not in rad50 or mre11) mutant cells drastically reduces DNA damage associated with the loss of Nbs suggesting that HP1a and Nbs work in concert to maintain chromosome integrity in flies. This study has also found that human HP1alpha and NBS1 interact with each other and that, similarly to Drosophila, siRNA-mediated inhibition of NBS1 reduces HP1alpha levels in human cultured cells. Surprisingly, fibroblasts from Nijmegen Breakage Syndrome (NBS) patients, carrying the 657del5 hypomorphic mutation in NBS1 and expressing the p26 and p70 NBS1 fragments, accumulate HP1alpha indicating that, differently from NBS1 knockout cells, the presence of truncated NBS1 extends HP1alpha turnover and/or promotes its stability. Remarkably, an siRNA-mediated reduction of HP1alpha in NBS fibroblasts decreases the hypersensitivity to irradiation, a characteristic of the NBS syndrome. Overall, these data provide an unanticipated evidence of a close interaction between HP1 and NBS1 that is essential for genome stability and point up HP1alpha as a potential target to counteract chromosome instability in NBS patient cells.
Wang, S. H. and Elgin, S. C. R. (2019). The impact of genetic background and cell lineage on the level and pattern of gene expression in position effect variegation. Epigenetics Chromatin 12(1): 70. PubMed ID: 31722719
Chromatin-based transcriptional silencing is often described as a stochastic process, largely because of the mosaic expression observed in position effect variegation (PEV), where a euchromatic reporter gene is silenced in some cells as a consequence of juxtaposition with heterochromatin. High levels of variation in PEV phenotypes are commonly observed in reporter stocks. To ascertain whether background mutations are the major contributors to this variation, it was asked how much of the variation is determined by genetic variants segregating in the population. Using selective breeding of a fourth chromosome PEV reporter line, 39C-12, two inbred lines were isolated exhibiting contrasting degrees of variegation (A1: low expression, D1: high expression). Within each inbred population, remarkable similarity is observed in the degree of variegation: 90% of the variation between the two inbred lines in the degree of silencing can be explained by genotype. Further analyses suggest that this result reflects the combined effect of multiple independent trans-acting loci. The results indicate that background genetic variants play the major role in determining the variable degrees of PEV commonly observed in laboratory stocks. Interestingly, not only does the degree of variegation become consistent in inbred lines, the patterns of variegation also appear similar. Combining these observations with the spreading model for local heterochromatin formation, an augmented stochastic model is proposed to describe PEV in which the genetic background drives the overall level of silencing, working with the cell lineage-specific regulatory environment to determine the on/off probability at the reporter locus in each cell. This model acknowledges cell type-specific events in the context of broader genetic impacts on heterochromatin formation.
Macinkovic, I., Theofel, I., Hundertmark, T., Kovac, K., Awe, S., Lenz, J., Forne, I., Lamp, B., Nist, A., Imhof, A., Stiewe, T., Renkawitz-Pohl, R., Rathke, C. and Brehm, A. (2019). Distinct CoREST complexes act in a cell-type-specific manner. Nucleic Acids Res. PubMed ID: 31701127
CoREST has been identified as a subunit of several protein complexes that generate transcriptionally repressive chromatin structures during development. However, a comprehensive analysis of the CoREST interactome has not been carried out. This study used proteomic approaches to define the interactomes of two dCoREST isoforms, dCoREST-L and dCoREST-M, in Drosophila. Three distinct histone deacetylase complexes built around a common dCoREST/dRPD3 core were identified: A dLSD1/dCoREST complex, the LINT complex and a dG9a/dCoREST complex. The latter two complexes can incorporate both dCoREST isoforms. By contrast, the dLSD1/dCoREST complex exclusively assembles with the dCoREST-L isoform. Genome-wide studies show that the three dCoREST complexes associate with chromatin predominantly at promoters. Transcriptome analyses in S2 cells and testes reveal that different cell lineages utilize distinct dCoREST complexes to maintain cell-type-specific gene expression programmes: In macrophage-like S2 cells, LINT represses germ line-related genes whereas other dCoREST complexes are largely dispensable. By contrast, in testes, the dLSD1/dCoREST complex prevents transcription of germ line-inappropriate genes and is essential for spermatogenesis and fertility, whereas depletion of other dCoREST complexes has no effect. This study uncovers three distinct dCoREST complexes that function in a lineage-restricted fashion to repress specific sets of genes thereby maintaining cell-type-specific gene expression programmes.
Becher, H., Jackson, B. C. and Charlesworth, B. (2019). Patterns of genetic variability in genomic regions with low rates of recombination. Curr Biol. PubMed ID: 31866366
The amount of DNA sequence variability in a genomic region is often positively correlated with its rate of crossing over (CO). This pattern is caused by selection acting on linked sites, which reduces genetic variability and biases the frequency distribution of segregating variants toward more rare variants than are expected without selection (skew). These effects may involve the spread of beneficial mutations (selective sweeps [SSWs]), the elimination of deleterious mutations (background selection [BGS]), or both, and are expected to be stronger with lower CO rates. However, in a recent study of human populations, the skew was reduced in the lowest CO regions compared with regions with somewhat higher CO rates. A low skew in very low CO regions, compared with theoretical predictions, is seen in the population genomic studies of Drosophila simulans described in this study and in other Drosophila species. This paper proposes an explanation for lower than expected skew in low CO regions, and validate it using computer simulations; explanations for higher skew with higher CO rates, as in D. simulans, will be explored elsewhere. Partially recessive, linked deleterious mutations can increase neutral variability when the product of the effective population size (Ne) and the selection coefficient against homozygous carriers of mutations (s) is </=1, i.e., there is associative overdominance (AOD) rather than BGS. AOD can operate in low CO regions, producing a lower skew than in its absence. This opens up a new perspective on how selection affects patterns of variability at linked sites.
Rosin, L. F., Crocker, O., Isenhart, R. L., Nguyen, S. C., Xu, Z. and Joyce, E. F. (2019). Chromosome territory formation attenuates the translocation potential of cells. Elife 8. PubMed ID: 31682226
The formation and spatial arrangement of chromosome territories (CTs) in interphase has been posited to influence the outcome and frequency of genomic translocations. This is supported by correlations between the frequency of inter-chromosomal contacts and translocation events in myriad systems. However, it remains unclear if CT formation itself influences the translocation potential of cells. This question was addressed in Drosophila cells by modulating the level of Condensin II, which regulates CT organization. Using whole-chromosome Oligopaints to identify genomic rearrangements, this study finds that increased contact frequencies between chromosomes due to Condensin II knockdown leads to an increased propensity to form translocations following DNA damage. Moreover, Condensin II over-expression is sufficient to drive spatial separation of CTs and attenuate the translocation potential of cells. Together, these results provide the first causal evidence that proper CT formation can protect the genome from potentially deleterious translocations in the presence of DNA damage.
Carvajal-Garcia, J., Gales, E. R., Ramsden, D. A. and Sekelsky, J. (2020). The Drosophila melanogaster ortholog of RFWD3 functions independently of RAD51 during DNA repair. G3 (Bethesda). PubMed ID: 31900333
Repair of damaged DNA is required for the viability of all organisms. Fewer than half of the alleles identified in genetic screens have been mapped to a specific gene, leaving a potential for new discoveries in this field. This study shows that the previously uncharacterized mutagen sensitive gene mus302 codes for the Drosophila melanogaster ortholog of the E3 ubiquitin ligase RING finger and WD domain protein 3 (RFWD3). In human cells, RFWD3 promotes ubiquitylation of RPA and RAD51 to facilitate repair of collapsed replication forks and double-strand breaks through homologous recombination. Despite the high similarity in sequence to the human ortholog, the current evidence fails to support a role for Mus302 in the repair of these types of damage. Last, it was observed that the N-terminal third of RFWD3 is only found in mammals, but not in other vertebrates or invertebrates. It is proposed that the new N-terminal sequence accounts for the acquisition of a new biological function in mammals that explains the functional differences between the human and the fly orthologs, and that Drosophila Mus302 may retain the ancestral function of the protein.

Wednesday, January 8th - Adult Physiology

Videlier, M., Rundle, H. D. and Careau, V. (2019). Sex-specific among-individual covariation in locomotor activity and resting metabolic rate in Drosophila melanogaster. Am Nat 194(6): E164-e176. PubMed ID: 31738101
A key endeavor in evolutionary physiology is to identify sources of among- and within-individual variation in resting metabolic rate (RMR). Although males and females often differ in whole-organism RMR due to sexual size dimorphism, sex differences in RMR sometimes persist after conditioning on body mass, suggesting phenotypic differences between males and females in energy-expensive activities contributing to RMR. One potential difference is locomotor activity, yet its relationship with RMR is unclear and different energy budget models predict different associations. This study quantified locomotor activity (walking) over 24 h and RMR (overnight) in 232 male and 245 female Drosophila melanogaster that were either mated or maintained as virgins between two sets of measurements. Accounting for body mass, sex, and reproductive status, RMR and activity were significantly and moderately repeatable. RMR and activity were positively correlated among but not within individuals. Moreover, activity varied throughout the day and between the sexes. Partitioning the analysis by sex and activity by time of day revealed that all among-individual correlations were positive and significant in males but nonsignificant or even significantly negative in females. Such differences in the RMR-activity covariance suggest fundamental differences in how the sexes manage their energy budget.
Xi, J., Cai, J., Cheng, Y., Fu, Y., Wei, W., Zhang, Z., Zhuang, Z., Hao, Y., Lilly, M. A. and Wei, Y. (2019). The TORC1 inhibitor Nprl2 protects age-related digestive function in Drosophila. Aging (Albany NY) 11. PubMed ID: 31712450
Aging and age-related diseases occur in almost all organisms. Recently, it was discovered that the inhibition of target of rapamycin complex 1 (TORC1), a conserved complex that mediates nutrient status and cell metabolism, can extend an individual's lifespan and inhibit age-related diseases in many model organisms. However, the mechanism whereby TORC1 affects aging remains elusive. This study used a loss-of-function mutation in nprl2, a component of GATOR1 that mediates amino acid levels and inhibits TORC1 activity, to investigate the effect of increased TORC1 activity on the occurrence of age-related digestive dysfunction in Drosophila. The nprl2 mutation decreased Drosophila lifespan. Furthermore, the nprl2 mutant had a distended crop, with food accumulation at an early age. Interestingly, the inappropriate food distribution and digestion along with decreased crop contraction in nprl2 mutant can be rescued by decreasing TORC1 activity. In addition, nprl2-mutant flies exhibited age-related phenotypes in the midgut, including short gut length, a high rate of intestinal stem cell proliferation, and metabolic dysfunction, which could be rescued by inhibiting TORC1 activity. These findings showed that the gastrointestinal tract aging process is accelerated in nprl2-mutant flies, owing to high TORC1 activity, which suggested that TORC1 promotes digestive tract senescence.
Zhao, M. and Zhou, B. (2019). A distinctive sequence motif in the fourth transmembrane domain confers ZIP13 iron function in Drosophila melanogaster. Biochim Biophys Acta Mol Cell Res: 118607. PubMed ID: 31733261
The zinc/iron permease (ZIP/SLC39A) family plays an important role in metal ion transport and is essential for diverse physiological processes. Members of the ZIP family function primarily in the influx of transition metal ions zinc and iron, into cytoplasm from extracellular space or intracellular organelles. The molecular determinants defining metal ion selectivity among ZIP family members remain unclear. Specifically, it has been reported that the Drosophila ZIP family member ZIP13 (dZIP13), functions as an iron exporter and was responsible for pumping iron into the secretory pathway. ZIP13 protein is unique in that it differs from the other LIV-1 subfamily members at transmembrane domain IV (TM4), wherein relative positions of the conserved H and D residues in the HNXXD sequence motif are switched, generating a DNXXH motif. This study undertook an in vivo approach to explore the significance of this D/H exchange. Comparative functional analysis of mutants revealed that the relative positions of D and H are critical for the physiological roles of dZIP13 and its close homologue dZIP7. Swapping D/H position of this DNXXH sequence in dZIP13 resulted in loss of iron activity; normal dZIP13 could not complement dZIP7 loss, but swapping the two relative amino acid positions D and H in dZIP13 was sufficient to make it functionally analogous to its close homologue dZIP7. This work provides the first in vivo functional analysis of a structural motif required to differentiate different transporting functions of ZIPs.
Barrett, M., Fiocca, K., Waddell, E. A., McNair, C., O'Donnell, S. and Marenda, D. R. (2020). Larval mannitol diets increase mortality, prolong development and decrease adult body sizes in fruit flies (Drosophila melanogaster). Biol Open 8(12). PubMed ID: 31822472
The ability of polyols to disrupt holometabolous insect development has not been studied, and identifying compounds in food that affect insect development can further understanding of the pathways that connect growth rate, developmental timing and body size in insects. High-sugar diets prolong development and generate smaller adult body sizes in Drosophila melanogaster. Doncentration-dependent effects on development were tested when D. melanogaster larvae are fed mannitol, a polyalcohol sweetener. Tests were performed for amelioration of developmental effects if introduction to mannitol media is delayed past the third instar, as expected if there is a developmental sensitive-period for mannitol effects. Both male and female larvae had prolonged development and smaller adult body sizes when fed increasing concentrations of mannitol. Mannitol-induced increases in mortality were concentration dependent in 0 M to 0.8 M treatments with mortality effects beginning as early as 48 h post-hatching. Larval survival, pupariation and eclosion times were unaffected in 0.4 M mannitol treatments when larvae were first introduced to mannitol 72 h post-hatching (the beginning of the third instar); 72 h delay of 0.8 M mannitol introduction reduced the adverse mannitol effects. The developmental effects of a larval mannitol diet closely resemble those of high-sugar larval diets.
Zhou, S., Morgante, F., Geisz, M., Ma, J., Anholt, R. and Mackay, T. (2019). Systems genetics of the Drosophila metabolome. Genome Res. PubMed ID: 31694867
How effects of DNA sequence variants are transmitted through intermediate endophenotypes to modulate organismal traits remains a central question in quantitative genetics. This problem can be addressed through a systems approach in a population in which genetic polymorphisms, gene expression traits, metabolites and complex phenotypes can be evaluated on the same genotypes. This study focused on the metabolome, which represents the most proximal link between genetic variation and organismal phenotype, and quantified metabolite levels in 40 lines of the Drosophila melanogaster Genetic Reference Panel. Sex-specific modules of genetically correlated metabolites were identified, and networks constructed that integrate DNA sequence variation and variation in gene expression with variation in metabolites and organismal traits, including starvation stress resistance and male aggression. Finally, it was asked to what extent SNPs and metabolites can predict trait phenotypes and generated trait- and sex-specific prediction models that provide novel insights about the metabolomic underpinnings of complex phenotypes.
Bensard, C. L., Wisidagama, D. R., Olson, K. A., Berg, J. A., Krah, N. M., Schell, J. C., Nowinski, S. M., Fogarty, S., Bott, A. J., Wei, P., Dove, K. K., Tanner, J. M., Panic, V., Cluntun, A., Lettlova, S., Earl, C. S., Namnath, D. F., Vazquez-Arreguin, K., Villanueva, C. J., Tantin, D., Murtaugh, L. C., Evason, K. J., Ducker, G. S., Thummel, C. S. and Rutter, J. (2019). Regulation of tumor initiation by the mitochondrial pyruvate carrier. Cell Metab. PubMed ID: 31813825
Although metabolic adaptations have been demonstrated to be essential for tumor cell proliferation, the metabolic underpinnings of tumor initiation are poorly understood. This study found that the earliest stages of colorectal cancer (CRC) initiation are marked by a glycolytic metabolic signature, including downregulation of the mitochondrial pyruvate carrier (MPC), which couples glycolysis and glucose oxidation through mitochondrial pyruvate import. Genetic studies in Drosophila suggest that this downregulation is required because hyperplasia caused by loss of the Apc or Notch tumor suppressors in intestinal stem cells can be completely blocked by MPC overexpression. Moreover, in two distinct CRC mouse models, loss of Mpc1 prior to a tumorigenic stimulus doubled the frequency of adenoma formation and produced higher grade tumors. MPC loss was associated with a glycolytic metabolic phenotype and increased expression of stem cell markers. These data suggest that changes in cellular pyruvate metabolism are necessary and sufficient to promote cancer initiation.

Wednesday, January 8th - Signaling

Potter, S., Sifers, J., Yocom, E., Blumich, S. L. E., Potter, R., Nadolski, J., Harrison, D. A. and Cooper, R. L. (2019). Effects of inhibiting mTOR with rapamycin on behavior, development, neuromuscular physiology and cardiac function in larval Drosophila. Biol Open 8(11). PubMed ID: 31704693
Rapamycin and other mTOR inhibitors are being heralded as possible treatments for many human ailments. It is currently being utilized clinically as an immunomodulator after transplantation procedures and as a treatment for certain forms of cancer, but it has numerous potential clinical indications. Some studies have shown profound effects on life cycle and muscle physiology, but these issues have not been addressed in an organism undergoing developmental processes. This paper fills this void by examining the effect of mTOR inhibition by rapamycin on several different qualities of larval Drosophila. Various dosages of the compound were fed to second instar larvae. These larvae were monitored for pupae formation to elucidate possible life cycle effects, and a delay to pupation was quantified. Behavioral deficits were documented in rapamycin-treated larvae. Electrophysiological measurements were taken to discern changes in muscle physiology and synaptic signaling (i.e. resting membrane potential, amplitude of excitatory post-synaptic potentials, synaptic facilitation). Pupation delay and effects on behavior that are likely due to synaptic alterations within the central nervous system were discovered in rapamycin-fed larvae. These results allow for several conclusions as to how mTOR inhibition by rapamycin affects a developing organism. This could eventually allow for a more informed decision when using rapamycin and other mTOR inhibitors to treat human diseases, especially in children and adolescents, to account for known side effects.
Banavali, N. K. (2019). The mechanism of cholesterol modification of Hedgehog ligand. J Comput Chem. PubMed ID: 31823413
Hedgehog (Hh) proteins are important components of signal transduction pathways involved in animal development, and their defects are implicated in carcinogenesis. Their N-terminal domain (HhN) acts as a signaling ligand, and their C-terminal domain (HhC) performs an autocatalytic function of cleaving itself away, while adding a cholesterol moiety to HhN. HhC has two sub-domains: a hedgehog/intein (hint) domain that primarily performs the autocatalytic activity, and a sterol-recognition region (SRR) that binds to cholesterol and properly positions it with respect to HhN. The three-dimensional details of this autocatalytic mechanism remain unknown, as does the structure of the precursor Hh protein. In this study, a complete cholesterol-bound precursor form of the Drosophila Hh precursor is modeled using known crystal structures of HhN and the hint domain, and a hypothesized similarity of SRR to an unrelated but similar-sized cholesterol binding protein. The restrained geometries and topology switching (RGATS) strategy is then used to predict atomic-detail pathways for the full autocatalytic reaction starting from the precursor and ending in a cholesterol-linked HhN domain and a cleaved HhC domain. The RGATS explicit solvent simulations indicate the roles of individual HhC residues in facilitating the reaction, which can be confirmed through mutational experiments. These simulations also provide plausible structural models for the N/S acyl transfer intermediate and the product states of this reaction. This study thus provides a good framework for future computational and experimental studies to develop a full structural and dynamic understanding of Hh autoprocessing.
Bagci, H., Sriskandarajah, N., Robert, A., Boulais, J., Elkholi, I. E., Tran, V., Lin, Z. Y., Thibault, M. P., Dube, N., Faubert, D., Hipfner, D. R., Gingras, A. C. and Cote, J. F. (2019). Mapping the proximity interaction network of the Rho-family GTPases reveals signalling pathways and regulatory mechanisms. Nat Cell Biol. PubMed ID: 31871319
Guanine nucleotide exchange factors (RhoGEFs) and GTPase-activating proteins (RhoGAPs) coordinate the activation state of the Rho family of GTPases for binding to effectors. This study exploited proximity-dependent biotinylation to systematically define the Rho family proximity interaction network from 28 baits to produce 9,939 high-confidence proximity interactions in two cell lines. Exploiting the nucleotide states of Rho GTPases, the landscape was revealed of interactions with RhoGEFs and RhoGAPs. Effectors of Rho proteins were systematically defined to reveal candidates for classical and atypical Rho proteins. Optogenetics was used to demonstrate that KIAA0355 (termed GARRE here) is a RAC1 interactor. A functional screen of RHOG candidate effectors identified PLEKHG3 as a promoter of Rac-mediated membrane ruffling downstream of RHOG. Active RHOA was shown to bind the kinase SLK in Drosophila and mammalian cells to promote Ezrin-Radixin-Moesin phosphorylation. These proximity interactions data pave the way for dissecting additional Rho signalling pathways, and the approaches described here are applicable to the Ras family.
Weavers, H., Wood, W. and Martin, P. (2019). Injury activates a dynamic cytoprotective network to confer stress resilience and drive repair. Curr Biol 29(22): 3851-3862 PubMed ID: 31668626
In healthy individuals, injured tissues rapidly repair themselves following damage. Within a healing skin wound, recruited inflammatory cells release a multitude of bacteriocidal factors, including reactive oxygen species (ROS), to eliminate invading pathogens. Paradoxically, while these highly reactive ROS confer resistance to infection, they are also toxic to host tissues and may ultimately delay repair. Repairing tissues have therefore evolved powerful cytoprotective "resilience" machinery to protect against and tolerate this collateral damage. This study used in vivo time-lapse imaging and genetic manipulation in Drosophila to dissect the molecular and cellular mechanisms that drive tissue resilience to wound-induced stress. A dynamic, cross-regulatory network of stress-activated cytoprotective pathways was identified, linking calcium, JNK, Nrf2, and Gadd45, that act to both "shield" tissues from oxidative damage and promote efficient damage repair. Ectopic activation of these pathways confers stress protection to naive tissue, while their inhibition leads to marked delays in wound closure. Strikingly, the induction of cytoprotection is tightly linked to the pathways that initiate the inflammatory response, suggesting evolution of a fail-safe mechanism for tissue protection each time inflammation is triggered. A better understanding of these resilience mechanisms-their identities and precise spatiotemporal regulation-is of major clinical importance for development of therapeutic interventions for all pathologies linked to oxidative stress, including debilitating chronic non-healing wounds.
Wei, Y., Bettedi, L., Ting, C. Y., Kim, K., Zhang, Y., Cai, J. and Lilly, M. A. (2019). The GATOR complex regulates an essential response to meiotic double-stranded breaks in Drosophila. Elife 8. PubMed ID: 31650955
The TORC1 regulator GATOR1/SEACIT controls meiotic entry and early meiotic events in yeast. However, how metabolic pathways influence meiotic progression in metazoans remains poorly understood. This study examined the role of the TORC1 regulators GATOR1 and GATOR2 in the response to meiotic double-stranded breaks (DSB) during Drosophila oogenesis. In mutants of the GATOR2 component mio, meiotic DSBs trigger the constitutive downregulation of TORC1 activity and a permanent arrest in oocyte growth. Conversely, in GATOR1 mutants, high TORC1 activity results in the delayed repair of meiotic DSBs and the hyperactivation of p53. Unexpectedly, it was found that GATOR1 inhibits retrotransposon expression in the presence of meiotic DSBs in a pathway that functions in parallel to p53. Thus, these studies have revealed a link between oocyte metabolism, the repair of meiotic DSBs and retrotransposon expression.
Viswanathan, R., Necakov, A., Trylinski, M., Harish, R. K., Krueger, D., Esposito, E., Schweisguth, F., Neveu, P. and De Renzis, S. (2019). Optogenetic inhibition of Delta reveals digital Notch signalling output during tissue differentiation. EMBO Rep: e47999. PubMed ID: 31668010
Spatio-temporal regulation of signalling pathways plays a key role in generating diverse responses during the development of multicellular organisms. The role of signal dynamics in transferring signalling information in vivo is incompletely understood. This study employed genome engineering in Drosophila melanogaster to generate a functional optogenetic allele of the Notch ligand Delta (opto-Delta), which replaces both copies of the endogenous wild-type locus. Using clonal analysis, it was shown that optogenetic activation blocks Notch activation through cis-inhibition in signal-receiving cells. Signal perturbation in combination with quantitative analysis of a live transcriptional reporter of Notch pathway activity reveals differential tissue- and cell-scale regulatory modes. While at the tissue-level the duration of Notch signalling determines the probability with which a cellular response will occur, in individual cells Notch activation acts through a switch-like mechanism. Thus, time confers regulatory properties to Notch signalling that exhibit integrative digital behaviours during tissue differentiation.

Monday, January 6th - Disease Models

Arnes, M., Alaniz, M. E., Karam, C. S., Cho, J. D., Lopez, G., Javitch, J. A. and Santa-Maria, I. (2019). Role of Tau protein in remodeling of circadian neuronal circuits and sleep. Front Aging Neurosci 11: 320. PubMed ID: 31824299
Multiple neurological, physiological, and behavioral functions are synchronized by circadian clocks into daily rhythms. Neurodegenerative diseases such as Alzheimer's disease and related tauopathies are associated with a decay of circadian rhythms, disruption of sleep patterns, and impaired cognitive function but the mechanisms underlying these alterations are still unclear. Traditional approaches in neurodegeneration research have focused on understanding how pathology impinges on circadian function. Since in Alzheimer's disease and related tauopathies tau proteostasis is compromised, this study sought to understand the role of tau protein in neuronal circadian biology and related behavior. Considering molecular mechanisms underlying circadian rhythms are conserved from Drosophila to humans, advantage was taken of a recently developed tau-deficient Drosophila line to show that loss of tau promotes dysregulation of daily circadian rhythms and sleep patterns. Strikingly, tau deficiency dysregulates the structural plasticity of the small ventral lateral circadian pacemaker neurons by disrupting the temporal cytoskeletal remodeling of its dorsal axonal projections and by inducing a slight increase in the cytoplasmic accumulation of core clock proteins. Taken together, these results suggest that loss of tau function participates in the regulation of circadian rhythms by modulating the correct operation and connectivity of core circadian networks and related behavior.
Wen, D. T., Zheng, L., Li, J. X., Cheng, D., Liu, Y., Lu, K. and Hou, W. Q. (2019). Endurance exercise resistance to lipotoxic cardiomyopathy is associated with cardiac NAD(+)/dSIR2/PGC-1alpha pathway activation in old Drosophila. Biol Open 8(10). PubMed ID: 31624074
Lipotoxic cardiomyopathy is caused by excessive lipid accumulation in myocardial cells and it is a form of cardiac dysfunction. Cardiac PGC-1alpha overexpression prevents lipotoxic cardiomyopathy induced by a high-fat diet (HFD). The level of NAD(+) and Sir2 expression upregulate the transcriptional activity of PGC-1alpha. Exercise improves cardiac NAD(+) level and PGC-1alpha activity. However, the relationship between exercise, NAD(+)/dSIR2/PGC-1alpha pathway and lipotoxic cardiomyopathy remains unknown. In this study, flies were fed a HFD and exercised. The heart dSir2 gene was specifically expressed or knocked down by UAS/hand-Gal4 system. The results showed that either a HFD or dSir2 knockdown remarkably increased cardiac TG level and dFAS expression, reduced heart fractional shortening and diastolic diameter, increased arrhythmia index, and decreased heart NAD(+) level, dSIR2 protein, dSir2 and PGC-1alpha expression levels. Contrarily, either exercise or dSir2 overexpression remarkably reduced heart TG level, dFAS expression and arrhythmia index, and notably increased heart fractional shortening, diastolic diameter, NAD(+) level, dSIR2 level, and heart dSir2 and PGC-1alpha expression. Therefore, it is declared that exercise training could improve lipotoxic cardiomyopathy induced by a HFD or cardiac dSir2 knockdown in old Drosophila. The NAD(+)/dSIR2/PGC-1alpha pathway activation was an important molecular mechanism of exercise resistance against lipotoxic cardiomyopathy.
Tain, L. S., Jain, C., Nespital, T., Froehlich, J., Hinze, Y., Gronke, S. and Partridge, L. (2019). Longevity in response to lowered insulin signaling requires glycine N-methyltransferase-dependent spermidine production. Aging Cell: e13043. PubMed ID: 31721422
Reduced insulin/IGF signaling (IIS) extends lifespan in multiple organisms. Different processes in different tissues mediate this lifespan extension, with a set of interplays that remain unclear. This study shows that, in Drosophila, reduced IIS activity modulates methionine metabolism, through tissue-specific regulation of glycine N-methyltransferase (Gnmt), and that this regulation is required for full IIS-mediated longevity. Furthermore, fat body-specific expression of Gnmt was sufficient to extend lifespan. Targeted metabolomics showed that reducing IIS activity led to a Gnmt-dependent increase in spermidine levels. It was also shown that both spermidine treatment and reduced IIS activity are sufficient to extend the lifespan of Drosophila, but only in the presence of Gnmt. This extension of lifespan was associated with increased levels of autophagy. Finally, this study found that increased expression of Gnmt occurs in the liver of liver-specific IRS1 KO mice and is thus an evolutionarily conserved response to reduced IIS. The discovery of Gnmt and spermidine as tissue-specific modulators of IIS-mediated longevity may aid in developing future therapeutic treatments to ameliorate aging and prevent disease.
Arnes, M., Romero, N., Casas-Tinto, S., Acebes, A. and Ferrus, A. (2019). PI3K activation prevents Abeta42-induced synapse loss and favors insoluble amyloid deposits formation. Mol Biol Cell: mbcE19050303. PubMed ID: 31877058
Excess of Abeta42 peptide is considered a hallmark of the disease. This study expressed the human Abeta42 peptide to assay the neuroprotective effects of PI3K in adult Drosophila melanogaster. The neuronal expression of the human peptide elicits progressive toxicity in the adult fly. The pathological traits include reduced axonal transport, synapse loss, defective climbing ability and olfactory perception, as well as lifespan reduction. The Abeta42-dependent synapse decay does not involve transcriptional changes in the core synaptic protein encoding genes: bruchpilot, liprin and synaptobrevin. All toxicity features, however, are suppressed by the co-expression of PI3K. Moreover, PI3K activation induces a significant increase of 6E10 and Thioflavin-positive amyloid deposits. Mechanistically, it is suggested that Abeta42-Ser26 could be a candidate residue for direct or indirect phosphorylation by PI3K. Along with these in vivo experiments this study further analyzed Abeta42 toxicity and its suppression by PI3K activation in in vitro assays with SH-SY5Y human neuroblastoma cell cultures, where Abeta42 aggregation into large insoluble deposits is reproduced. Finally, it was shown that the Abeta42 toxicity syndrome includes the transcriptional shut down of PI3K expression. Taken together, these results uncover a potential novel pharmacological strategy against this disease through the restoration of PI3K activity.
Sim, J. P. L., Ziyin, W., Basil, A. H., Lin, S., Chen, Z., Zhang, C., Zeng, L., Cai, Y. and Lim, K. L. (2019). Identification of PP2A and S6 kinase as modifiers of Leucine-rich repeat kinase-induced neurotoxicity. Neuromolecular Med. PubMed ID: 31664682
Mutations in LRRK2 are currently recognized as the most common monogenetic cause of Parkinsonism. The elevation of kinase activity of LRRK2 that frequently accompanies its mutations is widely thought to contribute to its toxicity. Accordingly, many groups have developed LRRK2-specific kinase inhibitors as a potential therapeutic strategy. Given that protein phosphorylation is a reversible event, this study sought to elucidate the phosphatase(s) that can reverse LRRK2-mediated phosphorylation, with the view that targeting this phosphatase(s) may similarly be beneficial. Using an unbiased RNAi phosphatase screen conducted in a Drosophila LRRK2 model, PP2A was identified as a genetic modulator of LRRK2-induced neurotoxicity. Further, ribosomal S6 kinase (S6K), a target of PP2A, was also identified as a novel regulator of LRRK2 function. Finally, modulation of PP2A or S6K activities were shown to ameliorate LRRK2-associated disease phenotype in Drosophila.
Auxerre-Plantie, E., Nakamori, M., Renaud, Y., Huguet, A., Choquet, C., Dondi, C., Miquerol, L., Takahashi, M. P., Gourdon, G., Junion, G., Jagla, T., Zmojdzian, M. and Jagla, K. (2019). Straightjacket/alpha2delta3 deregulation is associated with cardiac conduction defects in myotonic dystrophy type 1. Elife 8. PubMed ID: 31829940
Cardiac conduction defects decrease life expectancy in myotonic dystrophy type 1 (DM1), a CTG repeat disorder involving misbalance between two RNA binding factors, MBNL1 and CELF1. However, how DM1 condition translates into conduction disorders remains poorly understood. This study simulated MBNL1 and CELF1 misbalance in the Drosophila heart and performed TU-tagging-based RNAseq of cardiac cells. Deregulations of several genes controlling cellular calcium levels were detected, including increased expression of straightjacket/ alpha2delta3, which encodes a regulatory subunit of a voltage-gated calcium channel. Straightjacket overexpression in the fly heart leads to asynchronous heartbeat, a hallmark of abnormal conduction, whereas cardiac straightjacket knockdown improves these symptoms in DM1 fly models. It was also shown that ventricular alpha2delta3 expression is significantly elevated in ventricular muscles from DM1 patients with conduction defects. These findings suggest that reducing ventricular straightjacket/alpha2delta3 levels could offer a strategy to prevent conduction defects in DM1.

Friday, January 3rd - Cytoskeleton and Junctions

Butts, A. R., Ojelade, S. A., Pronovost, E. D., Seguin, A., Merrill, C. B., Rodan, A. R. and Rothenfluh, A. (2019). Altered actin filament dynamics in the Drosophila mushroom bodies lead to fast acquisition of alcohol consumption preference. J Neurosci. PubMed ID: 31558618
Alcohol use is highly prevalent in the United States and across the world, and every year millions of people suffer from alcohol use disorders (AUDs). While the genetic contribution to developing AUDs is estimated to be 50-60%, many of the underlying molecular mechanisms remain unclear. Previous studies have revealed that Drosophila melanogaster lacking RhoGAP18B and Ras Suppressor 1 (Rsu1) display reduced sensitivity to ethanol-induced sedation. Both Rsu1 and RhoGAP18B are negative regulators of the small Rho-family GTPase, Rac1, a modulator of actin dynamics. This study investigated the role of Rac1 and its downstream target, the actin-severing protein cofilin, in alcohol consumption preference. These two regulators of actin dynamics can alter male experience-dependent alcohol preference in a bidirectional manner: expressing either activated Rac1 or dominant-negative cofilin in the mushroom bodies (MB) abolishes experience-dependent alcohol preference. Conversely, dominant-negative Rac1 or activated cofilin MB expression lead to faster acquisition of alcohol preference. These data show that Rac1 and cofilin activity are key to determining the rate of acquisition of alcohol preference, revealing a critical role of actin dynamics regulation in the development of voluntary self-administration in Drosophila
Qu, Y., Hahn, I., Lees, M., Parkin, J., Voelzmann, A., Dorey, K., Rathbone, A., Friel, C. T., Allan, V. J., Okenve-Ramos, P., Sanchez-Soriano, N. and Prokop, A. (2019). Efa6 protects axons and regulates their growth and branching by inhibiting microtubule polymerisation at the cortex. Elife 8. PubMed ID: 31718774
Cortical collapse factors affect microtubule (MT) dynamics at the plasma membrane. They play important roles in neurons. How cortical collapse factors influence axon growth is little understood. This study focussed on the function of Drosophila Efa6 in experimentally and genetically amenable fly neurons. First, it was shown that Drosophila Efa6 can inhibit MTs directly without interacting molecules via an N-terminal 18 amino acid motif (MT elimination domain/MTED) that binds tubulin and inhibits microtubule growth in vitro and cells. If N-terminal MTED-containing fragments are in the cytoplasm they abolish entire microtubule networks of mouse fibroblasts and whole axons of fly neurons. Full-length Efa6 is membrane-attached, hence primarily blocks MTs in the periphery of fibroblasts, and explorative MTs that have left axonal bundles in neurons. Accordingly, loss of Efa6 causes an increase of explorative MTs: in growth cones they enhance axon growth, in axon shafts they cause excessive branching, as well as atrophy through perturbations of MT bundles. Efa6 over-expression causes the opposite phenotypes. Taken together, this work conceptually links molecular and sub-cellular functions of cortical collapse factors to axon growth regulation and reveals new roles in axon branching and in the prevention of axonal atrophy. Furthermore, the MTED delivers a promising tool that can be used to inhibit MTs in a compartmentalised fashion when fusing it to specifically localising protein domains.
Melkov, A., Baskar, R., Shachal, R., Alcalay, Y. and Abdu, U. (2019). The organization of Golgi in Drosophila bristles requires microtubule motor protein function and a properly organized microtubule array. PLoS One 14(10): e0223174. PubMed ID: 31577833
This study used highly elongated Drosophila bristle cells to dissect the role of dynein heavy chain (Dhc64C) in Golgi organization. Whereas in the bristle "somal" region Golgi units are composed of cis-, medial, and trans-Golgi compartments ("complete Golgi"), the bristle shaft contains Golgi satellites that lack the trans-Golgi compartment (hereafter referred to as "incomplete Golgi") and which are static and localized at the base area. However, in Dhc64C mutants, the entire bristle shaft was filled with complete Golgi units containing ectopic trans-Golgi components. To further understand Golgi bristle organization, the roles of microtubule (MT) polarity and the Dhc-opposing motor, kinesin heavy chain (Khc), were tested. Surprisingly, in Khc and Ik2Dominant-negative (DN) flies in which the polarized organization of MTs is affected, the bristle shaft was filled with complete Golgi, similarly to what is seen in Dhc64C flies. Thus, this study demonstrated that MTs and the motor proteins Dhc and Khc are required for bristle Golgi organization. However, the fact that both Dhc64C and Khc flies showed similar Golgi defects calls for an additional work to elucidate the molecular mechanism describing why these factors are required for bristle Golgi organization.
Bonello, T. T., Choi, W. and Peifer, M. (2019). Scribble and Discs-large direct initial assembly and positioning of adherens junctions during the establishment of apical-basal polarity. Development 146(22). PubMed ID: 31628110
Apical-basal polarity is a fundamental property of animal tissues. Drosophila embryos provide an outstanding model for defining mechanisms that initiate and maintain polarity. Polarity is initiated during cellularization, when cell-cell adherens junctions are positioned at the future boundary of apical and basolateral domains. Polarity maintenance then involves complementary and antagonistic interplay between apical and basal polarity complexes. The Scribble/Dlg module is well-known for promoting basolateral identity during polarity maintenance. This study reports a surprising role for Scribble/Dlg in polarity initiation, placing it near the top of the network-positioning adherens junctions. Scribble and Dlg are enriched in nascent adherens junctions, are essential for adherens junction positioning and supermolecular assembly, and also play a role in basal junction assembly. The hypotheses were tested for the underlying mechanisms, exploring potential effects on protein trafficking, cytoskeletal polarity or Par-1 localization/function. The data suggest that the Scribble/Dlg module plays multiple roles in polarity initiation. Different domains of Scribble contribute to these distinct roles. Together, these data reveal novel roles for Scribble/Dlg as master scaffolds regulating assembly of distinct junctional complexes at different times and places.
Kobb, A. B., Rothenberg, K. E. and Fernandez-Gonzalez, R. (2019). Actin and myosin dynamics are independent during Drosophila embryonic wound repair. Mol Biol Cell: mbcE18110703. PubMed ID: 31553671
Collective cell movements play a central role in embryonic development, tissue repair, and metastatic disease. Cell movements are often coordinated by supracellular networks formed by the cytoskeletal protein actin and the molecular motor non-muscle myosin II. During wound closure in the embryonic epidermis, the cells around the wound migrate collectively into the damaged region. In Drosophila embryos, mechanical tension stabilizes myosin at the wound edge, facilitating the assembly of a supracellular myosin cable around the wound that coordinates cell migration. This study shows that actin is also stabilized at the wound edge. However, loss of tension or myosin activity does not affect the dynamics of actin at the wound margin. Conversely, pharmacological stabilization of actin does not affect myosin levels or dynamics around the wound. Together, these data suggest that actin and myosin regulation during embryonic wound closure in Drosophila are largely independent, thus conferring robustness to the embryonic response to wounds.
Yolland, L., Burki, M., Marcotti, S., Luchici, A., Kenny, F. N., Davis, J. R., Serna-Morales, E., Muller, J., Sixt, M., Davidson, A., Wood, W., Schumacher, L. J., Endres, R. G., Miodownik, M. and Stramer, B. M. (2019). Persistent and polarized global actin flow is essential for directionality during cell migration. Nat Cell Biol 21(11): 1370-1381. PubMed ID: 31685997
Cell migration is hypothesized to involve a cycle of behaviours beginning with leading edge extension. However, recent evidence suggests that the leading edge may be dispensable for migration, raising the question of what actually controls cell directionality. This study exploits the embryonic migration of Drosophila macrophages to bridge the different temporal scales of the behaviours controlling motility. This approach reveals that edge fluctuations during random motility are not persistent and are weakly correlated with motion. In contrast, flow of the actin network behind the leading edge is highly persistent. Quantification of actin flow structure during migration reveals a stable organization and asymmetry in the cell-wide flowfield that strongly correlates with cell directionality. This organization is regulated by a gradient of actin network compression and destruction, which is controlled by myosin contraction and cofilin-mediated disassembly. It is this stable actin-flow polarity, which integrates rapid fluctuations of the leading edge, that controls inherent cellular persistence.

Thursday, January 2nd - Adult Neural Development and Function

Wei, H., Kyung, H. Y., Kim, P. J. and Desplan, C. (2019). The diversity of lobula plate tangential cells (LPTCs) in the Drosophila motion vision system. J Comp Physiol A Neuroethol Sens Neural Behav Physiol. PubMed ID: 31709462
To navigate through the environment, animals rely on visual feedback to control their movements relative to their surroundings. In dipteran flies, visual feedback is provided by the wide-field motion-sensitive neurons in the visual system called lobula plate tangential cells (LPTCs). Understanding the role of LPTCs in fly behaviors can address many fundamental questions on how sensory circuits guide behaviors. The blowfly was estimated to have ~ 60 LPTCs, but only a few have been identified in Drosophila. A Gal4 driver screen was conducted and five LPTC subtypes were identified in Drosophila, based on their morphological characteristics: LPTCs have large arborizations in the lobula plate and project to the central brain. Their morphologies were compared to the blowfly LPTCs and were named after the most similar blowfly cells: CH, H1, H2, FD1 and FD3, and V1. Their pre- and post-synaptic organizations, as well as their neurotransmitter profiles, were further characterized. These anatomical features largely agree with the anatomy and function of their likely blowfly counterparts. Nevertheless, several anatomical details indicate the Drosophila LPTCs may have more complex functions. This characterization of these five LPTCs in Drosophila will facilitate further functional studies to understand their roles in the visual circuits that instruct fly behaviors.
Shinomiya, K., Horne, J. A., McLin, S., Wiederman, M., Nern, A., Plaza, S. M. and Meinertzhagen, I. A. (2019). The organization of the second optic chiasm of the Drosophila optic lobe. Front Neural Circuits 13: 65. PubMed ID: 31680879
Visual pathways from the compound eye of an insect relay to four neuropils, successively the lamina, medulla, lobula, and lobula plate in the underlying optic lobe. The medulla, lobula, and lobula plate are interconnected by the complex second optic chiasm, through which the anteroposterior axis undergoes an inversion between the medulla and lobula. By densely reconstructing axon trajectories using a volumetric scanning electron microscopy technique, this study reveals the three-dimensional structure of the second optic chiasm of Drosophila comprising interleaving bundles and sheets of axons insulated from each other by glial sheaths. These axon bundles invert their horizontal sequence in passing between the medulla and lobula. ~19,500 cells per hemisphere, about two thirds of the optic lobe neurons, contribute to the second chiasm, most being Tm cells, with an estimated additional 2,780 T4 and T5 cells each. The chiasm mostly comprises axons and cell body fibers, but also a few synaptic elements. It is proposed that a chiasmal structure between the neuropils is potentially advantageous for processing complex visual information in parallel. The EM reconstruction suggest that the projection patterns of the neurons comprising the chiasm may be determined by the proliferation centers from which the neurons develop. Such a complex wiring pattern could only have arisen in several evolutionary steps.
Schoborg, T. A., Smith, S. L., Smith, L. N., Morris, H. D. and Rusan, N. M. (2019). Micro-computed tomography as a platform for exploring Drosophila development. Development. PubMed ID: 31722883
Understanding how events at the molecular and cellular scales contribute to tissue form and function is key to uncovering mechanisms driving animal development, physiology, and disease. Elucidating these mechanisms has been enhanced through the study of model organisms and use of sophisticated genetic, biochemical, and imaging tools. This paper presents an accessible method for non-invasive imaging of Drosophila melanogaster at high resolution using micro-computed tomography (micro-CT). Rapid processing of intact animals at any developmental stage, provides precise quantitative assessment of tissue size and morphology, and permits analysis of inter-organ relationships. Micro-CT imaging was used to study growth defects in the Drosophila brain through the characterization of Abnormal spindle (asp) and WD Repeat Domain 62 (wdr62), orthologs of the two most commonly mutated genes in human microcephaly patients. This work demonstrates the power of combining micro-CT with traditional genetic, cellular, and developmental biology tools available in model organisms to address novel biological mechanisms that control animal development and disease.
Senapati, B., Tsao, C. H., Juan, Y. A., Chiu, T. H., Wu, C. L., Waddell, S. and Lin, S. (2019). A neural mechanism for deprivation state-specific expression of relevant memories in Drosophila. Nat Neurosci. PubMed ID: 31659341
Motivational states modulate how animals value sensory stimuli and engage in goal-directed behaviors. The motivational states of thirst and hunger are represented in the brain by shared and unique neuromodulatory systems. However, it is unclear how such systems interact to coordinate the expression of appropriate state-specific behavior. The activity of two brain neurons expressing leucokinin neuropeptide is elevated in thirsty and hungry flies, and leucokinin release is necessary for state-dependent expression of water- and sugar-seeking memories. Leucokinin inhibits two types of mushroom-body-innervating dopaminergic neurons (DANs) to promote thirst-specific water memory expression, whereas it activates other mushroom-body-innervating DANs to facilitate hunger-dependent sugar memory expression. Selection of hunger- or thirst-appropriate memory emerges from competition between leucokinin and other neuromodulatory hunger signals at the level of the DANs. Therefore, coordinated modulation of the dopaminergic system allows flies to prioritize the expression of the relevant state-dependent motivated behavior.
Sims, D. W., Humphries, N. E., Hu, N., Medan, V. and Berni, J. (2019). Optimal searching behaviour generated intrinsically by the central pattern generator for locomotion. Elife 8. PubMed ID: 31674911
Efficient searching for resources such as food by animals is key to their survival. It has been proposed that diverse animals from insects to sharks and humans adopt searching patterns that resemble a simple Levy random walk, which is theoretically optimal for 'blind foragers' to locate sparse, patchy resources. To test if such patterns are generated intrinsically, or arise via environmental interactions, free-moving Drosophila larvae were tracked with (and without) blocked synaptic activity in the brain, suboesophageal ganglion (SOG) and sensory neurons. In brain-blocked larvae, extended substrate exploration was found to emerge as multi-scale movement paths similar to truncated Levy walks. Strikingly, power-law exponents of brain/SOG/sensory-blocked larvae averaged 1.96, close to a theoretical optimum (micro congruent with 2.0) for locating sparse resources. Thus, efficient spatial exploration can emerge from autonomous patterns in neural activity. These results provide the strongest evidence so far for the intrinsic generation of Levy-like movement patterns.
Thane, M., Viswanathan, V., Meyer, T. C., Paisios, E. and Schleyer, M. (2019). Modulations of microbehaviour by associative memory strength in Drosophila larvae. PLoS One 14(10): e0224154. PubMed ID: 31634372
Finding food is a vital skill and a constant task for any animal, and associative learning of food-predicting cues gives an advantage in this daily struggle. This study investigated what impact the strength of an associative odour-sugar memory has on the microbehaviour of Drosophila larvae. Larvae were found to form stronger memories with increasing concentrations of sugar or odour, and these stronger memories manifest themselves in stronger modulations of two aspects of larval microbehaviour, the rate and the direction of lateral reorientation manoeuvres (so-called head casts). These two modulations of larval behaviour are found to be correlated to each other in every experiment performed, in line with a model that assumes that both modulations are controlled by a common motor output. These analyses can guide future research into the neuronal circuits underlying the translation of associative memories of different strength into behaviour, and may help to understand how these processes are organised in more complex systems.
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