What's hot today:
Current papers in developmental biology and gene function
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Monday May 31st, 2021 - Evolution
What's hot today
April 2019 Yue, L., Cao, L. J., Chen, J. C., Gong, Y. J., Lin, Y. H., Hoffmann, A. A. and Wei, S. J. (2021). Low levels of genetic differentiation with isolation by geography and environment in populations of Drosophila melanogaster from across China. Heredity (Edinb). PubMed ID: 33686193
The fruit fly, Drosophila melanogaster, is a model species in evolutionary studies. However, population processes of this species in East Asia are poorly studied. This study examined the population genetic structure of D. melanogaster across China. There were 14 mitochondrial haplotypes with 10 unique ones out of 23 known from around the globe. Pairwise F(ST) values estimated from 15 novel microsatellites ranged from 0 to 0.11, with geographically isolated populations showing the highest level of genetic uniqueness. STRUCTURE analysis identified high levels of admixture at both the individual and population levels. Mantel tests indicated a strong association between genetic distance and geographical distance as well as environmental distance. Full redundancy analysis (RDA) showed that independent effects of environmental conditions and geography accounted for 62.10% and 31.58% of the total explained genetic variance, respectively. When geographic variables were constrained in a partial RDA analysis, the environmental variables bio2 (mean diurnal air temperature range), bio13 (precipitation of the wettest month), and bio15 (precipitation seasonality) were correlated with genetic distance. This study suggests that demographic history, geographical isolation, and environmental factors have together shaped the population genetic structure of D. melanogaster after its introduction into China. Bath, E., Edmunds, D., Norman, J., Atkins, C., Harper, L., Rostant, W. G., Chapman, T., Wigby, S. and Perry, J. C. (2021). Sex ratio and the evolution of aggression in fruit flies. Proc Biol Sci 288(1947): 20203053. PubMed ID: 33726599
Aggressive behaviours are among the most striking displayed by animals, and aggression strongly impacts fitness in many species. Aggression varies plastically in response to the social environment, but direct tests of how aggression evolves in response to intra-sexual competition are lacking. This study investigated how aggression in both sexes evolves in response to the competitive environment, using populations of Drosophila melanogaster that were experimentally evolved under female-biased, equal, and male-biased sex ratios. After evolution in a female-biased environment-with less male competition for mates-males fought less often on food patches, although the total frequency and duration of aggressive behaviour did not change. In females, evolution in a female-biased environment-where female competition for resources is higher-resulted in more frequent aggressive interactions among mated females, along with a greater increase in post-mating aggression. These changes in female aggression could not be attributed solely to evolution either in females or in male stimulation of female aggression, suggesting that coevolved interactions between the sexes determine female post-mating aggression. Evidence was found consistent with a positive genetic correlation for aggression between males and females, suggesting a shared genetic basis. This study demonstrates the experimental evolution of a behaviour strongly linked to fitness, and the potential for the social environment to shape the evolution of contest behaviours.
Sarikaya, D. P., Rickelton, K., Cridland, J. M., Hatmaker, R., Sheehy, H. K., Davis, S., Khan, N., Kochummen, A. and Begun, D. J. (2021). Sex and tissue-specific evolution of developmental plasticity in Drosophila melanogaster. Ecol Evol 11(3): 1334-1341. PubMed ID: 33598134
Developmental plasticity influences the size of adult tissues in insects. Tissues can have unique responses to environmental perturbation during development; however, the prevalence of within species evolution of tissue-specific developmental plasticity remains unclear. To address this, the effects of temperature and nutrition were studied on wing and femur size in D. melanogaster populations from a temperate and tropical region. Wings were more sensitive to temperature, while wings and femurs were equally responsive to nutrition in both populations and sexes. The temperate population was larger under all conditions, except for femurs of starved females. In line with this, greater femur size plasticity was observed in response to starvation in temperate females, leading to differences in sexual dimorphism between populations such that the slope of the reaction norm of sexual dimorphism in the tropical population was double that of the temperate population. Lastly, a significant trend was observed for steeper slopes of reaction norms in temperate than in tropical females, but not in males. These findings highlight that plasticity divergence between populations can evolve heterogeneously across sexes and tissues and that nutritional plasticity can alter sexual dimorphism in D. melanogaster. Sztepanacz, J. L. and Houle, D. (2021). Allometry constrains the evolution of sexual dimorphism in Drosophila across 33 million years of divergence. Evolution. PubMed ID: 33638384
Sexual dimorphism is widely viewed as adaptive, reflecting the evolution of males and females towards divergent fitness optima. Its evolution, however, may often be constrained by the shared genetic architecture of the sexes, and by allometry. This study investigated the evolution of sexual size dimorphism, shape dimorphism, and their allometric relationship, in the wings of 82 taxa in the family Drosophilidae which have been diverging for at least 33 million years. Shape dimorphism among species was remarkably similar, with males characterized by longer thinner wings than females. There was, however, quantitative variation among species in both size and shape dimorphism, with evidence that they have adapted to different evolutionary optima in different clades on timescales of about 10 million years. Within species, shape dimorphism was predicted by size, and among species, there was a strong relationship between size dimorphism and shape dimorphism. Allometry constrained the evolution of shape dimorphism for the two most variable traits studied, but dimorphism was evolutionary labile in other traits. The keys for disentangling alternative explanations for dimorphism evolution are studies of natural and sexual selection, together with a deeper understanding of how microevolutionary parameters of evolvability relate to macroevolutionary patterns of divergence.
Cheng, C. and Kirkpatrick, M. (2021). Molecular evolution and the decline of purifying selection with age. Nat Commun 12(1): 2657. PubMed ID: 33976227
Life history theory predicts that the intensity of selection declines with age, and this trend should impact how genes expressed at different ages evolve. This study finds consistent relationships between a gene's age of expression and patterns of molecular evolution in two mammals (the human Homo sapiens and the mouse Mus musculus) and two insects (the malaria mosquito Anopheles gambiae and the fruit fly Drosophila melanogaster). When expressed later in life, genes fix nonsynonymous mutations more frequently, are more polymorphic for nonsynonymous mutations, and have shorter evolutionary lifespans, relative to those expressed early. The latter pattern is explained by a simple evolutionary model. Further, early-expressed genes tend to be enriched in similar gene ontology terms across species, while late-expressed genes show no such consistency. In humans, late-expressed genes are more likely to be linked to cancer and to segregate for dominant disease-causing mutations. Last, the effective strength of selection (N(e) s) decreases and the fraction of beneficial mutations increases with a gene's age of expression. These results are consistent with the diminishing efficacy of purifying selection with age, as proposed by Medawar's classic hypothesis for the evolution of senescence, and provide links between life history theory and molecular evolution. Betancourt, N. J., Rajpurohit, S., Durmaz, E., Fabian, D. K., Kapun, M., Flatt, T. and Schmidt, P. (2021). Allelic polymorphism at foxo contributes to local adaptation in Drosophila melanogaster. Mol Ecol. PubMed ID: 33914989
The insulin/insulin-like growth factor signaling pathway has been hypothesized as a major determinant of life-history profiles that vary adaptively in natural populations. In Drosophila melanogaster, multiple components of this pathway vary predictably with latitude; this includes foxo, a conserved gene that regulates insulin signaling and has pleiotropic effects on a variety of fitness-associated traits. It was hypothesized that allelic variation at foxo contributes to genetic variance for size-related traits that vary adaptively with latitude. Patterns of variation were examined among natural populations along a latitudinal transect in the eastern United States; thorax length, wing area, wing loading, and starvation tolerance were found to exhibit significant latitudinal clines for both males and females but that development time does not vary predictably with latitude. Recombinant outbred populations were generated, naturally occurring allelic variation at foxo, which exhibits stronger clinality than expected, were shown to be associated with the same traits that vary with latitude in the natural populations. These results suggest that allelic variation at foxo contributes to adaptive patterns of life-history variation in natural populations of this genetic model.
Friday, May 28th - Synapse and vesicles
Ramirez, J., Morales, M., Osinalde, N., Martinez-Padron, I., Mayor, U. and Ferrus, A. (2021). The ubiquitin ligase Ariadne-1 regulates neurotransmitter release via ubiquitination of NSF. J Biol Chem: 100408. PubMed ID: 33581113
Ariadne-1 (Ari-1) is an E3 ubiquitin-ligase essential for neuronal development, but whose neuronal substrates are yet to be identified. To search for putative Ari-1 substrates, this study used an in vivo ubiquitin biotinylation strategy coupled to quantitative proteomics of Drosophila heads. Sixteen candidates were identified that met the established criteria: a significant change of at least two-fold increase on ubiquitination, with at least two unique peptides identified. Amongst those candidates, Comatose (Comt), the homologue of the N-ethylmaleimide sensitive factor (NSF), which is involved in neurotransmitter release, was identified. Using a pulldown approach that relies on the overexpression and stringent isolation of a GFP-fused construct, Comt/NSF was validated to be an ubiquitination substrate of Ari-1 in fly neurons, resulting in the preferential monoubiquitination of Comt/NSF. The possible functional relevance of this modification was tested using Ari-1 loss of function mutants, which displayed a lower rate of spontaneous neurotransmitter release due to failures at the pre-synaptic side. By contrast, evoked release in Ari-1 mutants was enhanced compared to controls in a Ca(2+) dependent manner without modifications in the number of active zones, indicating that the probability of release per synapse is increased in these mutants. This phenotype distinction between spontaneous versus evoked release suggests that NSF activity may discriminate between these two types of vesicle fusion. These results thus provide a mechanism to regulate NSF activity in the synapse through Ari-1-dependent ubiquitination. Florian, J. R., DeMatte, S. J., Sweeder, D. M., Ordway, R. W. and Kawasaki, F. (2021). Genetic analysis of the Drosophila ESCRT-III complex protein, VPS24, reveals a novel function in lysosome homeostasis. PLoS One 16(5): e0251184. PubMed ID: 33956855
The ESCRT pathway is evolutionarily conserved across eukaryotes and plays key roles in a variety of membrane remodeling processes. A new Drosophila mutant recovered in a forward genetic screens for synaptic transmission mutants mapped to the vps24 gene encoding a subunit of the ESCRT-III complex. Molecular characterization indicated a loss of VPS24 function, however the mutant is viable and thus loss of VPS24 may be studied in a developed multicellular organism. The mutant exhibits deficits in locomotion and lifespan and, notably, these phenotypes are rescued by neuronal expression of wild-type VPS24. At the cellular level, neuronal and muscle cells exhibit marked expansion of a ubiquitin-positive lysosomal compartment, as well as accumulation of autophagic intermediates, and these phenotypes are rescued cell-autonomously. Moreover, VPS24 expression in glia suppressed the mutant phenotype in muscle, indicating a cell-nonautonomous function for VPS24 in protective intercellular signaling. Ultrastructural analysis of neurons and muscle indicated marked accumulation of the lysosomal compartment in the vps24 mutant. In the neuronal cell body, this included characteristic lysosomal structures associated with an expansive membrane compartment with a striking tubular network morphology. These findings further define the in vivo roles of VPS24 and the ESCRT pathway in lysosome homeostasis and their potential contributions to neurodegenerative diseases characterized by defective ESCRT or lysosome function.
Bhimreddy, M., Rushton, E., Kopke, D. L. and Broadie, K. (2021). Secreted C-type lectin regulation of neuromuscular junction synaptic vesicle dynamics modulates coordinated movement. J Cell Sci 134(9). PubMed ID: 33973638
The synaptic cleft manifests enriched glycosylation, with structured glycans coordinating signaling between presynaptic and postsynaptic cells. Glycosylated signaling ligands orchestrating communication are tightly regulated by secreted glycan-binding lectins. Using the Drosophila neuromuscular junction (NMJ) as a model glutamatergic synapse, this study identified a new Ca2+-binding (C-type) lectin, Lectin-galC1 (LGC1), which modulates presynaptic function and neurotransmission strength. LGC1 is enriched in motoneuron presynaptic boutons and secreted into the NMJ extracellular synaptomatrix. LGC1 limits locomotor peristalsis and coordinated movement speed, with a specific requirement for synaptic function, but not NMJ architecture. LGC1 controls neurotransmission strength by limiting presynaptic active zone (AZ) and postsynaptic glutamate receptor (GluR) aligned synapse number, reducing both spontaneous and stimulation-evoked synaptic vesicle (SV) release, and capping SV cycling rate. During high-frequency stimulation (HFS), mutants have faster synaptic depression and impaired recovery while replenishing depleted SV pools. Although LGC1 removal increases the number of glutamatergic synapses, it was found that LGC1-null mutants exhibit decreased SV density within presynaptic boutons, particularly SV pools at presynaptic active zones. Thus, LGC1 regulates NMJ neurotransmission to modulate coordinated movement. Saito, M., Nakayama, M., Fujita, K., Uchida, A., Yano, H., Goto, S., Okazawa, H. and Sone, M. (2021). Role of the Drosophila YATA protein in the proper subcellular localization of COPI revealed by in vivo analysis. Genes Genet Syst. PubMed ID: 33583916
yata mutants of Drosophila melanogaster exhibit phenotypes including progressive brain shrinkage, developmental abnormalities and shortened lifespan, whereas in mammals, null mutations of the yata ortholog Scyl1 result in motor neuron degeneration. yata mutation also causes defects in the anterograde intracellular trafficking of a subset of proteins including APPL, which is the Drosophila ortholog of mammalian APP, a causative molecule in Alzheimer's disease. SCYL1 binds and regulates the function of coat protein complex I (COPI) in secretory vesicles. This study reveals a role for the Drosophila YATA protein in the proper localization of COPI. Immunohistochemical analyses performed using confocal microscopy and structured illumination microscopy showed that YATA colocalizes with COPI and GM130, a cis-Golgi marker. Analyses using transgenically expressed YATA with a modified N-terminal sequence revealed that the N-terminal portion of YATA is required for the proper subcellular localization of YATA. Analysis using transgenically expressed YATA proteins in which the C-terminal sequence was modified revealed a function for the C-terminal portion of YATA in the subcellular localization of COPI. Notably, when YATA was mislocalized, it also caused the mislocalization of COPI, indicating that YATA plays a role in directing COPI to the proper subcellular site. Moreover, when both YATA and COPI were mislocalized, the staining pattern of GM130 revealed Golgi with abnormal elongated shapes. Thus, these in vivo data indicate that YATA plays a role in the proper subcellular localization of COPI.
Sidisky, J. M., Weaver, D., Hussain, S., Okumus, M., Caratenuto, R. and Babcock, D. (2021). Mayday sustains trans-synaptic BMP signaling required for synaptic maintenance with age. Elife 10. PubMed ID: 33667157
Maintaining synaptic structure and function over time is vital for overall nervous system function and survival. The processes that underly synaptic development are well understood. However, the mechanisms responsible for sustaining synapses throughout the lifespan of an organism are poorly understood. This study demonstrates that a previously uncharacterized gene, CG31475, regulates synaptic maintenance in adult Drosophila NMJs. CG31475 was named mayday due to the progressive loss of flight ability and synapse architecture with age. Mayday is functionally homologous to the human protein Cab45, which sorts secretory cargo from the Trans Golgi Network (TGN). This study finds that Mayday is required to maintain trans-synaptic BMP signaling at adult NMJs in order to sustain proper synaptic structure and function. Finally, this study shows that mutations in mayday result in the loss of both presynaptic motor neurons as well as postsynaptic muscles, highlighting the importance of maintaining synaptic integrity for cell viability. Zhang, Z., Luo, S., Barbosa, G. O., Bai, M., Kornberg, T. B. and Ma, D. K. (2021). The conserved transmembrane protein TMEM-39 coordinates with COPII to promote collagen secretion and regulate ER stress response. PLoS Genet 17(2): e1009317. PubMed ID: 33524011
Dysregulation of collagen production and secretion contributes to aging and tissue fibrosis of major organs. How procollagen proteins in the endoplasmic reticulum (ER) route as specialized cargos for secretion remains to be fully elucidated. This study reports that TMEM39, an ER-localized transmembrane protein, regulates production and secretory cargo trafficking of procollagen. The C. elegans ortholog TMEM-39 was identified from an unbiased RNAi screen; deficiency of tmem-39 leads to striking defects in cuticle collagen production and constitutively high ER stress response. RNAi knockdown of the tmem-39 ortholog in Drosophila causes similar defects in collagen secretion from fat body cells. The cytosolic domain of human TMEM39A binds to Sec23A, a vesicle coat protein that drives collagen secretion and vesicular trafficking. TMEM-39 regulation of collagen secretion is independent of ER stress response and autophagy. It is proposed that the roles of TMEM-39 in collagen secretion and ER homeostasis are likely evolutionarily conserved.
Thursday, May 27th - Signaling
Dong, Q., Zavortink, M., Froldi, F., Golenkina, S., Lam, T. and Cheng, L. Y. (2021). Glial Hedgehog signalling and lipid metabolism regulate neural stem cell proliferation in Drosophila. EMBO Rep 22(5): e52130. PubMed ID: 33751817
The final size and function of the adult central nervous system (CNS) are determined by neuronal lineages generated by neural stem cells (NSCs) in the developing brain. In Drosophila, NSCs called neuroblasts (NBs) reside within a specialised microenvironment called the glial niche. This study explored non-autonomous glial regulation of NB proliferation. Lipid droplets (LDs) which reside within the glial niche were shown to be closely associated with the signalling molecule Hedgehog (Hh). Under physiological conditions, cortex glial Hh is autonomously required to sustain niche chamber formation. Upon FGF-mediated cortex glial overgrowth, glial Hh non-autonomously activates Hh signalling in the NBs, which in turn disrupts NB cell cycle progression and its ability to produce neurons. Glial Hh's ability to signal to NB is further modulated by lipid storage regulator lipid storage droplet-2 (Lsd-2) and de novo lipogenesis gene fatty acid synthase 1 (Fasn1). Together, these data suggest that glial-derived Hh modified by lipid metabolism mechanisms can affect the neighbouring NB's ability to proliferate and produce neurons. Daiber, T., VanderZwan-Butler, C. J., Bashaw, G. J. and Evans, T. A. (2021). Conserved and divergent aspects of Robo receptor signaling and regulation between Drosophila Robo1 and C. elegans SAX-3. Genetics 217(3). PubMed ID: 33789352
The evolutionarily conserved Roundabout (Robo) family of axon guidance receptors control midline crossing of axons in response to the midline repellant ligand Slit in bilaterian animals including insects, nematodes, and vertebrates. This study used a transgenic approach to express the Robo family receptor SAX-3 from the nematode Caenorhabditis elegans in neurons of the fruit fly, Drosophila melanogaster. SAX-3's ability to repel Drosophila axons from the Slit-expressing midline was tested in gain of function assays, and SAX-3's ability to substitute for Drosophila Robo1 during fly embryonic development was tested in genetic rescue experiments. C. elegans SAX-3 was shown to be properly translated and localized to neuronal axons when expressed in the Drosophila embryonic CNS, and SAX-3 can signal midline repulsion in Drosophila embryonic neurons, although not as efficiently as Drosophila Robo1. Using a series of Robo1/SAX-3 chimeras, this study shows that the SAX-3 cytoplasmic domain can signal midline repulsion to the same extent as Robo1 when combined with the Robo1 ectodomain. SAX-3 is not subject to endosomal sorting by the negative regulator Commissureless (Comm) in Drosophila neurons in vivo, and peri-membrane and ectodomain sequences are both required for Comm sorting of Drosophila Robo1.
Esteban-Collado, J., Corominas, M. and Serras, F. (2021). Nutrition and PI3K/Akt signaling are required for p38-dependent regeneration. Development 148(8). PubMed ID: 33913483
Regeneration after damage requires early signals to trigger the tissue repair machinery. Reactive oxygen species (ROS) act as early signals that are sensed by the MAP3 kinase Ask1, which in turn activates by phosphorylation the MAP kinases p38 and JNK. The sustained or high activation of these kinases can result in apoptosis, whereas short or low activation can promote regeneration. Using the Ask1-dependent regeneration program, this study demonstrated in Drosophila wing that PI3K/Akt signaling is necessary for Ask1 to activate p38, but not JNK. In addition, nutrient restriction or mutations that target Ser83 of the Drosophila Ask1 protein, a PI3K/Akt-sensitive residue, block regeneration. However, these effects can be reversed by the ectopic activation of p38, but not of JNK. These results demonstrate that Ask1 controls the activation of p38 through Ser83, and that the phosphorylation of p38 during regeneration is nutrient sensitive. This mechanism is important for discriminating between p38 and JNK in the cells involved in tissue repair and regenerative growth. Dong, Y. L., Vadla, G. P., Lu, J. J., Ahmad, V., Klein, T. J., Liu, L. F., Glazer, P. M., Xu, T. and Chabu, C. Y. (2021). Cooperation between oncogenic Ras and wild-type p53 stimulates STAT non-cell autonomously to promote tumor radioresistance. Commun Biol 4(1): 374. PubMed ID: 33742110
Oncogenic RAS mutations are associated with tumor resistance to radiation therapy. Cell-cell interactions in the tumor microenvironment (TME) profoundly influence therapy outcomes. However, the nature of these interactions and their role in Ras tumor radioresistance remain unclear. This study used Drosophila oncogenic Ras tissues and human Ras cancer cell radiation models to address these questions. It was discovered that cellular response to genotoxic stress cooperates with oncogenic Ras to activate JAK/STAT non-cell autonomously in the TME. Specifically, p53 is heterogeneously activated in Ras tumor tissues in response to irradiation. This mosaicism allows high p53-expressing Ras clones to stimulate JAK/STAT cytokines, which activate JAK/STAT in the nearby low p53-expressing surviving Ras clones, leading to robust tumor re-establishment. Blocking any part of this cell-cell communication loop re-sensitizes Ras tumor cells to irradiation. These findings suggest that coupling STAT inhibitors to radiotherapy might improve clinical outcomes for Ras cancer patients.
Wang, M., Luan, X., Yan, Y., Zheng, Q., Chen, W. and Fang, J. (2021). Wnt6 regulates the homeostasis of the stem cell niche via Rac1-and Cdc42-mediated noncanonical Wnt signalling pathways in Drosophila testis. Exp Cell Res: 112511. PubMed ID: 33582096
The homeostasis of the stem cell niche is regulated by both intrinsic and extrinsic factors, and the complex and ordered molecular and cellular regulatory mechanisms need to be further explored. In Drosophila testis, germline stem cells (GSCs) rely on hub cells for self-renewal and physical attachment. GSCs are also in contact with somatic cyst stem cells (CySCs). Utilizing genetic manipulation in Drosophila, this study investigated the role of Wnt6 in vivo and in vitro. In Drosophila testis, Wnt6 was found to be required for GSC differentiation and CySC self-renewal. In Schneider 2 (S2) cells, Wnt6 was found to regulate cell proliferation and apoptosis. Mechanistically, it was demonstrated that Wnt6 can downregulate the expression levels of Arm, Rac1 and Cdc42 in S2 cells. Notably, Rac1 and Cdc42, which act downstream of the noncanonical Wnt signalling pathway, imitated the phenotypes of Wnt6 in Drosophila testis. Thus, the newly discovered Wnt6-Rac1/Cdc42 signal axis is required for the homeostasis of the stem cell niche in the Drosophila testis. Yu, S., Luo, F. and Jin, L. H. (2021). Rab5 and Rab11 maintain hematopoietic homeostasis by restricting multiple signaling pathways in Drosophila. Elife 10. PubMed ID: 33560224
The hematopoietic system of Drosophila is a powerful genetic model for studying hematopoiesis, and vesicle trafficking is important for signal transduction during various developmental processes; however, its interaction with hematopoiesis is currently largely unknown. Three endosome markers, Rab5, Rab7, and Rab11, were selected for study that play a key role in membrane trafficking, and it was determined whether they participate in hematopoiesis. Inhibiting Rab5 or Rab11 in hemocytes or the cortical zone (CZ) significantly induced cell overproliferation and lamellocyte formation in circulating hemocytes and lymph glands and disrupted blood cell progenitor maintenance. Lamellocyte formation involves the JNK, Toll, and Ras/EGFR signaling pathways. Notably, lamellocyte formation was also associated with JNK-dependent autophagy. In conclusion, Rab5 and Rab11 were identified as novel regulators of hematopoiesis, and the results advance the understanding of the mechanisms underlying the maintenance of hematopoietic homeostasis as well as the pathology of blood disorders such as leukemia.
Wednesday, May 26th - Chromatin
Tian, Y. and Smith-Bolton, R. K. (2021). Regulation of growth and cell fate during tissue regeneration by the two SWI/SNF chromatin-remodeling complexes of Drosophila. Genetics 217(1): 1-16. PubMed ID: 33683366
To regenerate, damaged tissue must heal the wound, regrow to the proper size, replace the correct cell types, and return to the normal gene-expression program. However, the mechanisms that temporally and spatially control the activation or repression of important genes during regeneration are not fully understood. To determine the role that chromatin modifiers play in regulating gene expression after tissue damage, ablation was induced in Drosophila melanogaster imaginal wing discs, and a screen was carried out for chromatin regulators that are required for epithelial tissue regeneration. Many of these genes are shown to be important for promoting or constraining regeneration. Specifically, the two SWI/SNF chromatin-remodeling complexes play distinct roles in regulating different aspects of regeneration. The PBAP (see Polybromo) complex regulates regenerative growth and developmental timing, and is required for the expression of JNK signaling targets and the growth promoter Myc. By contrast, the BAP complex (see Osa) ensures correct patterning and cell fate by stabilizing the expression of the posterior gene engrailed. Thus, both SWI/SNF complexes are essential for proper gene expression during tissue regeneration, but they play distinct roles in regulating growth and cell fate. Climent-Canto, P., Carbonell, A., Tamirisa, S., Henn, L., Perez-Montero, S., Boros, I. M. and Azorin, F. (2021). The tumour suppressor brain tumour (Brat) regulates linker histone dBigH1 expression in the Drosophila female germline and the early embryo. Open Biol 11(5): 200408. PubMed ID: 33947246
Linker histones H1 are essential chromatin components that exist as multiple developmentally regulated variants. In metazoans, specific H1s are expressed during germline development in a tightly regulated manner. However, the mechanisms governing their stage-dependent expression are poorly understood. This question was addressed in Drosophila, which encodes for a single germline-specific dBigH1 linker histone. During female germline lineage differentiation, dBigH1 is expressed in germ stem cells and cystoblasts, becomes silenced during transit-amplifying (TA) cystocytes divisions to resume expression after proliferation stops and differentiation starts, when it progressively accumulates in the oocyte. This study finds that dBigH1 silencing during TA divisions is post-transcriptional and depends on the tumour suppressor Brain tumour (Brat), an essential RNA-binding protein that regulates mRNA translation and stability. Like other oocyte-specific variants, dBigH1 is maternally expressed during early embryogenesis until it is replaced by somatic dH1 at the maternal-to-zygotic transition (MZT). Brat also mediates dBigH1 silencing at MZT. Finally, the situation in testes is discussed, where Brat is not expressed, but dBigH1 is translationally silenced too.
Mahadevaraju, S., Fear, J. M., Akeju, M., Galletta, B. J., Pinheiro, M., Avelino, C. C., Cabral-de-Mello, D. C., Conlon, K., Dell'Orso, S., Demere, Z., Mansuria, K., Mendonca, C. A., Palacios-Gimenez, O. M., Ross, E., Savery, M., Yu, K., Smith, H. E., Sartorelli, V., Yang, H., Rusan, N. M., Vibranovski, M. D., Matunis, E. and Oliver, B. (2021). Dynamic sex chromosome expression in Drosophila male germ cells. Nat Commun 12(1): 892. PubMed ID: 33563972
Given their copy number differences and unique modes of inheritance, the evolved gene content and expression of sex chromosomes is unusual. In many organisms the X and Y chromosomes are inactivated in spermatocytes, possibly as a defense mechanism against insertions into unpaired chromatin. In addition to current sex chromosomes, Drosophila has a small gene-poor X-chromosome relic (4(th)) that re-acquired autosomal status. This study used single cell RNA-Seq on fly larvae to demonstrate that the single X and pair of 4(th) chromosomes are specifically inactivated in primary spermatocytes, based on measuring all genes or a set of broadly expressed genes in testis that were identified. In contrast, genes on the single Y chromosome become maximally active in primary spermatocytes. Reduced X transcript levels are due to failed activation of RNA-Polymerase-II by phosphorylation of Serine 2 and 5. On, K., Crevel, G., Cotterill, S., Itoh, M. and Kato, Y. (2021). Drosophila telomere capping protein HOAP interacts with DSB sensor proteins Mre11 and Nbs. Genes Cells PubMed ID: 33556205
In eukaryotes, specific DNA-protein structures called telomeres exist at linear chromosome ends. Telomere stability is maintained by a specific capping protein complex. This capping complex is essential for the inhibition of the DNA damage response (DDR) at telomeres and contributes to genome integrity. In Drosophila the central factors of telomere capping complex are HOAP and HipHop. Furthermore, a DDR protein complex Mre11-Rad50-Nbs (MRN) is known to be important for the telomere association of HOAP and HipHop. However, whether MRN interacts with HOAP and HipHop, and the telomere recognition mechanisms of HOAP and HipHop are poorly understood. This study shows that Nbs interacts with Mre11 and transports the Mre11-Rad50 complex from the cytoplasm to the nucleus. In addition, this study reports that HOAP interacts with both Mre11 and Nbs. The N-terminal region of HOAP is essential for its co-localization with HipHop. Finally, it is revealed that Nbs interacts with the N-terminal region of HOAP.
Ota, R., Hayashi, M., Morita, S., Miura, H. and Kobayashi, S. (2021). Absence of X-chromosome dosage compensation in the primordial germ cells of Drosophila embryos. Sci Rep 11(1): 4890. PubMed ID: 33649478
Dosage compensation is a mechanism that equalizes sex chromosome gene expression between the sexes. In Drosophila, individuals with two X chromosomes (XX) become female, whereas males have one X chromosome (XY). In males, dosage compensation of the X chromosome in the soma is achieved by five proteins and two non-coding RNAs, which assemble into the male-specific lethal (MSL) complex to upregulate X-linked genes twofold. By contrast, it remains unclear whether dosage compensation occurs in the germline. To address this issue, transcriptome analysis was performed of male and female primordial germ cells (PGCs). The expression levels of X-linked genes were approximately twofold higher in female PGCs than in male PGCs. Acetylation of lysine residue 16 on histone H4 (H4K16ac), which is catalyzed by the MSL complex, was undetectable in these cells. In male PGCs, hyperactivation of X-linked genes and H4K16ac were induced by overexpression of the essential components of the MSL complex, which were expressed at very low levels in PGCs. Together, these findings indicate that failure of MSL complex formation results in the absence of X-chromosome dosage compensation in male PGCs. Regadas, I., Dahlberg, O., Vaid, R., Ho, O., Belikov, S., Dixit, G., Deindl, S., Wen, J. and Mannervik, M. (2021). A unique histone 3 lysine 14 chromatin signature underlies tissue-specific gene regulation. Mol Cell. PubMed ID: 33631105
Organismal development and cell differentiation critically depend on chromatin state transitions. However, certain developmentally regulated genes lack histone 3 lysine 9 and 27 acetylation (H3K9ac and H3K27ac, respectively) and histone 3 lysine 4 (H3K4) methylation, histone modifications common to most active genes. This study describes a chromatin state featuring unique histone 3 lysine 14 acetylation (H3K14ac) peaks in key tissue-specific genes in Drosophila and human cells. Replacing H3K14 in Drosophila demonstrates that H3K14 is essential for expression of genes devoid of canonical histone modifications in the embryonic gut and larval wing imaginal disc, causing lethality and defective wing patterning. The SWI/SNF protein Brahma (Brm) recognizes H3K14ac, that brm acts in the same genetic pathway as H3K14R, and that chromatin accessibility at H3K14ac-unique genes is decreased in H3K14R mutants. These results show that acetylation of a single lysine is essential at genes devoid of canonical histone marks and uncover an important requirement for H3K14 in tissue-specific gene regulation.
Tuesday, May 25th - Methods
Chen, X. and Dickman, D. (2021). Tissue-Specific Ribosome Profiling in Drosophila. Methods Mol Biol 2252: 175-188. PubMed ID: 33765275
Robust mechanisms exist that serve to dynamically regulate the translation of mRNA into proteins across heterogeneous tissues. These processes ensure timely generation of proteins in quantities that scale with the demands of specific cell types. Importantly, this translational regulation occurs with spatiotemporal precision and is capable of recalibration as conditions change. Aberrant regulation of translation contributes to and exacerbates a wide range of diseases. Although dynamic control of translation is an essential and fundamental process shared by organisms, specific tissues and cell types can be differentially impacted by circumstances that challenge and impair basal translation, highlighting the heterogeneous nature of translational regulation. To understand how translation is differentially regulated during changing environments and across specific cells and tissues, methods capable of profiling translation in specific tissues and cells are crucial. This paper describes a method for profiling genome-wide translation in specific tissues or cell types in Drosophila melanogaster, in which ribosome affinity purification is combined with ribosome profiling to enable a simplified protocol for robust analysis of translation in specific tissues. Shaw, H. S., Larkin, J. and Rao, Y. (2020). Phototactic T-maze Behavioral Assay for Comparing the Functionality of Color-sensitive Photoreceptor Subtypes in the Drosophila Visual System. Bio Protoc 10(6): e3558. PubMed ID: 33659530
The Drosophila retina contains light-sensitive photoreceptors (R cells) with distinct spectral sensitivities that allow them to distinguish light by its spectral composition. R7 and R8 photoreceptors are important for color vision, and can be further classified into pale (p) or yellow (y) subtypes depending on the rhodopsin expressed. While both R7y and R7p are sensitive to UV light, R8y and R8p detect light in the green and blue spectrum, respectively. The ability of R7 and R8 photoreceptors to distinguish different spectral sensitivities and the natural preference for Drosophila towards light sources (phototaxis), allow for the development of a phototactic T-maze assay that compares the functionality of different R7 and R8 subtypes. A "UV vs. blue" choice can compare the functionalities of R7p and R8p photoreceptors, while a "UV vs. green" choice can compare the functionalities of R7y and R8y photoreceptors. Additionally, a "blue vs. green" choice could be used to compare R8p and R8y photoreceptors, while a "dark vs. light" choice could be used to determine overall vision functionality. The phototactic T-maze assay presented in this study is a robust, straight-forward and an inexpensive method to study genetic and developmental factors that contribute to the individual functionality of R7 and R8 photoreceptors, and is especially useful when performing large-scale genetic screens.
Pun, P., Brown, J., Cobb, T., Wessells, R. J. and Kim, D. H. (2021). Navigation of a Freely Walking Fruit Fly in Infinite Space Using a Transparent Omnidirectional Locomotion Compensator (TOLC). Sensors (Basel) 21(5). PubMed ID: 33673520
Animal behavior is an essential element in behavioral neuroscience study. However, most behavior studies in small animals such as fruit flies (Drosophila melanogaster) have been performed in a limited spatial chamber or by tethering the fly's body on a fixture, which restricts its natural behavior. This study has developed the Transparent Omnidirectional Locomotion Compensator (TOLC) for a freely walking fruit fly without tethering, which enables its navigation in infinite space. The TOLC maintains a position of a fruit fly by compensating its motion using the transparent sphere. The TOLC is capable of maintaining the position error < 1 mm for 90.3% of the time and the heading error < 5° for 80.2% of the time. The inverted imaging system with a transparent sphere secures the space for an additional experimental apparatus. Because the proposed TOLC allows observation of a freely walking fly without physical tethering, there is no potential injury during the experiment. Thus, the TOLC will offer a unique opportunity to investigate longitudinal studies of a wide range of behavior in an unrestricted walking Drosophila. Terradas, G., Buchman, A. B., Bennett, J. B., Shriner, I., Marshall, J. M., Akbari, O. S. and Bier, E. (2021). Inherently confinable split-drive systems in Drosophila. Inherently confinable split-drive systems in Drosophila
CRISPR-based gene-drive systems, which copy themselves via gene conversion mediated by the homology-directed repair (HDR) pathway, have the potential to revolutionize vector control. However, mutant alleles generated by the competing non-homologous end-joining (NHEJ) pathway, resistant to Cas9 cleavage, can interrupt the spread of gene-drive elements. It was hypothesized that drives targeting genes essential for viability or reproduction, also carrying recoded sequences that restore endogenous gene functionality, should benefit from dominantly-acting maternal clearance of NHEJ alleles combined with recessive Mendelian culling processes. This study tested split gene-drive (sGD) systems in Drosophila melanogaster that are inserted into essential genes required for viability (rab5, rab11, prosalpha2) or fertility (spo11). In single generation crosses, sGDs copy with variable efficiencies and display sex-biased transmission. In multigenerational cage trials, sGDs follow distinct drive trajectories reflecting their differential tendencies to induce target chromosome damage and/or lethal/sterile mosaic Cas9-dependent phenotypes, leading to inherently confinable drive outcomes.
Reddish, F. N., Miller, C. L., Deng, X., Dong, B., Patel, A. A., Ghane, M. A., Mosca, B., McBean, C., Wu, S., Solntsev, K. M., Zhuo, Y., Gadda, G., Fang, N., Cox, D. N., Mabb, A. M., Treves, S., Zorzato, F. and Yang, J. J. (2021). Rapid subcellular calcium responses and dynamics by calcium sensor G-CatchER. iScience 24(3): 102129. PubMed ID: 33665552
The precise spatiotemporal characteristics of subcellular calcium (Ca(2+)) transients are critical for the physiological processes. This study reports a green Ca(2+) sensor called "G-CatchER(+)" using a protein design to report rapid local ER Ca(2+) dynamics with significantly improved folding properties. G-CatchER(+) exhibits a superior Ca(2+) on rate to G-CEPIA1er and has a Ca(2+)-induced fluorescence lifetimes increase. G-CatchER(+) also reports agonist/antagonist triggered Ca(2+) dynamics in several cell types including primary neurons that are orchestrated by IP(3)Rs, RyRs, and SERCAs with an ability to differentiate expression. Upon localization to the lumen of the RyR channel (G-CatchER(+)-JP45), a rapid local Ca(2+) release occurs that is likely due to calsequestrin. Transgenic expression of G-CatchER(+) in Drosophila muscle demonstrates its utility as an in vivo reporter of stimulus-evoked SR local Ca(2+) dynamics. G-CatchER(+) will be an invaluable tool to examine local ER/SR Ca(2+) dynamics and facilitate drug development associated with ER dysfunction. Sun, R., Brogan, D., Buchman, A., Yang, T. and Akbari, O. S. (2021). Ubiquitous and Tissue-specific RNA Targeting in Drosophila Melanogaster using CRISPR/CasRx. J Vis Exp(168). PubMed ID: 33616113
CasRx, a member of the RNA-targeting Cas13 family, is a promising new addition of the CRISPR/Cas technologies in efficient gene transcript reduction with an attractive off-target profile at both cellular and organismal levels. It has been reported that the CRISPR/CasRx system can be used to achieve ubiquitous and tissue-specific gene transcript reduction in Drosophila melanogaster. This paper details the methods from the recent work, consisting of three parts: 1) ubiquitous in vivo endogenous RNA targeting using a two-component CasRx system; 2) ubiquitous in vivo exogenous RNA targeting using a three-component CasRx system; and 3) tissue-specific in vivo RNA targeting using a three-component CasRx system. The effects of RNA targeting observed include targeted gene specific phenotypic changes, targeted RNA transcript reduction, and occasional lethality phenotypes associated with high expression of CasRx protein and collateral activity. Overall, these results showed that the CasRx system is capable of target RNA transcript reduction at the organismal level in a programmable and efficient manner, demonstrating that in vivo transcriptome targeting, and engineering is feasible and lays the foundation for future in vivo CRISPR-based RNA targeting technologies.
Monday, May 24th - Immune Response
Balog, J., Honti, V., Kurucz, E., Kari, B., Pusas, L. G., Ando, I. and Szebeni, G. J. (2021). Immunoprofiling of Drosophila Hemocytes by Single-cell Mass Cytometry. Genomics Proteomics Bioinformatics. PubMed ID: 33713850
Single-cell mass cytometry (SCMC) combines features of traditional flow cytometry (FACS) with mass spectrometry, making it possible to measure several parameters at the single-cell level for a complex analysis of biological regulatory mechanisms. SCMC was optimized to analyze hemocytes of the Drosophila innate immune system. Metal-conjugated antibodies (H2, H3, H18, L1, L4, and P1 at the cell surface, intracellular 3A5 and L2) and anti-IgM (L6 at the cell surface) were used to detect the levels of antigens, while anti-GFP was used to detect crystal cells in the immune induced samples. This study investigated the antigen expression profile of single cells and hemocyte populations in naive states, in immune induced states, in tumorous mutants bearing a driver mutation in the Drosophila homologue of Janus kinase (hopTum) and carrying deficiency of a tumor suppressor l(3)mbn1 gene, as well as in stem cell maintenance-defective hdcΔ84) mutant larvae. Multidimensional analysis enabled the discrimination of the functionally different major hemocyte subsets for lamellocytes, plasmatocytes, and crystal cells, and delineated the unique immunophenotype of Drosophila mutants. Subpopulations of L2(+)/P1(+) (l(3)mbn1), L2(+)/L4(+)/P1(+) hopTum) transitional phenotype cells were identified in the tumorous strains and a subpopulation of L4(+)/P1(+) cells was identified upon immune induction. These results demonstrated for the first time that SCMC, combined with multidimensional bioinformatic analysis, represents a versatile and powerful tool to deeply analyze the regulation of cell-mediated immunity of Drosophila. Du, B. B., Liu, L. and Zhu, Y. Y. (2020). RNA-binding protein Roquin negatively regulates STING-dependent innate immune response in Drosophila. Yi Chuan 42(12): 1201-1210. PubMed ID: 33509784
Drosophila melanogaster utilizes innate immune response to defend against exogenous pathogens. The molecular regulation mechanism of the process is evolutionarily conserved. Research of the regulatory mechanisms of Drosophila innate immunity is greatly significant for understanding the modulation of the human innate immunity and the pathogenesis of related diseases. To explore novel regulators in the STING-dependent innate immune response in Drosophila, the double-stranded RNA-mediated gene expression silencing technique and the dual-luciferase reporter system were used in knockdown experiments on 9 genes encoding the ubiquitin ligase such as echinus (CG2904), usp16 (CG4165), smurf (CG4943), pellino (CG5212), usp47 (CG5486), diap2 (CG8293), dtraf2 (CG10961), roquin (CG16807) and usp10 (CG32479) in the S2 cells in vitro. The results suggested a negative correlation between CG16807 (roquin) and the STING signaling pathway. Further studies showed that over-expression of roquin in S2 cells significantly inhibited STING innate immune signaling. Meanwhile, Listeria infection experiments showed that knocking down of roquin markedly elevated the expression levels of anti-microbial peptides and inhibited the proliferation of Listeria, thus increasing the survival rates post pathogenic infection. Taken together, these results suggested that the RNA-binding protein Roquin negatively regulates the STING-dependent innate immune response in Drosophila. In view of the high correlation between Drosophila genes and human genes, this study provides a theoretical basis for further development of treatments for STING-related innate immune diseases in humans.
Bartolo, G., Gonzalez, L. O., Levitin, A. and Martchenko Shilman, M. (2021). Drosophila melanogaster Y Chromosome Genes Affect Male Sensitivity to Microbial Infections. Insects 12(1). PubMed ID: 33466347
The genders of Drosophila melanogaster vary in their sensitivities to microbial pathogens. While many of the immunity-related genes are located on the X chromosome, the polymorphisms within the Y chromosome were also shown to affect the immunity of flies. This study investigated the necessity of individual genes on the Y chromosome (Y-genes) for male sensitivity to microbes. Several Y-genes were identified whose genetic inactivation either increases or decreases the sensitivity of males to gastrointestinal infections with fungal Saccharomyces cerevisiae and bacterial Serratia liquefaciens. Specifically, the loss of function mutations in fly kl-5 and Ppr-Y Y-genes lead to increased and decreased sensitivity of males to fungal challenge, respectively, compared to female sensitivity. In contrast, mutations in Drosophila Pp1-Y1, kl-5, kl-3, Ppr-Y, CCY, and FDY Y-genes lead to increased sensitivity of males to bacterial infection, compared to females. Moreover, while these Y-genes are necessary, the Y chromosome is not sufficient for the sensitivity of males to microbes, since the sensitivity of XXY females to fungal and bacterial challenges was not different from the sensitivity of wild-type female flies, compared to males. This study assigns a new immunity-related function to numerous Y-genes in D. melanogaster. Trainor, J. E., Kr, P. and Mortimer, N. T. (2021). Immune Cell Production Is Targeted by Parasitoid Wasp Virulence in a Drosophila-Parasitoid Wasp Interaction. Pathogens 10(1). PubMed ID: 33429864
The interactions between Drosophila melanogaster and the parasitoid wasps that infect Drosophila species provide an important model for understanding host-parasite relationships. Following parasitoid infection, D. melanogaster larvae mount a response in which immune cells (hemocytes) form a capsule around the wasp egg, which then melanizes, leading to death of the parasitoid. Previous studies have found that host hemocyte load; the number of hemocytes available for the encapsulation response; and the production of lamellocytes, an infection induced hemocyte type, are major determinants of host resistance. Parasitoids have evolved various virulence mechanisms to overcome the immune response of the D. melanogaster host, including both active immune suppression by venom proteins and passive immune evasive mechanisms. This study identified a previously undescribed parasitoid species, Asobara sp. AsDen, which utilizes an active virulence mechanism to infect D. melanogaster hosts. Asobara sp. AsDen infection inhibits host hemocyte expression of msn, a member of the JNK signaling pathway, which plays a role in lamellocyte production. Asobara sp. AsDen infection restricts the production of lamellocytes as assayed by hemocyte cell morphology and altered msn expression. These findings suggest that Asobara sp. AsDen infection alters host signaling to suppress immunity.
Cattenoz, P. B., Monticelli, S., Pavlidaki, A. and Giangrande, A. (2021). Toward a Consensus in the Repertoire of Hemocytes Identified in Drosophila. Front Cell Dev Biol 9: 643712. PubMed ID: 33748138
The catalog of the Drosophila immune cells was until recently limited to three major cell types, based on morphology, function and few molecular markers. Three recent single cell studies highlight the presence of several subgroups, revealing a large diversity in the molecular signature of the larval immune cells. Since these studies rely on somewhat different experimental and analytical approaches, this study compared the datasets and identify eight common, robust subgroups associated to distinct functions such as proliferation, immune response, phagocytosis or secretion. Similar comparative analyses with datasets from different stages and tissues disclose the presence of larval immune cells resembling embryonic hemocyte progenitors and the expression of specific properties in larval immune cells associated with peripheral tissues. Kumar, J. R., Smith, J. P., Kwon, H. and Smith, R. C. (2021). Use of Clodronate Liposomes to Deplete Phagocytic Immune Cells in Drosophila melanogaster and Aedes aegypti. Front Cell Dev Biol 9: 627976. PubMed ID: 33604338
The innate immune system is the primary defense response to limit invading pathogens for all invertebrate species. In insects, immune cells are central to both cellular and humoral immune responses, however few genetic resources exist beyond Drosophila to study immune cell function. Therefore, the development of innovative tools that can be widely applied to a variety of insect systems is of importance to advance the study of insect immunity. This study has adapted the use of clodronate liposomes (CLD; a hydrophilic molecule that can be encapsulated within phospholipid bilayers) to deplete phagocytic immune cells in the vinegar fly, Drosophila melanogaster, and the yellow fever mosquito, Aedes aegypti. Through microscopy and molecular techniques, the depletion of phagocytic cell populations was validated in both insect species, and the integral role of phagocytes in combating bacterial pathogens demonstrated. Together, these data demonstrate the wide utility of CLD in insect systems to advance the study of phagocyte function in insect innate immunity.
Friday, May 21st - Disease Models
Belhorma, K., Darwish, N., Benn-Hirsch, E., Duenas, A., Gates, H., Sanghera, N., Wu, J. and French, R. L. (2021). Developmental ethanol exposure causes central nervous system dysfunction and may slow the aging process in a Drosophila model of fetal alcohol spectrum disorder. Alcohol. PubMed ID: 33961967
Alcohol is a known teratogen, and developmental exposure to ethanol results in Fetal Alcohol Spectrum Disorder (FASD). Children born with FASD can exhibit a range of symptoms including low birth weight, microcephaly, and neurobehavioral problems. Treatment of patients with FASD is estimated to cost 4 billion dollars per year in the United States alone, and 2 million dollars per affected individual's lifetime. This study has established Drosophila melanogaster as a model organism for the study of FASD. This study reports that mutations in Dementin (Dmtn), the Drosophila ortholog of the Alzheimer Disease associated protein TMCC2, convey sensitivity to developmental ethanol exposure, and provide evidence that Dmtn expression is disrupted by ethanol. In addition, it was found that flies reared on ethanol exhibit mild climbing defects suggestive of neurodegeneration. Surprisingly, the data also suggest that flies reared on ethanol age more slowly than control animals, and a number of slow-aging mutants were found to be sensitive to developmental ethanol exposure. Finally, this study found that flies reared on ethanol showed a persistent upregulation of genes encoding antioxidant enzymes, which may contribute to a reduced rate of central nervous system aging. Thus, in addition to the well-documented negative effects of developmental alcohol exposure on the nervous system, there may be a previously-unsuspected neuroprotective effect in adult animals. Bhat, S. A., Yousuf, A., Mushtaq, Z., Kumar, V. and Qurashi, A. (2021). Fragile X Premutation rCGG Repeats Impair Synaptic Growth and Synaptic Transmission at Drosophila larval Neuromuscular Junction. Hum Mol Genet. PubMed ID: 33772546
Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset neurodegenerative disease that develops in some premutation (PM) carriers of the FMR1 gene with alleles bearing 55-200 CGG repeats. The discovery of a broad spectrum of clinical and cell developmental abnormalities among PM carriers with or without FXTAS and in model systems suggests that neurodegeneration seen in FXTAS could be the inevitable end-result of pathophysiological processes set during early development. Hence, it is imperative to trace early PM-induced pathological abnormalities. Previous studies have shown that transgenic Drosophila carrying PM-length CGG repeats are sufficient to cause neurodegeneration. This study used the same transgenic model to understand the effect of CGG repeats on the structure and function of the developing nervous system. Presynaptic expression of CGG repeats restricts synaptic growth, reduces the number of synaptic boutons, leads to aberrant presynaptic varicosities, and impairs synaptic transmission at the larval neuromuscular junctions. The postsynaptic analysis shows that both glutamate receptors and subsynaptic reticulum proteins were normal. However, a high percentage of boutons show a reduced density of Bruchpilot protein, a key component of presynaptic active zones required for vesicle release. The electrophysiological analysis shows a significant reduction in quantal content, a measure of total synaptic vesicles released per excitation potential. Together, these findings suggest that synapse perturbation caused by rCGG repeats mediates presynaptically during larval NMJ development. It is also suggested that the stress-activated c-Jun N-terminal kinase protein Basket and CIDE-N protein Drep-2 positively mediate Bruchpilot active zone defects caused by rCGG repeats.
Cabasso, O., Paul, S., Maor, G., Pasmanik-Chor, M., Kallemeijn, W., Aerts, J. and Horowitz, M. (2021). The Uncovered Function of the Drosophila GBA1a-Encoded Protein. Cells 10(3). PubMed ID: 33809074
Human GBA1 encodes lysosomal acid β-glucocerebrosidase (GCase), which hydrolyzes cleavage of the beta-glucosidic linkage of glucosylceramide (GlcCer). Mutations in this gene lead to reduced GCase activity, accumulation of glucosylceramide and glucosylsphingosine, and development of Gaucher disease (GD). Drosophila melanogaster has two GBA1 orthologs. Thus far, GBA1b was documented as a bone fide GCase-encoding gene, while the role of GBA1a encoded protein remained unclear. The present study characterized a mutant variant of the fly GBA1a, which underwent ERAD and mildly activated the UPR machinery. RNA-seq analyses of homozygous mutant flies revealed upregulation of inflammation-associated as well as of cell-cycle related genes and reduction in programmed cell death (PCD)-associated genes, which was confirmed by qRT-PCR. Compromised cell death in the midgut of homozygous larvae and a reduction in pupation were also observed. These results strongly indicated that GBA1a-encoded protein plays a role in midgut maturation during larvae development. Baek, M., Choe, Y. J., Bannwarth, S., Kim, J., Maitra, S., Dorn, G. W., Taylor, J. P., Paquis-Flucklinger, V. and Kim, N. C. (2021). TDP-43 and PINK1 mediate CHCHD10(S59L) mutation-induced defects in Drosophila and in vitro. Nat Commun 12(1): 1924. PubMed ID: 33772006
Mutations in coiled-coil-helix-coiled-coil-helix domain containing 10 (CHCHD10) can cause amyotrophic lateral sclerosis and frontotemporal dementia (ALS-FTD). However, the underlying mechanisms are unclear. This study generate CHCH10(S59L)-mutant Drosophila melanogaster and HeLa cell lines to model CHCHD10-associated ALS-FTD. The CHCHD10(S59L) mutation results in cell toxicity in several tissues and mitochondrial defects. CHCHD10(S59L) independently affects the TDP-43 and PINK1 pathways. CHCHD10(S59L) expression increases TDP-43 insolubility and mitochondrial translocation. Blocking TDP-43 mitochondrial translocation with a peptide inhibitor reduced CHCHD10(S59L)-mediated toxicity. While genetic and pharmacological modulation of PINK1 expression and activity of its substrates rescues and mitigates the CHCHD10(S59L)-induced phenotypes and mitochondrial defects, respectively, in both Drosophila and HeLa cells. These findings suggest that CHCHD10(S59L)-induced TDP-43 mitochondrial translocation and chronic activation of PINK1-mediated pathways result in dominant toxicity, providing a mechanistic insight into the CHCHD10 mutations associated with ALS-FTD.
Bangi, E., Smibert, P., Uzilov, A. V., Teague, A. G., Gopinath, S., Antipin, Y., Chen, R., Hecht, C., Gruszczynski, N., Yon, W. J., Malyshev, D., Laspina, D., Selkridge, I., Wang, H., Gomez, J., Mascarenhas, J., Moe, A. S., Lau, C. Y., Taik, P., Pandya, C., Sung, M., Kim, S., Yum, K., Sebra, R., Donovan, M., Misiukiewicz, K., Ang, C., Schadt, E. E., Posner, M. R. and Cagan, R. L. (2021). PubMed ID: 33733072 . A Drosophila platform identifies a novel, personalized therapy for a patient with adenoid cystic carcinoma. iScience 24(3): 102212
Adenoid cystic carcinoma (ACC) is a rare cancer type that originates in the salivary glands. Tumors commonly invade along nerve tracks in the head and neck, making surgery challenging. Follow-up treatments for recurrence or metastasis including chemotherapy and targeted therapies have shown limited efficacy, emphasizing the need for new therapies. This study reports a Drosophila-based therapeutic approach for a patient with advanced ACC disease. A patient-specific Drosophila transgenic line was developed to model the five major variants associated with the patient's disease. Robotics-based screening identified a three-drug cocktail-vorinostat, pindolol, tofacitinib-that rescued transgene-mediated lethality in the Drosophila patient-specific line. Patient treatment led to a sustained stabilization and a partial metabolic response of 12 months. Subsequent resistance was associated with new genomic amplifications and deletions. Given the lack of options for patients with ACC, these data suggest that this approach may prove useful for identifying novel therapeutic candidates. Bawa, S., Piccirillo, R. and Geisbrecht, E. R. (2021). TRIM32: A Multifunctional Protein Involved in Muscle Homeostasis, Glucose Metabolism, and Tumorigenesis. Biomolecules 11(3). PubMed ID: 33802079
Human tripartite motif family of proteins 32 (TRIM32) is a ubiquitous multifunctional protein that has demonstrated roles in differentiation, muscle physiology and regeneration, and tumor suppression. Mutations in TRIM32 result in two clinically diverse diseases. A mutation in the B-box domain gives rise to Bardet-Biedl syndrome (BBS), a disease whose clinical presentation shares no muscle pathology, while mutations in the NHL (NCL-1, HT2A, LIN-41) repeats of TRIM32 causes limb-girdle muscular dystrophy type 2H (LGMD2H). TRIM32 also functions as a tumor suppressor, but paradoxically is overexpressed in certain types of cancer. Recent evidence supports a role for TRIM32 in glycolytic-mediated cell growth, thus providing a possible mechanism for TRIM32 in the accumulation of cellular biomass during regeneration and tumorigenesis, including in vitro and in vivo approaches, to understand the broad spectrum of TRIM32 functions. A special emphasis is placed on the utility of the Drosophila model, a unique system to study glycolysis and anabolic pathways that contribute to the growth and homeostasis of both normal and tumor tissues.
Thursday, May 20th - RNA and Transposons
van der Graaf, K., Jindrich, K., Mitchell, R. and White-Cooper, H. (2021). Roles for RNA export factor, Nxt1, in ensuring muscle integrity and normal RNA expression in Drosophila. G3 (Bethesda) 11(1). PubMed ID: 33561245
The mRNA export pathway is responsible for the transport of mRNAs from the nucleus to the cytoplasm, and thus is essential for protein production and normal cellular functions. A partial loss of function allele of the mRNA export factor Nxt1 in Drosophila shows reduced viability and sterility. A previous study has shown that the male fertility defect is due to a defect in transcription and RNA stability, indicating the potential for this pathway to be implicated in processes beyond the known mRNA transport function. This study investigated the reduced viability of Nxt1 partial loss of function mutants and describes a defect in growth and maintenance of the larval muscles, leading to muscle degeneration. RNA-seq revealed reduced expression of a set of mRNAs, particularly from genes with long introns in Nxt1 mutant carcass. Differential expression was detected of circRNA, and significantly fewer distinct circRNAs expressed in the mutants. Despite the widespread defects in gene expression, muscle degeneration was rescued by increased expression of the costamere component tn (abba) in muscles. This is the first report of a role for the RNA export pathway gene Nxt1 in the maintenance of muscle integrity. These data also links the mRNA export pathway to a specific role in the expression of mRNA and circRNA from common precursor genes, in vivo. Takemura, M., Bowden, N., Lu, Y. S., Nakato, E., O'Connor, M. B. and Nakato, H. (2021). Drosophila MOV10 regulates the termination of midgut regeneration. Genetics. PubMed ID: 33693718
The molecular mechanisms by which stem cell proliferation is precisely controlled during the course of regeneration are poorly understood. Namely, how a damaged tissue senses when to terminate the regeneration process, inactivates stem cell mitotic activity, and organizes ECM integrity remain fundamental unanswered questions. The Drosophila midgut intestinal stem cell (ISC) offers an excellent model system to study the molecular basis for stem cell inactivation. This study shows that a novel gene, CG6967 or dMOV10, is induced at the termination stage of midgut regeneration, and shows an inhibitory effect on ISC proliferation. dMOV10 encodes a putative component of the microRNA (miRNA) gene silencing complex (miRISC). The data, along with previous studies on the mammalian MOV10, suggest that dMOV10 is not a core member of miRISC, but modulates miRISC activity as an additional component. Further analyses identified direct target mRNAs of dMOV10-containing miRISC, including Daughter against Dpp (Dad), a known inhibitor of BMP/TGF-β signaling. RNAi knockdown of Dad significantly impaired ISC division during regeneration. Six miRNAs were identified that are induced at the termination stage and their potential target transcripts. One of these miRNAs, mir-1, is required for proper termination of ISC division at the end of regeneration. It is proposed that miRNA-mediated gene regulation contributes to the precise control of Drosophila midgut regeneration.
Siudeja, K., van den Beek, M., Riddiford, N., Boumard, B., Wurmser, A., Stefanutti, M., Lameiras, S. and Bardin, A. J. (2021). Unraveling the features of somatic transposition in the Drosophila intestine. Embo j: e106388. PubMed ID: 33634906
Transposable elements (TEs) play a significant role in evolution, contributing to genetic variation. However, TE mobilization in somatic cells is not well understood. This study addressed the prevalence of transposition in a somatic tissue, exploiting the Drosophila midgut as a model. Using whole-genome sequencing of in vivo clonally expanded gut tissue, hundreds of high-confidence somatic TE integration sites were mapped genome-wide. Somatic retrotransposon insertions were shown to be associated with inactivation of the tumor suppressor Notch, likely contributing to neoplasia formation. Moreover, applying Oxford Nanopore long-read sequencing technology, evidence is provided for tissue-specific differences in retrotransposition. Comparing somatic TE insertional activity with transcriptomic and small RNA sequencing data, this study demonstrates that transposon mobility cannot be simply predicted by whole tissue TE expression levels or by small RNA pathway activity. Finally, it was revealed that somatic TE insertions in the adult fly intestine are enriched in genic regions and in transcriptionally active chromatin. Together, these findings provide clear evidence of ongoing somatic transposition in Drosophila and delineate previously unknown features underlying somatic TE mobility in vivo. Titus, M. B., Wright, E. G., Bono, J. M., Poliakon, A. K., Goldstein, B. R., Super, M. K., Young, L. A., Manaj, M., Litchford, M., Reist, N. E., Killian, D. J. and Olesnicky, E. C. (2021). The conserved alternative splicing factor caper regulates neuromuscular phenotypes during development and aging. Dev Biol 473: 15-32. PubMed ID: 33508255
RNA-binding proteins play an important role in the regulation of post-transcriptional gene expression throughout the nervous system. This is underscored by the prevalence of mutations in genes encoding RNA splicing factors and other RNA-binding proteins in a number of neurodegenerative and neurodevelopmental disorders. The highly conserved alternative splicing factor Caper is widely expressed throughout the developing embryo and functions in the development of various sensory neural subtypes in the Drosophila peripheral nervous system. This study found that caper dysfunction leads to aberrant neuromuscular junction morphogenesis, as well as aberrant locomotor behavior during larval and adult stages. Despite its widespread expression, the results indicate that caper function is required to a greater extent within the nervous system, as opposed to muscle, for neuromuscular junction development and for the regulation of adult locomotor behavior. Moreover, Caper was found to interact with the RNA-binding protein Fmrp to regulate adult locomotor behavior. Finally, it was shown that caper dysfunction leads to various phenotypes that have both a sex and age bias, both of which are commonly seen in neurodegenerative disorders in humans.
Tsai, S. Y. and Huang, F. (2021). Acetyltransferase Enok regulates transposon silencing and piRNA cluster transcription. PLoS Genet 17(2): e1009349. PubMed ID: 33524038
The piRNA pathway is a highly conserved mechanism to repress transposon activation in the germline in Drosophila and mammals. This pathway starts from transcribing piRNA clusters to generate long piRNA precursors. The majority of piRNA clusters lack conventional promoters, and utilize heterochromatin- and HP1D/Rhino-dependent noncanonical mechanisms for transcription. However, information regarding the transcriptional regulation of piRNA clusters is limited. This study reports that the Drosophila acetyltransferase Enok, which can activate transcription by acetylating H3K23, is critical for piRNA production from 54% of piRNA clusters including 42AB, the major piRNA source. Surprisingly, it was found that Enok not only promotes rhino expression by acetylating H3K23, but also directly enhances transcription of piRNA clusters by facilitating Rhino recruitment. Taken together, this study provides novel insights into the regulation of noncanonical transcription at piRNA clusters and transposon silencing. Bouska, M. J. and Bai, H. (2021). Long noncoding RNA regulation of spermatogenesis via the spectrin cytoskeleton in Drosophila. G3 (Bethesda) 11(5). PubMed ID: 33720346
The spectrin cytoskeleton has been shown to be critical in diverse processes such as axon development and degeneration, myoblast fusion, and spermatogenesis. Spectrin can be modulated in a tissue specific manner through junctional protein complexes, however, it has not been shown that long noncoding RNAs (lncRNAs) interact with and modulate spectrin. This study provides evidence of a lncRNA CR45362 that interacts with α-Spectrin, is required for spermatid nuclear bundling during Drosophila spermatogenesis. It was observed that CR45362 showed high expression in the cyst cells at the basal testis, and CRISPR-mediated knockout of CR45362 led to sterile male, unbundled spermatid nuclei, and disrupted actin cones. Through chromatin isolation by RNA precipitation-mass spectrometry (ChIRP-MS), actin-spectrin cytoskeletal components were observed tophysically interact with the lncRNA CR45362. Genetic screening on identified cytoskeletal factors revealed that cyst cell-specific knockdown of α-Spectrin phenocopied CR45362 mutants and resulted in spermatid nuclear bundle defects. Consistently, CR45362 knockout disrupted the co-localization of α-Spectrin and spermatid nuclear bundles in the head cyst cells at the basal testis. Thus, this study uncovered a novel lncRNA CR45362 that interacts with α-Spectrin to stabilize spermatid nuclear bundles during spermatid maturation.
Wednesday, May 19th - Adult Neural Function
Zhang, Y., Zhou, Y., Zhang, X., Wang, L. and Zhong, Y. (2021). Clock neurons gate memory extinction in Drosophila. Curr Biol. PubMed ID: 33545046
Memory forms when a previously neutral stimulus (CS+) becomes competent to predict a biologically potent stimulus (US). However, if the CS+ is repeatedly presented without the US after the memory formation, this memory will be suppressed by newly formed extinction memory. The striking feature of extinction learning is that it requires repeated trials to robustly form extinction. Extended repetition only yields memories that remain transient in nature, thus imposing challenges in understanding the underlying mechanisms of extinction learning. This study took advantage of the versatile genetic tools and the well-characterized circadian system of Drosophila to link these unique features to clock neurons. Inhibiting the activity of clock neurons blocks the formation of extinction memory. Further investigation attributes this role to a subset of cryptochrome-positive dorsal neurons 1 (DN1s) and their downstream SIFamide neurons. The requirement of clock neurons from a gating mechanism of extinction for a single extinction learning trial robustly causes typical extinction when coupled with acute activation of DN1s, as marked by the initially enhanced but eventually diminished memory suppression. Accordingly, specific neural responses were detected to extinction training in a few DN1s via calcium imaging fulfilled by the TRIC tool, but not in dorsal neurons 2 or dorsolateral neurons. Based on these findings, it is proposed that in extinction of appetitive long-term memory, multiple trials of extinction learning robustly activate DN1 clock neurons to open the gate of extinction, which may contribute to the transient nature of extinction memory. Bohme, M. A., McCarthy, A. W., Blaum, N., Berezeckaja, M., Ponimaskine, K., Schwefel, D. and Walter, A. M. (2021). Glial Synaptobrevin mediates peripheral nerve insulation, neural metabolic supply, and is required for motor function. Glia. PubMed ID: 33811396
Peripheral nerves contain sensory and motor neuron axons coated by glial cells whose interplay ensures function, but molecular details are lacking. SNARE-proteins mediate the exchange and secretion of cargo by fusing vesicles with target organelles, but how glial SNAREs contribute to peripheral nerve function is largely unknown. This study identified non-neuronal Synaptobrevin (Syb) as the essential vesicular SNARE in Drosophila peripheral glia to insulate and metabolically supply neurons. Tetanus neurotoxin light chain (TeNT-LC), which potently inhibits SNARE-mediated exocytosis from neurons, also impairs peripheral nerve function when selectively expressed in glia, causing nerve disintegration, defective axonal transport, tetanic muscle hyperactivity, impaired locomotion, and lethality. While TeNT-LC disrupts neural function by cleaving neuronal Synaptobrevin (nSyb), it targets non-neuronal Synaptobrevin (Syb) in glia, which it cleaves at low rates: Glial knockdown of Syb (but not nSyb) phenocopied glial TeNT-LC expression whose effects were reverted by a TeNT-LC-insensitive Syb mutant. This study linked Syb-necessity to two distinct glial subtypes: Impairing Syb function in subperineurial glia disrupted nerve morphology, axonal transport, and locomotion, likely, because nerve-isolating septate junctions (SJs) could not form as essential SJ components (like the cell adhesion protein Neurexin-IV) were mistargeted. Interference with Syb in axon-encircling wrapping glia left nerve morphology and locomotion intact but impaired axonal transport. This study identifies crucial roles of Syb in various glial subtypes to ensure glial-glial and glial-neural interplay needed for proper nerve function, animal motility, and survival.
Baltruschat, L., Prisco, L., Ranft, P., Lauritzen, J. S., Fiala, A., Bock, D. D. and Tavosanis, G. (2021). Circuit reorganization in the Drosophila mushroom body calyx accompanies memory consolidation. Cell Rep 34(11): 108871. PubMed ID: 33730583
The formation and consolidation of memories are complex phenomena involving synaptic plasticity, microcircuit reorganization, and the formation of multiple representations within distinct circuits. To gain insight into the structural aspects of memory consolidation, this study focused on the calyx of the Drosophila mushroom body. In this essential center, essential for olfactory learning, second- and third-order neurons connect through large synaptic microglomeruli, which this study dissected at the electron microscopy level. Focusing on microglomeruli that respond to a specific odor, it was revealed that appetitive long-term memory results in increased numbers of precisely those functional microglomeruli responding to the conditioned odor. Hindering memory consolidation by non-coincident presentation of odor and reward, by blocking protein synthesis, or by including memory mutants suppress these structural changes, revealing their tight correlation with the process of memory consolidation. Thus, olfactory long-term memory is associated with input-specific structural modifications in a high-order center of the fly brain. Ackerman, S. D., Perez-Catalan, N. A., Freeman, M. R. and Doe, C. Q. (2021). Astrocytes close a motor circuit critical period. Nature 592(7854): 414-420. PubMed ID: 33828296
Critical periods-brief intervals during which neural circuits can be modified by activity-are necessary for proper neural circuit assembly. Extended critical periods are associated with neurodevelopmental disorders; however, the mechanisms that ensure timely critical period closure remain poorly understood. This study defined a critical period in a developing Drosophila motor circuit and identified astrocytes as essential for proper critical period termination. During the critical period, changes in activity regulate dendrite length, complexity and connectivity of motor neurons. Astrocytes invaded the neuropil just before critical period closure, and astrocyte ablation prolonged the critical period. Finally, a genetic screen was used to identify astrocyte-motor neuron signalling pathways that close the critical period, including Neuroligin-Neurexin signalling. Reduced signalling destabilized dendritic microtubules, increased dendrite dynamicity and impaired locomotor behaviour, underscoring the importance of critical period closure. Previous work defined astroglia as regulators of plasticity at individual synapses; this study shows that astrocytes also regulate motor circuit critical period closure to ensure proper locomotor behaviour.
Bennett, J. E. M., Philippides, A. and Nowotny, T. (2021). Learning with reinforcement prediction errors in a model of the Drosophila mushroom body. Nat Commun 12(1): 2569. PubMed ID: 33963189
Effective decision making in a changing environment demands that accurate predictions are learned about decision outcomes. In Drosophila, such learning is orchestrated in part by the mushroom body, where dopamine neurons signal reinforcing stimuli to modulate plasticity presynaptic to mushroom body output neurons. Building on previous mushroom body models, in which dopamine neurons signal absolute reinforcement, it is proposed instead that dopamine neurons signal reinforcement prediction errors by utilising feedback reinforcement predictions from output neurons. Plasticity rules were formulated that minimise prediction errors, verify that output neurons learn accurate reinforcement predictions in simulations, and postulate connectivity that explains more physiological observations than an experimentally constrained model. The constrained and augmented models reproduce a broad range of conditioning and blocking experiments, and this study demonstrated that the absence of blocking does not imply the absence of prediction error dependent learning. These results provide five predictions that can be tested using established experimental methods. Bornstein, B., Meltzer, H., Adler, R., Alyagor, I., Berkun, V., Cummings, G., Reh, F., Keren-Shaul, H., David, E., Riemensperger, T. and Schuldiner, O. (2021). Transneuronal Dpr12/DIP-delta interactions facilitate compartmentalized dopaminergic innervation of Drosophila mushroom body axons. Embo J: e105763. PubMed ID: 33847376
The mechanisms controlling wiring of neuronal networks are not completely understood. The stereotypic architecture of the Drosophila mushroom body (MB) offers a unique system to study circuit assembly. The adult medial MB γ-lobe is comprised of a long bundle of axons that wire with specific modulatory and output neurons in a tiled manner, defining five distinct zones. The immunoglobulin superfamily protein Dpr12 is cell-autonomously required in γ-neurons for their developmental regrowth into the distal γ4/5 zones, where both Dpr12 and its interacting protein, DIP-δ, are enriched. DIP-δ functions in a subset of dopaminergic neurons that wire with γ-neurons within the γ4/5 zone. During metamorphosis, these dopaminergic projections arrive to the γ4/5 zone prior to γ-axons, suggesting that γ-axons extend through a prepatterned region. Thus, Dpr12/DIP-γ transneuronal interaction is required for γ4/5 zone formation. This study sheds light onto molecular and cellular mechanisms underlying circuit formation within subcellular resolution.
Tuesday - May 18th - Cytoskeleton and junctions
Yoon, J., Wu, H., Hung, R. J. and Terman, J. R. (2021). Enhanced Production of the Mical Redox Domain for Enzymology and F-actin Disassembly Assays. Int J Mol Sci 22(4). PubMed ID: 33671465
To change their behaviors, cells require actin proteins to assemble together into long polymers/filaments-and so a critical goal is to understand the factors that control this actin filament (F-actin) assembly and stability. This study has identified a family of unusual actin regulators, the MICALs, which are flavoprotein monooxygenase/hydroxylase enzymes that associate with flavin adenine dinucleotide (FAD) and use the co-enzyme nicotinamide adenine dinucleotide phosphate (NADPH) in Redox reactions. F-actin is a specific substrate for these MICAL Redox enzymes, which oxidize specific amino acids within actin to destabilize actin filaments. Furthermore, this MICAL-catalyzed reaction is reversed by another family of Redox enzymes (SelR/MsrB enzymes)-thereby revealing a reversible Redox signaling process and biochemical mechanism regulating actin dynamics. Interestingly, in addition to the MICALs' Redox enzymatic portion through which MICALs covalently modify and affect actin, MICALs have multiple other domains. Less is known about the roles of these other MICAL domains. This study provides approaches for obtaining high levels of recombinant protein for the Redox only portion of Mical and demonstrate its catalytic and F-actin disassembly activity. These results provide a ground state for future work aimed at defining the role of the other domains of Mical - including characterizing their effects on Mical's Redox enzymatic and F-actin disassembly activity. Alhadyian, H., Shoaib, D. and Ward, R. E. (2021). Septate junction proteins are required for egg elongation and border cell migration during oogenesis in Drosophila. G3 (Bethesda). PubMed ID: 33871584
Protein components of the invertebrate occluding junction-known as the septate junction (SJ) - are required for morphogenetic developmental events during embryogenesis in Drosophila melanogaster. In order to determine whether SJ proteins are similarly required for morphogenesis during other developmental stages, this study investigated the localization and requirement of four representative SJ proteins during oogenesis: Contactin, Macroglobulin complement-related, Neurexin IV, and Coracle. A number of morphogenetic processes occur during oogenesis, including egg elongation, formation of dorsal appendages, and border cell migration. All four SJ proteins are expressed in egg chambers throughout oogenesis, with the highest and most sustained levels in the follicular epithelium (FE). In the FE, SJ proteins localize along the lateral membrane during early and mid-oogenesis, but become enriched in an apical-lateral domain (the presumptive SJ) by stage 10B. SJ protein relocalization requires the expression of other SJ proteins, as well as Rab5 and Rab11 in a manner similar to SJ biogenesis in the embryo. Knocking down the expression of these SJ proteins in follicle cells throughout oogenesis results in egg elongation defects and abnormal dorsal appendages. Similarly, reducing the expression of SJ genes in the border cell cluster results in border cell migration defects. Together, these results demonstrate an essential requirement for SJ genes in morphogenesis during oogenesis, and suggests that SJ proteins may have conserved functions in epithelial morphogenesis across developmental stages.
Tavares, L., Gracio, P., Ramos, R., Traquete, R., Relvas, J. B. and Pereira, P. S. (2021). The Pebble/Rho1/Anillin pathway controls polyploidization and axonal wrapping activity in the glial cells of the Drosophila eye. Dev Biol 473: 90-96. PubMed ID: 33581137
During development glial cells are crucially important for the establishment of neuronal networks. Proliferation and migration of glial cells can be modulated by neurons, and in turn glial cells can differentiate to assume key roles such as axonal wrapping and targeting. To explore the roles of actin cytoskeletal rearrangements in glial cells, the function of Rho1 was studied in Drosophila developing visual system. The Pebble (RhoGEF)/Rho1/Anillin pathway is required for glia proliferation and to prevent the formation of large polyploid perineurial glial cells, which can still migrate into the eye disc if generated. Surprisingly, this Rho1 pathway is not necessary to establish the total glial membrane area or for the differentiation of the polyploid perineurial cells. The resulting polyploid wrapping glial cells are able to initiate wrapping of axons in the basal eye disc, however the arrangement and density of glia nuclei and membrane processes in the optic stalk are altered and the ensheathing of the photoreceptor axonal fascicles is reduced. Shoda, T., Yamazoe, K., Tanaka, Y., Asano, Y. and Inoue, Y. H. (2021). Orbit/CLASP determines centriole length by antagonising Klp10A in Drosophila spermatocytes. J Cell Sci. PubMed ID: 33674447
After centrosome duplication, centrioles elongate before the M phase. To identify genes required for this process and understand the regulatory mechanism, the centrioles in Drosophila premeiotic spermatocytes, expressing fluorescently tagged centrioles, were investigated. An essential microtubule polymerisation factor, Orbit/CLASP, was demonstrated to accumulate at the distal end of centrioles and was required for the elongation. Conversely, a microtubule severing factor, Klp10A, shortened the centrioles. Genetic analyses revealed that these two proteins functioned antagonistically for determining centriole length. Furthermore, Cp110 in the distal tip complex was closely associated with the factors involved in centriolar dynamics at the distal end. Loss of centriole integrity, including fragmentation of centrioles and earlier separation of the centriole pairs was observed in Cp110 null mutant cells either overexpressing Orbit or harbouring Klp10A depletion. Excess centriole elongation in the absence of the distal tip complex resulted in the loss of centriole integrity, leading to the formation of multipolar spindle microtubules emanating from centriole fragments, even when they are unpaired. These findings contribute to understanding the mechanism of centriole integrity, leading to chromosome instability in cancer cells.
Tian, Y., Wei, C., He, J., Yan, Y., Pang, N., Fang, X., Liang, X. and Fu, J. (2021). Superresolution characterization of core centriole architecture. J Cell Biol 220(4). PubMed ID: 33533934
The centrosome is the main microtubule-organizing center in animal cells. It comprises of two centrioles and the surrounding pericentriolar material. Protein organization at the outer layer of the centriole and outward has been studied extensively; however, an overall picture of the protein architecture at the centriole core has been missing. This study reports a direct view of Drosophila centriolar proteins at ∼50-nm resolution. This reveals a Sas6 ring at the C-terminus, where it overlaps with the C-terminus of Cep135. The ninefold symmetrical pattern of Cep135 is further conveyed through Ana1-Asterless axes that extend past the microtubule wall from between the blades. Ana3 and Rcd4, whose termini are close to Cep135, are arranged in ninefold symmetry that does not match the above axes. During centriole biogenesis, Ana3 and Rcd4 are sequentially loaded on the newly formed centriole and are required for centriole-to-centrosome conversion through recruiting the Cep135-Ana1-Asterless complex. Together, these results provide a spatiotemporal map of the centriole core and implications of how the structure might be built. Badmos, H., Cobbe, N., Campbell, A., Jackson, R. and Bennett, D. (2021). Drosophila USP22/nonstop polarizes the actin cytoskeleton during collective border cell migration. J Cell Biol 220(7). PubMed ID: 33988679
Polarization of the actin cytoskeleton is vital for the collective migration of cells in vivo. During invasive border cell migration in Drosophila, actin polarization is directly controlled by the Hippo signaling complex, which resides at contacts between border cells in the cluster. This study identified, in a genetic screen for deubiquitinating enzymes involved in border cell migration, an essential role for nonstop/USP22 in the expression of Hippo pathway components expanded and merlin. Loss of nonstop function consequently leads to a redistribution of F-actin and the polarity determinant Crumbs, loss of polarized actin protrusions, and tumbling of the border cell cluster. Nonstop is a component of the Spt-Ada-Gcn5-acetyltransferase (SAGA) transcriptional coactivator complex, but SAGA's histone acetyltransferase module, which does not bind to Expanded or Merlin, is dispensable for migration. Taken together, these results uncover novel roles for SAGA-independent nonstop/USP22 in collective cell migration, which may help guide studies in other systems where USP22 is necessary for cell motility and invasion.
Monday, May 17th - Disease Models
Abreha, M. H., Ojelade, S., Dammer, E. B., McEachin, Z. T., Duong, D. M., Gearing, M., Bassell, G. J., Lah, J. J., Levey, A. I., Shulman, J. M. and Seyfried, N. T. (2021). TBK1 interacts with tau and enhances neurodegeneration in tauopathy. J Biol Chem: 100760. PubMed ID: 33965374
One of the defining pathological features of Alzheimer's Disease (AD) is the deposition of neurofibrillary tangles (NFTs) composed of hyperphosphorylated tau (see Drosophila Tau) in the brain. Aberrant activation of kinases in AD has been suggested to enhance phosphorylation and toxicity of tau, making the responsible tau kinases attractive therapeutic targets. The full complement of tau interacting kinases in AD brain and their activity in disease remains incompletely defined. In this study, immunoaffinity enrichment coupled with mass spectrometry (MS) identified TANK-binding kinase 1 (TBK1) as a tau-interacting partner in human AD cortical brain tissues. This interaction was validated in human AD, familial frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) caused by mutations in MAPT (R406W & P301L) and corticobasal degeneration (CBD) postmortem brain tissues as well as human cell lines. Further, this study documented increased TBK1 activation in both AD and FTDP-17 and map TBK1 phosphorylation sites on tau based on in vitro kinase assays coupled to MS. Lastly, in a Drosophila tauopathy model, activating expression of a conserved TBK1 ortholog (I-kappaB kinase ε) triggers tau hyperphosphorylation and enhanced neurodegeneration, whereas knockdown had the reciprocal effect, suppressing tau toxicity. Collectively, these findings suggest that increased TBK1 activation may promote tau hyperphosphorylation and neuronal loss in AD and related tauopathies. Akan, I., Halim, A., Vakhrushev, S. Y., Clausen, H. and Hanover, J. A. (2021). Drosophila O-GlcNAcase Mutants Reveal an Expanded Glycoproteome and Novel Growth and Longevity Phenotypes. Cells 10(5). PubMed ID: 33925313
The reversible posttranslational O-GlcNAc modification of serine or threonine residues of intracellular proteins is involved in many cellular events from signaling cascades to epigenetic and transcriptional regulation. O-GlcNAcylation is a conserved nutrient-dependent process involving two enzymes, with O-GlcNAc transferase (OGT) adding O-GlcNAc and with O-GlcNAcase (OGA) removing it in a manner that's protein- and context-dependent. O-GlcNAcylation is essential for epigenetic regulation of gene expression through its action on Polycomb and Trithorax and COMPASS complexes. However, the important role of O-GlcNAc in adult life and health span has been largely unexplored, mainly due the lack of available model systems. Cataloging the O-GlcNAc proteome has proven useful in understanding the biology of this modification in vivo. In this study, a recently developed oga knockout fly mutant was leveraged to identify the O-GlcNAcylated proteins in adult Drosophila melanogaster. The adult O-GlcNAc proteome revealed many proteins related to cell and organismal growth, development, differentiation, and epigenetics. Many O-GlcNAcylated proteins were identified that play a role in increased growth and decreased longevity, including HCF, SIN3A, LOLA, KISMET, ATX2, SHOT, and FOXO. Interestingly, oga mutant flies are larger and have a shorter life span compared to wild type flies, suggesting increased O-GlcNAc results in increased growth. These results suggest that O-GlcNAc alters the function of many proteins related to transcription, epigenetic modification and signaling pathways that regulate growth rate and longevity. Therefore, these findings highlight the importance of O-GlcNAc in growth and life span in adult Drosophila.
Atilano, M. L., Grpnke, S., Niccoli, T., Kempthorne, L., Hahn, O., Moron-Oset, J., Hendrich, O., Dyson, M., Adams, M. L., Hull, A., Salcher-Konrad, M. T., Monaghan, A., Bictash, M., Glaria, I., Isaacs, A. M. and Partridge, L. (2021). Enhanced insulin signalling ameliorates C9orf72 hexanucleotide repeat expansion toxicity in Drosophila. Elife 10. PubMed ID: 33739284
G4C2 repeat expansions within the C9orf72 gene are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The repeats undergo repeat-associated non-ATG translation to generate toxic dipeptide repeat proteins. This study shows that insulin/IGF signalling is reduced in fly models of C9orf72 repeat expansion using RNA sequencing of adult brain. It was further demonstrated that activation of insulin/IGF signalling can mitigate multiple neurodegenerative phenotypes in flies expressing either expanded G4C2 repeats or the toxic dipeptide repeat protein poly-GR. Levels of poly-GR are reduced when components of the insulin/IGF signalling pathway are genetically activated in the diseased flies, suggesting a mechanism of rescue. Modulating insulin signalling in mammalian cells also lowers poly-GR levels. Remarkably, systemic injection of insulin improves the survival of flies expressing G4C2 repeats. Overall, these data suggest that modulation of insulin/IGF signalling could be an effective therapeutic approach against C9orf72 ALS/FTD. Yeom, E., Shin, H., Yoo, W., Jun, E., Kim, S., Hong, S. H., Kwon, D. W., Ryu, T. H., Suh, J. M., Kim, S. C., Lee, K. S. and Yu, K. (2021). Tumour-derived Dilp8/INSL3 induces cancer anorexia by regulating feeding neuropeptides via Lgr3/8 in the brain. Nat Cell Biol 23(2): 172-183. PubMed ID: 33558728
In patients with advanced-stage cancer, cancer-associated anorexia affects treatment success and patient survival. However, the underlying mechanism is poorly understood. This study shows that Dilp8, a Drosophila homologue of mammalian insulin-like 3 peptide (INSL3), is secreted from tumour tissues and induces anorexia through the Lgr3 receptor in the brain. Activated Dilp8-Lgr3 signalling upregulated anorexigenic nucleobinding 1 (NUCB1) and downregulated orexigenic short neuropeptide F (sNPF) and NPF expression in the brain. In the cancer condition, the protein expression of Lgr3 and NUCB1 was significantly upregulated in neurons expressing sNPF and NPF. INSL3 levels were increased in tumour-implanted mice and INSL3-treated mouse hypothalamic cells showed Nucb2 upregulation and Npy downregulation. Food consumption was significantly reduced in intracerebrospinal INSL3-injected mice. In patients with pancreatic cancer, higher serum INSL3 levels increased anorexia. These results indicate that tumour-derived Dilp8/INSL3 induces cancer anorexia by regulating feeding hormones through the Lgr3/Lgr8 receptor in Drosophila and mammals.
Wei, T., Ji, X., Gao, Y., Zhu, X. and Xiao, G. (2021). ZnT7 RNAi favors Raf(GOF)scrib(-/-)-induced tumor growth and invasion in Drosophila through JNK signaling pathway. Oncogene. PubMed ID: 33649534
The disruption of zinc homeostasis has been identified in patients suffering from various cancers, but a causative relationship has not yet been established. Drosophila melanogaster has become a powerful model to study cancer biology. Using a Drosophila model of malignant tumor Raf(GOF)scrib(-/-), it was observed that the tumor growth, invasion and migration were enhanced by silencing dZnT7, a zinc transporter localized on the Golgi apparatus. Further study indicated that the zinc deficiency in Golgi of dZnT7 RNAi resulted in ER stress which could activate the c-Jun-N-terminal Kinase (JNK) signaling and this process is mediated by Atg9. Lastly, it was demonstrated that the exacerbation of dZnT7 RNAi on tumor was promoted by JNK signaling-dependent cell autonomous and non-autonomous autophagy. These findings suggest that zinc homeostasis in secretory compartments may provide a new therapeutic target for tumor treatment. Yatsenko, A. S., Kucherenko, M. M., Xie, Y., Urlaub, H. and Shcherbata, H. R. (2021). Exocyst-mediated membrane trafficking of the lissencephaly-associated ECM receptor dystroglycan is required for proper brain compartmentalization. Elife 10. PubMed ID: 33620318
To assemble a brain, differentiating neurons must make proper connections and establish specialized brain compartments. Abnormal levels of cell adhesion molecules disrupt these processes. Dystroglycan (Dg) is a major non-integrin cell adhesion receptor, deregulation of which is associated with dramatic neuroanatomical defects such as lissencephaly type II or cobblestone brain. The previously established Drosophila model for cobblestone lissencephaly was used to understand how Dg is regulated in the brain. During development, Dg has a spatiotemporally dynamic expression pattern, fine-tuning of which is crucial for accurate brain assembly. In addition, mass spectrometry analyses identified numerous components associated with Dg in neurons, including several proteins of the exocyst complex. Data show that exocyst-based membrane trafficking of Dg allows its distinct expression pattern, essential for proper brain morphogenesis. Further studies of the Dg neuronal interactome will allow identification of new factors involved in the development of dystroglycanopathies and advance disease diagnostics in humans.
Friday, May 14th - Behavior
Ruiz, C. and Theobald, J. C. (2021). Stabilizing responses to sideslip disturbances in Drosophila melanogaster are modulated by the density of moving elements on the ground. Biol Lett 17(3): 20200748. PubMed ID: 33653094
Stabilizing responses to sideslip disturbances are a critical part of the flight control system in flies. While strongly mediated by mechanoreception, much of the final response results from the wide-field motion detection system associated with vision. In order to be effective, these responses must match the disturbance they are aimed to correct. To do this, flies must estimate the velocity of the disturbance, although it is not known how they accomplish this task when presented with natural images or dot fields. The recent finding, that motion parallax in dot fields can modulate stabilizing responses only if perceived below the fly, raises the question of whether other image statistics are also processed differently between eye regions. One such parameter is the density of elements moving in translational optic flow. Depending on the habitat, there might be strong differences in the density of elements providing information about self-motion above and below the fly, which in turn could act as selective pressures tuning the visual system to process this parameter on a regional basis. By presenting laterally moving dot fields of different densities this study found that, in Drosophila melanogaster, the amplitude of the stabilizing response is significantly affected by the number of elements in the field of view. Flies countersteer strongly within a relatively low and narrow range of element densities. But this effect is exclusive to the ventral region of the eye, and dorsal stimuli elicit an unaltered and stereotypical response regardless of the density of elements in the flow. This highlights local specialization of the eye and suggests the lower region may play a more critical role in translational flight stabilization. Polak, M., Hurtado-Gonzales, J. L., Benoit, J. B., Hooker, K. J. and Tyler, F. (2021). Positive genetic covariance between male sexual ornamentation and fertilizing capacity. Curr Biol. PubMed ID: 33567290
Postcopulatory sexual selection results from variation in competitive fertilization success among males and comprises powerful evolutionary forces that operate after the onset of mating. Theoretical advances in the field of sexual selection addressing the buildup and coevolutionary consequences of genetic coupling motivate the hypothesis that indirect postcopulatory sexual selection may promote evolution of male secondary sexual traits-those traits traditionally ascribed to mate choice and male fighting. A crucial prediction of this hypothesis is genetic covariance between trait expression and competitive fertilization success, which has been predicted to arise, for example, when traits subject to pre- and postcopulatory sexual selection are under positive correlational selection. This study imposed bidirectional artificial selection on male ornament (sex comb) size in Drosophila bipectinata and demonstrated increased competitive fertilization success as a correlated evolutionary response to increasing ornament size. Transcriptional analyses revealed that levels of specific seminal fluid proteins repeatedly shifted in response to this selection, suggesting that properties of the ejaculate, rather than the enlarged sex comb itself, contributed fertilizing capacity. Ultraprecise laser surgery was used to reduce ornament size of high-line males and found that their fertilizing superiority persisted despite the size reduction, reinforcing the transcriptional results. The data support the existence of positive genetic covariance between a male secondary sexual trait and competitive fertilization success, and suggest the possibility that indirect postcopulatory sexual selection may, under certain conditions, magnify net selection on ornamental trait expression.
Wan, G., Hayden, A. N., Iiams, S. E. and Merlin, C. (2021). Cryptochrome 1 mediates light-dependent inclination magnetosensing in monarch butterflies. Nat Commun 12(1): 771. PubMed ID: 33536422
Many animals use the Earth's geomagnetic field for orientation and navigation. Yet, the molecular and cellular underpinnings of the magnetic sense remain largely unknown. A biophysical model proposed that magnetoreception can be achieved through quantum effects of magnetically-sensitive radical pairs formed by the photoexcitation of cryptochrome (CRY) proteins. Studies in Drosophila are the only ones to date to have provided compelling evidence for the ultraviolet (UV)-A/blue light-sensitive type 1 CRY (CRY1) involvement in animal magnetoreception, and surprisingly extended this discovery to the light-insensitive mammalian-like type 2 CRYs (CRY2s) of both monarchs and humans. This study shows that monarchs respond to a reversal of the inclination of the Earth's magnetic field in an UV-A/blue light and CRY1, but not CRY2, dependent manner. It was further demonstrated that both antennae and eyes, which express CRY1, are magnetosensory organs. This work argues that only light-sensitive CRYs function in animal light-dependent inclination-based magnetic sensing. Walkowicz, L., Krzeptowski, W., Krzeptowska, E., Warzecha, K., Salek, J., Gorska-Andrzejak, J. and Pyza, E. (2021). Glial expression of DmMANF is required for the regulation of activity, sleep and circadian rhythms in the visual system of Drosophila melanogaster. Eur J Neurosci. PubMed ID: 33666288
DmMANF, Drosophila melanogaster mesencephalic astrocyte-derived neurotrophic factor, is an evolutionarily conserved orthologue of mammalian MANF. This neurotrophic factor exerts many functions in the Drosophila brain, particularly those dependent on glial cells. An earlier study found that downregulation of DmMANF in glia induces degeneration of glial cells in the first optic neuropil (lamina) where DmMANF abundance is especially high. The present study observed that changes in the level of DmMANF in two types of glia, astrocyte-like glia (AlGl) and ensheathing glia (EnGl), affect activity and sleep of flies. Interestingly, a proper level of DmMANF in AlGl seems to be important in guiding processes of pigment dispersing factor (PDF)-expressing clock neurons. This is supported by THE finding that DmMANF overexpression in AlGl leads to structural changes in the architecture of the PDF-positive arborisation in the brain. Finally, it was detected that DmMANF also affects rhythms in glia themselves, since circadian oscillations in expression of the catalytic α subunit of the sodium pump in the lamina epithelial glia were abolished after DmMANF silencing. DmMANF-expressed in AlGL and EnGl seems to affect activity of neurons leading to changes in behaviour.
Zhu, M. L., Herrera, K. J., Vogt, K. and Bahl, A. (2021). Navigational strategies underlying temporal phototaxis in Drosophila larvae. J Exp Biol. PubMed ID: 33954778
Navigating across light gradients is essential for survival for many animals. However, there is still a poor understanding of the algorithms that underlie such behaviors. This study developed a novel closed-loop phototaxis assay for Drosophila larvae in which light intensity is always spatially uniform but updates depending on the location of the animal in the arena. Even though larvae can only rely on temporal cues during runs, this study finds that they are capable of finding preferred areas of low light intensity. Further detailed analysis of their behavior reveals that larvae turn more frequently and that heading angle changes increase when they experience brightness increments over extended periods of time. It is suggested that temporal integration of brightness change during runs is an important - and so far largely unexplored - element of phototaxis. Rauscher, M. J. and Fox, J. L. (2021). Haltere and visual inputs sum linearly to predict wing (but not gaze) motor output in tethered flying Drosophila. Proc Biol Sci 288(1943): 20202374. PubMed ID: 33499788
In the true flies (Diptera), the hind wings have evolved into specialized mechanosensory organs known as halteres, which are sensitive to gyroscopic and other inertial forces. Together with the fly's visual system, the halteres direct head and wing movements through a suite of equilibrium reflexes that are crucial to the fly's ability to maintain stable flight. As in other animals (including humans), this presents challenges to the nervous system as equilibrium reflexes driven by the inertial sensory system must be integrated with those driven by the visual system in order to control an overlapping pool of motor outputs shared between the two of them. This study introduced an experimental paradigm for reproducibly altering haltere stroke kinematics and used it to quantify multisensory integration of wing and gaze equilibrium reflexes. Multisensory wing-steering responses reflect a linear superposition of haltere-driven and visually driven responses, but multisensory gaze responses are not well predicted by this framework. These models, based on populations, extend also to the responses of individual flies.
Thursday, May 13th - Gonad Development
Weaver, L. N. and Drummond-Barbosa, D. (2021). Hormone receptor 4 is required in muscles and distinct ovarian cell types to regulate specific steps of Drosophila oogenesis. Development 148(5). PubMed ID: 33547134
The conserved nuclear receptor superfamily has crucial roles in many processes, including reproduction. Nuclear receptors with known roles in oogenesis have been studied mostly in the context of their ovary-intrinsic requirement. Recent studies in Drosophila, however, have begun to reveal new roles of nuclear receptor signaling in peripheral tissues in controlling reproduction. This study identified Hormone receptor 4 (Hr4) as an oogenesis regulator required in the ovary and muscles. Global Hr4 knockdown leads to increased germline stem cell (GSC) loss, reduced GSC proliferation, early germline cyst death, slowed follicle growth and vitellogenic follicle degeneration. Tissue-specific knockdown experiments uncovered ovary-intrinsic and peripheral tissue requirements for Hr4. In the ovary, Hr4 is required in the niche for GSC proliferation and in the germline for GSC maintenance. Hr4 functions in muscles to promote GSC maintenance and follicle growth. The specific tissues that require Hr4 for survival of early germline cysts and vitellogenic follicles remain unidentified. These results add to the few examples of muscles controlling gametogenesis and expand understanding of the complexity of nuclear receptor regulation of various aspects of oogenesis. Zhang, C., Daubnerova, I., Jang, Y. H., Kondo, S., Zitnan, D. and Kim, Y. J. (2021). The neuropeptide allatostatin C from clock-associated DN1p neurons generates the circadian rhythm for oogenesis. Proc Natl Acad Sci U S A 118(4). PubMed ID: 33479181
The link between the biological clock and reproduction is evident in most metazoans. The fruit fly Drosophila melanogaster, a key model organism in the field of chronobiology because of its well-defined networks of molecular clock genes and pacemaker neurons in the brain, shows a pronounced diurnal rhythmicity in oogenesis. Still, it is unclear how the circadian clock generates this reproductive rhythm. A subset of the group of neurons designated "posterior dorsal neuron 1" (DN1p), which are among the ∼150 pacemaker neurons in the fly brain, produces the neuropeptide allatostatin C (AstC-DN1p). This study reports that six pairs of AstC-DN1p send inhibitory inputs to the brain insulin-producing cells, which express two AstC receptors, star1 and AICR2. Consistent with the roles of insulin/insulin-like signaling in oogenesis, activation of AstC-DN1p suppresses oogenesis through the insulin-producing cells. This study shows evidence that AstC-DN1p activity plays a role in generating an oogenesis rhythm by regulating juvenile hormone and vitellogenesis indirectly via insulin/insulin-like signaling. AstC is orthologous to the vertebrate neuropeptide somatostatin (SST). Like AstC, SST inhibits gonadotrophin secretion indirectly through gonadotropin-releasing hormone neurons in the hypothalamus. The functional and structural conservation linking the AstC and SST systems suggest an ancient origin for the neural substrates that generate reproductive rhythms.
Palacios, V., Kimble, G. C., Tootle, T. L. and Buszczak, M. (2021). Importin-9 regulates chromosome segregation and packaging in Drosophila germ cells. J Cell Sci. PubMed ID: 33632744
Germ cells undergo distinct nuclear processes as they differentiate into gametes. While these events must be coordinated to ensure proper maturation, the stage-specific transport of proteins in and out of germ cell nuclei remains incompletely understood. Efforts to genetically characterize Drosophila genes that exhibit enriched expression in germ cells led to the finding that loss of the highly-conserved Importin β/karyopherin family member Importin-9 (Ipo9) results in female and male sterility. Immunofluorescence and fluorescent in situ hybridization (FISH) revealed that Ipo9 (KO) mutants display chromosome condensation and segregation defects during meiosis. In addition, Ipo9 (KO) mutant males form abnormally structured sperm and fail to properly exchange histones for protamines. Ipo9 physically interacts with proteasome proteins and Ipo9 mutant males exhibit disruption of the nuclear localization of several proteasome components. Thus, Ipo9 coordinates the nuclear import of functionally related factors necessary for the completion of gametogenesis. McDonough-Goldstein, C. E., Pitnick, S. and Dorus, S. (2021). Drosophila oocyte proteome composition covaries with female mating status. Sci Rep 11(1): 3142. PubMed ID: 33542461
Oocyte composition can directly influence offspring fitness, particularly in oviparous species such as most insects, where it is the primary form of parental investment. Oocyte production is also energetically costly, dependent on female condition and responsive to external cues. This study investigated whether mating influences mature oocyte composition in Drosophila melanogaster using a quantitative proteomic approach. The analyses robustly identified 4,485 oocyte proteins and revealed that stage-14 oocytes from mated females differed significantly in protein composition relative to oocytes from unmated females. Proteins forming a highly interconnected network enriched for translational machinery and transmembrane proteins were increased in oocytes from mated females, including calcium binding and transport proteins. This mating-induced modulation of oocyte maturation was also significantly associated with proteome changes that are known to be triggered by egg activation. It is proposed that these compositional changes are likely to have fitness consequences and adaptive implications given the importance of oocyte protein composition, rather than active gene expression, to the maternal-to-zygotic transition and early embryogenesis.
Shapiro-Kulnane, L., Bautista, O. and Salz, H. K. (2021). An RNA-interference screen in Drosophila to identify ZAD-containing C2H2 zinc finger genes that function in female germ cells. G3 (Bethesda) 11(1). PubMed ID: 33561227
The zinc finger-associated domain (ZAD) is present in over 90 C2H2 zinc finger (ZNF) proteins. Despite their abundance, only a few ZAD-ZNF genes have been characterized to date. This study systematically analyzed the function of 68 ZAD-ZNF genes in Drosophila female germ cells by performing an in vivo RNA-interference screen. Eight ZAD-ZNF genes required for oogenesis were identified, and based on further characterization of the knockdown phenotypes, defects broadly consistent with functions in germ cell specification and/or survival, early differentiation, and egg chamber maturation were uncovered. These results provide a candidate pool for future studies aimed at functionalization of this large but poorly characterized gene family. Van De Bor, V., Loreau, V., Malbouyres, M., Cerezo, D., Placenti, A., Ruggiero, F. and Noselli, S. (2021). A dynamic and mosaic basement membrane controls cell intercalation in Drosophila ovaries. Development 148(4). PubMed ID: 33526583
Basement membranes (BM) are extracellular matrices assembled into complex and highly organized networks essential for organ morphogenesis and function. However, little is known about the tissue origin of BM components and their dynamics in vivo, This study unravel the assembly and role of the BM main component, Collagen type IV (ColIV), in Drosophila ovarian stalk morphogenesis. Stalks are short strings of cells assembled through cell intercalation that link adjacent follicles and maintain ovarian integrity. Stalk ColIV has multiple origins and is assembled following a regulated pattern leading to a unique BM organisation. Absence of ColIV leads to follicle fusion, as observed upon ablation of stalk cells. ColIV and integrins are both required to trigger cell intercalation and maintain mechanically strong cell-cell attachment within the stalk. These results show how the dynamic assembly of a mosaic BM controls complex tissue morphogenesis and integrity.
Wednesday, May 12th - Adult neural development and function
Wu, J., Ji, X., Gu, Q., Liao, B., Dong, W. and Han, J. (2021). Parallel Synaptic Acetylcholine Signals Facilitate Large Monopolar Cell Repolarization and Modulate Visual Behavior in Drosophila. J Neurosci 41(10): 2164-2176. PubMed ID: 33468565
Appropriate termination of the photoresponse in image-forming photoreceptors and downstream neurons is critical for an animal to achieve high temporal resolution. Although the cellular and molecular mechanisms of termination in image-forming photoreceptors have been extensively studied in Drosophila, the underlying mechanism of termination in their downstream large monopolar cells remains less explored. This study shows that synaptic ACh signaling, from both amacrine cells (ACs) and L4 neurons, facilitates the rapid repolarization of L1 and L2 neurons. Intracellular recordings in female flies show that blocking synaptic ACh output from either ACs or L4 neurons leads to slow repolarization of L1 and L2 neurons. Genetic and electrophysiological studies in both male and female flies determine that L2 neurons express ACh receptors and directly receive ACh signaling. Moreover, the results demonstrate that synaptic ACh signaling from both ACs and L4 neurons simultaneously facilitates ERG termination. Finally, visual behavior studies in both male and female flies show that synaptic ACh signaling, from either ACs or L4 neurons to L2 neurons, is essential for the optomotor response of the flies in high-frequency light stimulation. This study identifies parallel synaptic ACh signaling for repolarization of L1 and L2 neurons and demonstrates that synaptic ACh signaling facilitates L1 and L2 neuron repolarization to maintain the optomotor response of the fly on high-frequency light stimulation. Zandawala, M., Nguyen, T., Balanya Segura, M., Johard, H. A. D., Amcoff, M., Wegener, C., Paluzzi, J. P. and Nassel, D. R. (2021). A neuroendocrine pathway modulating osmotic stress in Drosophila. PLoS Genet 17(3): e1009425. PubMed ID: 33684132
This study identified a peptidergic pathway that links these types of regulatory functions. The neuropeptide Corazonin (Crz), previously implicated in responses to metabolic stress, as a neuroendocrine factor that inhibits the release of a diuretic hormone, CAPA, and thereby modulates the tolerance to osmotic and ionic stress. Both knockdown of Crz and acute injections of Crz peptide impact desiccation tolerance and recovery from chill-coma. Mapping of the Crz receptor (CrzR) expression identified three pairs of Capa-expressing neurons (Va neurons) in the ventral nerve cord that mediate these effects of Crz. Crz acts to restore water/ion homeostasis by inhibiting release of CAPA neuropeptides via inhibition of cAMP production in Va neurons. Knockdown of CrzR in Va neurons affects CAPA signaling, and consequently increases tolerance for desiccation, ionic stress and starvation, but delays chill-coma recovery. Optogenetic activation of Va neurons stimulates excretion and simultaneous activation of Crz and CAPA-expressing neurons reduces this response, supporting the inhibitory action of Crz. Thus, Crz inhibits Va neurons to maintain osmotic and ionic homeostasis, which in turn affects stress tolerance. Earlier work demonstrated that systemic Crz signaling restores nutrient levels by promoting food search and feeding. It is additionally proposed that Crz signaling also ensures osmotic homeostasis by inhibiting release of CAPA neuropeptides and suppressing diuresis. Thus, Crz ameliorates stress-associated physiology through systemic modulation of both peptidergic neurosecretory cells and the fat body in Drosophila.
Vogt, K., Zimmerman, D. M., Schlichting, M., Hernandez-Nunez, L., Qin, S., Malacon, K., Rosbash, M., Pehlevan, C., Cardona, A. and Samuel, A. D. T. (2021). Internal state configures olfactory behavior and early sensory processing in Drosophila larvae. Sci Adv 7(1). PubMed ID: 33523854
Animals exhibit different behavioral responses to the same sensory cue depending on their internal state at a given moment. How and where in the brain are sensory inputs combined with state information to select an appropriate behavior? This study investigated how food deprivation affects olfactory behavior in Drosophila larvae. Certain odors repel well-fed animals but attract food-deprived animals and feeding state flexibly alters neural processing in the first olfactory center, the antennal lobe. Hunger differentially modulates two output pathways required for opposing behavioral responses. Upon food deprivation, attraction-mediating uniglomerular projection neurons show elevated odor-evoked activity, whereas an aversion-mediating multiglomerular projection neuron receives odor-evoked inhibition. The switch between these two pathways is regulated by the lone serotonergic neuron in the antennal lobe, CSD. These findings demonstrate how flexible behaviors can arise from state-dependent circuit dynamics in an early sensory processing center. Tasman, K., Hidalgo, S., Zhu, B., Rands, S. A. and Hodge, J. J. L. (2021). Neonicotinoids disrupt memory, circadian behaviour and sleep. Sci Rep 11(1): 2061. PubMed ID: 33479461
Globally, neonicotinoids are the most used insecticides, despite their well-documented sub-lethal effects on beneficial insects. Neonicotinoids are nicotinic acetylcholine receptor agonists. Memory, circadian rhythmicity and sleep are essential for efficient foraging and pollination and require nicotinic acetylcholine receptor signalling. The effect of field-relevant concentrations of the European Union-banned neonicotinoids: imidacloprid, clothianidin, thiamethoxam and thiacloprid were tested on Drosophila memory, circadian rhythms and sleep. Field-relevant concentrations of imidacloprid, clothianidin and thiamethoxam disrupted learning, behavioural rhythmicity and sleep whilst thiacloprid exposure only affected sleep. Exposure to imidacloprid and clothianidin prevented the day/night remodelling and accumulation of pigment dispersing factor (PDF) neuropeptide in the dorsal terminals of clock neurons. Knockdown of the neonicotinoid susceptible Dα1 and Dβ2 nicotinic acetylcholine receptor subunits in the mushroom bodies or clock neurons recapitulated the neonicotinoid like deficits in memory or sleep/circadian behaviour respectively. Disruption of learning, circadian rhythmicity and sleep are likely to have far-reaching detrimental effects on beneficial insects in the field.
Takakura, M., Nakagawa, R., Ota, T., Kimura, Y., Ng, M. Y., Alia, A. G., Okuno, H. and Hirano, Y. (2021). Rpd3/CoRest-mediated activity-dependent transcription regulates the flexibility in memory updating in Drosophila. Nat Commun 12(1): 628. PubMed ID: 33504795
Consolidated memory can be preserved or updated depending on the environmental change. Although such conflicting regulation may happen during memory updating, the flexibility of memory updating may have already been determined in the initial memory consolidation process. This study explored the gating mechanism for activity-dependent transcription in memory consolidation, which is unexpectedly linked to the later memory updating in Drosophila. Through proteomic analysis, it was discovered that the compositional change in the transcriptional repressor, which contains the histone deacetylase Rpd3 and CoRest, acts as the gating mechanism that opens and closes the time window for activity-dependent transcription. Opening the gate through the compositional change in Rpd3/CoRest is required for memory consolidation, but closing the gate through Rpd3/CoRest is significant to limit future memory updating. These data indicate that the flexibility of memory updating is determined through the initial activity-dependent transcription, providing a mechanism involved in defining memory state. Takechi, H., Hakeda-Suzuki, S., Nitta, Y., Ishiwata, Y., Iwanaga, R., Sato, M., Sugie, A. and Suzuki, T. (2021). Glial insulin regulates cooperative or antagonistic Golden goal/Flamingo interactions during photoreceptor axon guidance. Elife 10. PubMed ID: 33666170
Transmembrane protein Golden goal (Gogo) interacts with atypical cadherin Flamingo to direct R8 photoreceptor axons in the Drosophila visual system. However, the precise mechanisms underlying Gogo regulation during columnar- and layer-specific R8 axon targeting are unknown. These studies demonstrated that the insulin secreted from surface and cortex glia switches the phosphorylation status of Gogo, thereby regulating its two distinct functions. Non-phosphorylated Gogo mediates the initial recognition of the glial protrusion in the center of the medulla column, whereas phosphorylated Gogo suppresses radial filopodia extension by counteracting Flamingo to maintain a one axon to one column ratio. Later, Gogo expression ceases during the midpupal stage, thus allowing R8 filopodia to extend vertically into the M3 layer. These results demonstrate that the long- and short-range signaling between the glia and R8 axon growth cones regulates growth cone dynamics in a stepwise manner, and thus shape the entire organization of the visual system.
Tuesday May 11th - Adult Physiology
Rohde, P. D., Kristensen, T. N., Sarup, P., Munoz, J. and Malmendal, A. (2021). Prediction of complex phenotypes using the Drosophila melanogaster metabolome. Heredity (Edinb). PubMed ID: 33510469
Understanding the genotype-phenotype map and how variation at different levels of biological organization is associated are central topics in modern biology. Fast developments in sequencing technologies and other molecular omic tools enable researchers to obtain detailed information on variation at DNA level and on intermediate endophenotypes, such as RNA, proteins and metabolites. This can facilitate understanding of the link between genotypes and molecular and functional organismal phenotypes. This study used the Drosophila melanogaster Genetic Reference Panel and nuclear magnetic resonance (NMR) metabolomics to investigate the ability of the metabolome to predict organismal phenotypes. NMR metabolomics was performed on four replicate pools of male flies from each of 170 different isogenic lines. The results show that metabolite profiles are variable among the investigated lines and that this variation is highly heritable. Second, genes associated with metabolome variation were identified. Third, using the metabolome gave better prediction accuracies than genomic information for four of five quantitative traits analyzed. This comprehensive characterization of population-scale diversity of metabolomes and its genetic basis illustrates that metabolites have large potential as predictors of organismal phenotypes. This finding is of great importance, e.g., in human medicine, evolutionary biology and animal and plant breeding. Perry, T., Chen W., Ghazali, R., Yang, Y. T., Christesen, D., Martelli, F., Lumb, C., Bao Luong, H. N., Mitchell, J., Holien, J. K., Parker, M. W., Sparks, T. C. and Batterham, P. (2021). Role of nicotinic acetylcholine receptor subunits in the mode of action of neonicotinoid, sulfoximine and spinosyn insecticides in Drosophila melanogaster. Insect Biochem Mol Biol 131: 103547. PubMed ID: 33548485
This study study systematically investigated the nicotinic acetylcholine receptor (nAChR) gene family, in order to identify the receptor subunits critical to the insect response to insecticides from three distinct chemical classes (neonicotinoids, spinosyns and sulfoximines). Applying the CRISPR/Cas9 gene editing technology in D. melanogaster, it was possible to generate and maintain homozygous mutants for eight nAChR subunit genes. A ninth gene (Dβ1) was investigated using somatic CRISPR in neural cells to overcome the low viability of the homozygous germline knockout mutant. These findings highlight the specificity of the spinosyn class insecticide, spinosad, to receptors containing the Dalpha6 subunit. By way of contrast, neonicotinoids are likely to target multiple receptor subtypes, beyond those receptor subunit combinations previously identified. Significant differences in the impacts of specific nAChR subunit deletions on the resistance level of flies to neonicotinoids imidacloprid and nitenpyram indicate that the receptor subtypes they target do not completely overlap. While an R81T mutation in β1 subunits has revealed residues co-ordinating binding of sulfoximines and neonicotinoids differ, the resistance profiles of a deletion of Dβ1 examined in this study provide new insights into the mode of action of sulfoxaflor (sulfoximine) and identify Dβ1 as a key component of nAChRs targeted by both these insecticide classes.
Watanabe, L. P. and Riddle, N. C. (2021). GWAS reveal a role for the central nervous system in regulating weight and weight change in response to exercise. Sci Rep 11(1): 5144. PubMed ID: 33664357
Body size and weight show considerable variation both within and between species. This variation is controlled in part by genetics, but also strongly influenced by environmental factors including diet and the level of activity experienced by the individual. Due to the increasing obesity epidemic in much of the world, there is considerable interest in the genetic factors that control body weight and how weight changes in response to exercise treatments. This study addressed this question in the Drosophila model system, utilizing 38 strains of the Drosophila Genetics Reference Panel. GWAS was used to identify the molecular pathways that control weight and weight changes in response to exercise. This study found that there is a complex set of molecular pathways controlling weight, with many genes linked to the central nervous system (CNS). The CNS also plays a role in the weight change with exercise, in particular, signaling from the CNS. Additional analyses revealed that weight in Drosophila is driven by two factors, animal size, and body composition, as the amount of fat mass versus lean mass impacts the density. Thus, while the CNS appears to be important for weight and exercise-induced weight change, signaling pathways are particularly important for determining how exercise impacts weight. Parker, D. J., Envall, T., Ritchie, M. G. and Kankare, M. (2021). Sex-specific responses to cold in a very cold-tolerant, northern Drosophila species. Heredity (Edinb). PubMed ID: 33510465
Organisms can plastically alter resource allocation in response to changing environmental factors. For example, in harsh conditions, organisms are expected to shift investment from reproduction toward survival; however, the factors and mechanisms that govern the magnitude of such shifts are relatively poorly studied. This study compared the impact of cold on males and females of the highly cold-tolerant species Drosophila montana at the phenotypic and transcriptomic levels. Although both sexes showed similar changes in cold tolerance and gene expression in response to cold treatment, indicating that the majority of changes are concordant between the sexes, a clear reduction was identified in sexually dimorphic gene expression, suggesting that preparing for the colder season involves reducing investment in sex-specific traits. This reduction was larger in males than females, as expected if male sexual traits are more condition-dependent than female traits, as predicted by theory. Gene expression changes were primarily associated with shifts in metabolic profile, which likely play a role in increasing cold tolerance. Finally, it was found that the expression of immune genes was reduced following cold treatment, suggesting that reduced investment in costly immune function may be important in helping flies survive colder periods.
Zanco, B., Mirth, C. K., Sgro, C. M. and Piper, M. D. (2021). A dietary sterol trade-off determines lifespan responses to dietary restriction in Drosophila melanogaster females. Elife 10. PubMed ID: 33494859
Diet plays a significant role in maintaining lifelong health. In particular, lowering the dietary protein: carbohydrate ratio can improve lifespan. This has been interpreted as a direct effect of these macronutrients on physiology. Using Drosophila melanogaster, this study shows that the role of protein and carbohydrate on lifespan is indirect, acting by altering the partitioning of limiting amounts of dietary sterols between reproduction and lifespan. Shorter lifespans in flies fed on high protein: carbohydrate diets can be rescued by supplementing their food with cholesterol. Not only does this fundamentally alter the way the mechanisms of lifespan extension by dietary restriction are interpreted, these data highlight the important principle that life histories can be affected by nutrient-dependent trade-offs that are indirect and independent of the nutrients (often macronutrients) that are the focus of study. This brings closer an understanding of the mechanistic basis of dietary restriction. van Alphen, B., Semenza, E. R., Yap, M., van Swinderen, B. and Allada, R. (2021). A deep sleep stage in Drosophila with a functional role in waste clearance. Sci Adv 7(4). PubMed ID: 33523916
Sleep is a highly conserved state, suggesting that sleep's benefits outweigh the increased vulnerability it brings. Yet, little is known about how sleep fulfills its functions. This study used video tracking in tethered flies to identify a discrete deep sleep stage in Drosophila, termed proboscis extension sleep, that is defined by repeated stereotyped proboscis extensions and retractions. Proboscis extension sleep is accompanied by highly elevated arousal thresholds and decreased brain activity, indicative of a deep sleep state. Preventing proboscis extensions increases injury-related mortality and reduces waste clearance. Sleep deprivation reduces waste clearance and during subsequent rebound sleep, sleep, proboscis extensions, and waste clearance are increased. Together, these results provide evidence of a discrete deep sleep stage that is linked to a specific function and suggest that waste clearance is a core and ancient function of deep sleep.
Vita, D. J., Meier, C. J. and Broadie, K. (2021). Neuronal fragile X mental retardation protein activates glial insulin receptor mediated PDF-Tri neuron developmental clearance. Nat Commun 12(1): 1160. PubMed ID: 33608547
Glia engulf and phagocytose neurons during neural circuit developmental remodeling. Disrupting this pruning process contributes to Fragile X syndrome (FXS), a leading cause of intellectual disability and autism spectrum disorder in mammals. Utilizing a Drosophila FXS model central brain circuit, two glial classes responsible for Draper-dependent elimination of developmentally transient PDF-Tri neurons were identified. Neuronal Fragile X Mental Retardation Protein (FMRP) was found to drive insulin receptor activation in glia, promotes glial Draper engulfment receptor expression, and negatively regulates membrane-molding ESCRT-III Shrub function during PDF-Tri neuron clearance during neurodevelopment in Drosophila. In this context, this study demonstrated genetic interactions between FMRP and insulin receptor signaling, FMRP and Draper, and FMRP and Shrub in PDF-Tri (a transient pair of PDF positive tritocerebral neurons) elimination. FMRP is required within neurons, not glia, for glial engulfment, indicating FMRP-dependent neuron-to-glia signaling mediates neuronal clearance. It is conclude neuronal FMRP drives glial insulin receptor activation to facilitate Draper- and Shrub-dependent neuronal clearance during neurodevelopment in Drosophila. Pragati and Sarkar, S. (2021). Shaggy functions downstream of dMyc and their concurrent downregulation confers additive rescue against tau toxicity in Drosophila. Biofactors. PubMed ID: 33651466
Neurodegenerative tauopathies such as Alzheimer's and Parkinson's diseases are characterized by hyperphosphorylation of tau protein (see Drosophila Tau) and their subsequent aggregation in the forms of paired helical filaments and/or neurofibrillary tangles in specific areas of the brain. Despite several attempts, it remains a challenge to develop reliable biomarkers or effective drugs against tauopathies. Multitargets based combinatorial approach(s) have been suggested to provide an efficient rescue against tauopathies. It has been reported earlier that targeted downregulation of dmyc (a Drosophila homolog of human cmyc proto-oncogene) restricts tau etiology by limiting tau hyperphosphorylation and heterochromatin loss. Although, dmyc generates a significant rescue; however, it is not proficient enough to provide a complete alleviation against tauopathies. This study reports that tissue-specific concurrent downregulation of dmyc and gsk3β conveys a near-complete rescue against tau toxicity in Drosophila. It is noted that combinatorial downregulation of dmyc and gsk3β reduces tau hyperphosphorylation, restricts the formation of neurofibrillary tangles, and restores heterochromatin loss to the physiological level. Subsequent investigations revealed that dmyc regulates gsk3β via protein phosphatase 2A (dPP2A) in a dose-dependent manner to regulate tau pathogenesis. It is proposed that dmyc and gsk3β candidates can be utilized in a synergistic manner for the development of an efficient combinatorial therapeutic approach against the devastating human tauopathies.
Recasens-Alvarez, C., Alexandre, C., Kirkpatrick, J., Nojima, H., Huels, D. J., Snijders, A. P. and Vincent, J. P. (2021). Ribosomopathy-associated mutations cause proteotoxic stress that is alleviated by TOR inhibition. Nat Cell Biol 23(2): 127-135. PubMed ID: 33495632
Ribosomes are multicomponent molecular machines that synthesize all of the proteins of living cells. Most of the genes that encode the protein components of ribosomes are therefore essential. A reduction in gene dosage is often viable albeit deleterious and is associated with human syndromes, which are collectively known as ribosomopathies. The cell biological basis of these pathologies has remained unclear. This study modelled human ribosomopathies in Drosophila and found widespread apoptosis and cellular stress in the resulting animals. This is not caused by insufficient protein synthesis, as reasonably expected. Instead, ribosomal protein deficiency elicits proteotoxic stress, which is suggested to be caused by the accumulation of misfolded proteins that overwhelm the protein degradation machinery. Dampening the integrated stress response or autophagy increases the harm inflicted by ribosomal protein deficiency, suggesting that these activities could be cytoprotective. Inhibition of TOR activity-which decreases ribosomal protein production, slows down protein synthesis and stimulates autophagy-reduces proteotoxic stress in the ribosomopathy model. Interventions that stimulate autophagy, combined with means of boosting protein quality control, could form the basis of a therapeutic strategy for this class of diseases. Singh, A., Hulsmeier, J., Kandi, A. R., Pothapragada, S. S., Hillebrand, J., Petrauskas, A., Agrawal, K., Rt, K., Thiagarajan, D., Jayaprakashappa, D., VijayRaghavan, K., Ramaswami, M. and Bakthavachalu, B. (2021). Antagonistic roles for Ataxin-2 structured and disordered domains in RNP condensation. Elife 10. PubMed ID: 33689682
Ataxin-2 (Atx2) is a translational control molecule mutated in spinocerebellar ataxia type II and Als. While intrinsically disordered domains (IDRs) of Atx2 facilitate mRNP condensation into granules, how IDRs work with structured domains to enable positive and negative regulation of target mRNAs remains unclear. Using the Targets of RNA-Binding Proteins Identified by Editing technology, this study identified an extensive data set of Atx2-target mRNAs in the Drosophila brain and S2 cells. Atx2 interactions with AU-rich elements in 3'UTRs appear to modulate stability/turnover of a large fraction of these target mRNAs. Further genomic and cell biological analyses of Atx2 domain deletions demonstrate that Atx2 (1) interacts closely with target mRNAs within mRNP granules, (2) contains distinct protein domains that drive or oppose RNP-granule assembly, and (3) has additional essential roles outside of mRNP granules. These findings increase the understanding of neuronal translational control mechanisms and inform strategies for Atx2-based interventions under development for neurodegenerative disease.
Wang, X., Wu, H., Zhao, L., Liu, Z., Qi, M., Jin, Y. and Liu, W. (2021). FLCN regulates transferrin receptor 1 transport and iron homeostasis. J Biol Chem: 100426. PubMed ID: 33609526
Birt-Hogg-Dubé (BHD) syndrome is a multiorgan disorder caused by inactivation of the folliculin (FLCN) protein. Previous work identified FLCN as a binding protein of Rab11A, a key regulator of the endocytic recycling pathway. This finding implies that the abnormal localization of specific proteins whose transport requires the FLCN-Rab11A complex may contribute to BHD. This study used human kidney-derived HEK293 cells as a model, and it is reported that FLCN promotes the binding of Rab11A with transferrin receptor 1 (TfR1), which is required for iron uptake through continuous trafficking between the cell surface and the cytoplasm. Loss of FLCN attenuated the Rab11A-TfR1 interaction, resulting in delayed recycling transport of TfR1. This delay caused an iron deficiency condition that induced hypoxia-inducible factor (HIF) activity, which was reversed by iron supplementation. In a Drosophila model of BHD syndrome, it was further demonstrated that the phenotype of BHD mutant larvae was substantially rescued by an iron-rich diet. These findings reveal a conserved function of FLCN in iron metabolism and may help to elucidate the mechanisms driving BHD syndrome. Tallo, C. A., Duncan, L. H., Yamamoto, A. H., Slaydon, J. D., Arya, G. H., Turlapati, L., Mackay, T. F. C. and Carbone, M. A. (2021). Heat shock proteins and small nucleolar RNAs are dysregulated in a Drosophila model for feline hypertrophic cardiomyopathy. G3 (Bethesda) 11(1). PubMed ID: 33561224
In cats, mutations in myosin binding protein C (encoded by the MYBPC3 gene) have been associated with hypertrophic cardiomyopathy (HCM). However, the molecular mechanisms linking these mutations to HCM remain unknown. This study establish Drosophila melanogaster as a model to understand this connection by generating flies harboring MYBPC3 missense mutations (A31P and R820W) associated with feline HCM. The A31P and R820W flies displayed cardiovascular defects in their heart rates and exercise endurance. RNA-seq was used to determine which processes are misregulated in the presence of mutant MYBPC3 alleles. Transcriptome analysis revealed significant downregulation of genes encoding small nucleolar RNA (snoRNAs) in exercised female flies harboring the mutant alleles compared to flies that harbor the wild-type allele. Other processes that were affected included the unfolded protein response and immune/defense responses. These data show that mutant MYBPC3 proteins have widespread effects on the transcriptome of co-regulated genes. Transcriptionally differentially expressed genes are also candidate genes for future evaluation as genetic modifiers of HCM as well as candidate genes for genotype by exercise environment interaction effects on the manifestation of HCM; in cats as well as humans.
Friday, May 7th - Signal Transduction
Taira, Y., Wada, H., Hayashi, S. and Kageyama, Y. (2021). polished rice mediates ecdysone-dependent control of Drosophila embryonic organogenesis. Genes Cells. PubMed ID: 33621395
In many animals, progression of developmental stages is temporally controlled by steroid hormones. In Drosophila, the level of ecdysone titer oscillates and developmental stage transitions, such as larval molting and metamorphosis, are induced at each of ecdysone peaks. Ecdysone titer also peaks at the stage of mid-embryogenesis and the embryonic ecdysone is necessary for morphogenesis of several organs, although the regulatory mechanisms of embryonic organogenesis dependent on ecdysone signaling are still open questions. This study found that absence or interruption of embryonic ecdysone signaling caused multiple defects in the tracheal system, including decrease of luminal protein deposition, uneven dilation of the dorsal trunk and loss of terminal branches. It was also revealed that an ecdysone-inducible gene polished rice (pri) is essential for tip cell fate decision in dorsal branches. As overexpression of pri can restore the defects caused by disturbance of ecdysone biosynthesis, pri functions as one of the major mediators of embryonic ecdysone signal in tracheogenesis. These results demonstrate that ecdysone and its downstream target pri play essential roles in tracheal development by modulating cell fate decision. Wainwright, S. M., et al. (2021). Drosophila Sex Peptide controls the assembly of lipid microcarriers in seminal fluid. Proc Natl Acad Sci U S A 118(5). PubMed ID: 33495334
Seminal fluid plays an essential role in promoting male reproductive success and modulating female physiology and behavior. In the fruit fly, Drosophila melanogaster, Sex Peptide (SP) is the best-characterized protein mediator of these effects. It is secreted from the paired male accessory glands (AGs), which, like the mammalian prostate and seminal vesicles, generate most of the seminal fluid contents. After mating, SP binds to spermatozoa and is retained in the female sperm storage organs. It is gradually released by proteolytic cleavage and induces several long-term postmating responses, including increased ovulation, elevated feeding, and reduced receptivity to remating, primarily signaling through the SP receptor (SPR). This study demonstrates a previously unsuspected SPR-independent function for SP. In the AG lumen, SP and secreted proteins with membrane-binding anchors are carried on abundant, large neutral lipid-containing microcarriers. These microcarriers are transferred to females during mating where they rapidly disassemble. Remarkably, SP is a key microcarrier assembly and disassembly factor. Males expressing nonfunctional SP mutant proteins that affect SP's binding to and release from sperm in females also do not produce normal microcarriers. These data therefore reveal a role for SP in formation of seminal macromolecular assemblies.
Tokamov, S. A., Su, T., Ullyot, A. and Fehon, R. G. (2021). Negative feedback couples Hippo pathway activation with Kibra degradation independent of Yorkie-mediated transcription. Elife 10. PubMed ID: 33555257
The Hippo (Hpo) pathway regulates tissue growth in many animals. Multiple upstream components promote Hpo pathway activity, but the organization of these different inputs, the degree of crosstalk between them, and whether they are regulated in a distinct manner is not well understood. Kibra (Kib) activates the Hpo pathway by recruiting the core Hpo kinase cassette to the apical cortex. This study shows that the Hpo pathway downregulates Drosophila Kib levels independently of Yorkie-mediated transcription. Hpo signaling complex formation promotes Kib degradation via SCF(Slimb)-mediated ubiquitination. This effect requires Merlin, Salvador, Hpo, and Warts, and this mechanism functions independently of other upstream Hpo pathway activators. Moreover, Kib degradation appears patterned by differences in mechanical tension across the wing. It is proposed that Kib degradation mediated by Hpo pathway components and regulated by cytoskeletal tension serves to control Kib-driven Hpo pathway activation and ensure optimally scaled and patterned tissue growth. Saad, F. and Hipfner, D. R. (2021). Extensive crosstalk of G protein-coupled receptors with the Hedgehog signalling pathway. Development. PubMed ID: 33653875
Hedgehog (Hh) ligands orchestrate tissue patterning and growth by acting as morphogens, dictating different cellular responses depending on ligand concentration. Cellular sensitivity to Hh ligands is influenced by heterotrimeric G protein activity, which controls production of the second messenger 3',5'-cyclic adenosine monophosphate (cAMP). cAMP in turn activates Protein kinase A (PKA), which functions as an inhibitor and (uniquely in Drosophila) an activator of Hh signalling. A few mammalian Gαi- and Gαs-coupled G protein-coupled receptors (GPCRs) have been shown to influence Sonic Hh (Shh) responses in this way. To determine if this is a more general phenomenon, an RNAi screen targeting GPCRs was carried out in Drosophila. RNAi-mediated depletion of more than 40% of GPCRs tested either decreased or increased Hh responsiveness in the developing Drosophila wing, closely matching the effects of Gαs and Gαi depletion, respectively. Genetic analysis indicated that the orphan GPCR Mthl5 lowers cAMP levels to attenuate Hh responsiveness. These results identify Mthl5 as a new Hh signalling pathway modulator in Drosophila and suggest that many GPCRs may crosstalk with the Hh pathway in mammals.
Steinmetz, E. L., Dewald, D. N. and Walldorf, U. (2021). Drosophila Homeodomain-Interacting Protein Kinase (Hipk) Phosphorylates the Hippo/Warts Signalling Effector Yorkie. Int J Mol Sci 22(4). PubMed ID: 33668437
Developmental growth and patterning are regulated by an interconnected signalling network of several pathways. In Drosophila, the Warts (Wts) kinase, a component of the Hippo signalling pathway, plays an essential role in regulating transcription and growth by phosphorylating its substrate Yorkie (Yki). The phosphorylation of Yki critically influences its localisation and activity as a transcriptional coactivator. This study identified the homeodomain-interacting protein kinase (Hipk) as another kinase that phosphorylates Yki and mapped several sites of Yki phosphorylated by Hipk, using in vitro analysis: Ser168, Ser169/Ser172 and Ser255. These sites might provide auxiliary input for Yki regulation in vivo, as transgenic flies with mutations in these show prominent phenotypes; Hipk, therefore, represents an additional upstream regulator of Yki that works in concert with Wts. Sun, J., Wang, X., Xu, R. G., Mao, D., Shen, D., Wang, X., Qiu, Y., Han, Y., Lu, X., Li, Y., Che, Q., Zheng, L., Peng, P., Kang, X., Zhu, R., Jia, Y., Wang, Y., Liu, L. P., Chang, Z., Ji, J. Y., Wang, Z., Liu, Q., Li, S., Sun, F. L. and Ni, J. Q. (2021). HP1c regulates development and gut homeostasis by suppressing Notch signaling through Su(H). EMBO Rep: e51298. PubMed ID: 33594776
Notch signaling and epigenetic factors are known to play critical roles in regulating tissue homeostasis in most multicellular organisms, but how Notch signaling coordinates with epigenetic modulators to control differentiation remains poorly understood. This study identified heterochromatin protein 1c (HP1c) as an essential epigenetic regulator of gut homeostasis in Drosophila. Specifically, it was observe that HP1c loss-of-function phenotypes resemble those observed after Notch signaling perturbation and that HP1c interacts genetically with components of the Notch pathway. HP1c represses the transcription of Notch target genes by directly interacting with Suppressor of Hairless (Su(H)), the key transcription factor of Notch signaling. Moreover, phenotypes caused by depletion of HP1c in Drosophila can be rescued by expressing human HP1γ, suggesting that HP1γ functions similar to HP1c in Drosophila. Taken together, these findings reveal an essential role of HP1c in normal development and gut homeostasis by suppressing Notch signaling.
Thursday, May 6th - Apoptosis and autophagy
Cho, D. G., Lee, S. S. and Cho, K. O. (2021). Anastral Spindle 3/Rotatin Stabilizes Sol narae and Promotes Cell Survival in Drosophila melanogaster. Mol Cells 44(1): 13-25. PubMed ID: 33510049
Apoptosis and compensatory proliferation, two intertwined cellular processes essential for both development and adult homeostasis, are often initiated by the mis-regulation of centrosomal proteins, damaged DNA, and defects in mitosis. Fly Anastral spindle 3 (Ana3) is a member of the pericentriolar matrix proteins and known as a key component of centriolar cohesion and basal body formation. This study reports that ana3m19 is a suppressor of lethality induced by the overexpression of Sol narae (Sona), a metalloprotease in a disintegrin and metalloprotease with thrombospondin motif (ADAMTS) family. ana3m19 has a nonsense mutation that truncates the highly conserved carboxyl terminal region containing multiple Armadillo repeats. Lethality induced by Sona overexpression was completely rescued by knockdown of Ana3, and the small and malformed wing and hinge phenotype induced by the knockdown of Ana3 was also normalized by Sona overexpression, establishing a mutually positive genetic interaction between ana3 and sona. p35 inhibited apoptosis and rescued the small wing and hinge phenotype induced by knockdown of ana3. Furthermore, overexpression of Ana3 increased the survival rate of irradiated flies and reduced the number of dying cells, demonstrating that Ana3 actively promotes cell survival. Knockdown of Ana3 decreased the levels of both intra- and extracellular Sona in wing discs, while overexpression of Ana3 in S2 cells dramatically increased the levels of both cytoplasmic and exosomal Sona due to the stabilization of Sona in the lysosomal degradation pathway. It is proposed that one of the main functions of Ana3 is to stabilize Sona for cell survival and proliferation. Laczko-Dobos, H., Maddali, A. K., Jipa, A., Bhattacharjee, A., Vegh, A. G. and Juhasz, G. (2021). Lipid profiles of autophagic structures isolated from wild type and Atg2 mutant Drosophila. Biochim Biophys Acta Mol Cell Biol Lipids 1866(3): 158868. PubMed ID: 33333179
Autophagy is mediated by membrane-bound organelles and it is an intrinsic catabolic and recycling process of the cell, which is very important for the health of organisms. The biogenesis of autophagic membranes is still incompletely understood. In vitro studies suggest that Atg2 protein transports lipids presumably from the ER to the expanding autophagic structures. Autophagy research has focused heavily on proteins and very little is known about the lipid composition of autophagic membranes. This study describes a method for immunopurification of autophagic structures from Drosophila melanogaster (an excellent model to study autophagy in a complete organism) for subsequent lipidomic analysis. Western blots of several organelle markers indicate the high purity of the isolated autophagic vesicles, visualized by various microscopy techniques. Mass spectrometry results show that phosphatidylethanolamine (PE) is the dominant lipid class in wild type (control) membranes. In Atg2 mutants (Atg2-), phosphatidylinositol (PI), negatively charged phosphatidylserine (PS), and phosphatidic acid (PA) with longer fatty acyl chains accumulate on stalled, negatively charged phagophores. Tandem mass spectrometry analysis of lipid species composing the lipid classes reveal the enrichment of unsaturated PE and phosphatidylcholine (PC) in controls versus PI, PS and PA species in Atg2-. Significant differences in the lipid profiles of control and Atg2- flies suggest that the lipid composition of autophagic membranes dynamically changes during their maturation. These lipidomic results also point to the in vivo lipid transport function of the Atg2 protein, pointing to its specific role in the transport of short fatty acyl chain PE species.
Zhu, J. Y., Hannan, S. B., Drager, N. M., Vereshchagina, N., Krahl, A. C., Fu, Y., Elliott, C. J. H., Han, Z., Jahn, T. R. and Rasse, T. M. (2021). Autophagy inhibition rescues structural and functional defects caused by the loss of mitochondrial chaperone Hsc70-5 in Drosophila. Autophagy. PubMed ID: 33404278
This study investigated in larval and adult Drosophila models whether loss of the mitochondrial chaperone Hsc70-5 is sufficient to cause pathological alterations commonly observed in Parkinson disease. At affected larval neuromuscular junctions, no effects on terminal size, bouton size or number, synapse size, or number were observed, suggesting that an early stage of pathogenesis was studied. At this stage, a loss of synaptic vesicle proteins and active zone components was observed, delayed synapse maturation, reduced evoked and spontaneous excitatory junctional potentials, increased synaptic fatigue, and cytoskeleton rearrangements. The adult model displayed ATP depletion, altered body posture, and susceptibility to heat-induced paralysis. Adult phenotypes could be suppressed by knockdown of dj-1β, Lrrk, DCTN2-p50, DCTN1-p150, Atg1, Atg101, Atg5, Atg7, and Atg12. The knockdown of components of the macroautophagy/autophagy machinery or overexpression of human HSPA9 broadly rescued larval and adult phenotypes, while disease-associated HSPA9 variants did not. Overexpression of Pink1 or promotion of autophagy exacerbated defects. Guntur, A. R., Venkatanarayan, A., Gangula, S. and Lundell, M. J. (2021). Zfh-2 facilitates Notch-induced apoptosis in the CNS and appendages of Drosophila melanogaster. Dev Biol. PubMed ID: 33705738
Apoptosis is a fundamental remodeling process for most tissues during development. This study examined a pro-apoptotic function for the Drosophila DNA binding protein Zfh-2 during development of the central nervous system (CNS) and appendages. In the CNS it was found that a loss-of-function zfh-2 allele gives an overall reduction of apoptotic cells in the CNS, and an altered pattern of expression for the axonal markers 22C10 and FasII. This same loss-of-function zfh-2 allele causes specific cells in the NB7-3 lineage of the CNS that would normally undergo apoptosis to be inappropriately maintained, whereas a gain-of-function zfh-2 allele has the opposite effect, resulting in a loss of normal NB 7-3 progeny. It was also demonstrated that Zfh-2 and Hunchback reciprocally repress each other's gene expression which limits apoptosis to later born progeny of the NB7-3 lineage. Apoptosis is also required for proper segmentation of the fly appendages. Zfh-2 co-localizes with apoptotic cells in the folds of the imaginal discs and presumptive cuticular joints. A reduction of Zfh-2 levels with RNAi inhibits expression of the pro-apoptotic gene reaper, and produces abnormal joints in the leg, antenna and haltere. Apoptosis has previously been shown to be activated by Notch signaling in both the NB7-3 CNS lineage and the appendage joints. These results indicate that Zfh-2 facilitates Notch-induced apoptosis in these structures.
Tang, H. W., Weng, J. H., Lee, W. X., Hu, Y., Gu, L., Cho, S., Lee, G., Binari, R., Li, C., Cheng, M. E., Kim, A. R., Xu, J., Shen, Z., Xu, C., Asara, J. M., Blenis, J. and Perrimon, N. (2021). mTORC1-chaperonin CCT signaling regulates m(6)A RNA methylation to suppress autophagy. Proc Natl Acad Sci U S A 118(10). PubMed ID: 33649236
Mechanistic Target of Rapamycin Complex 1 (mTORC1) is a central regulator of cell growth and metabolism that senses and integrates nutritional and environmental cues with cellular responses. Recent studies have revealed critical roles of mTORC1 in RNA biogenesis and processing. This study finds that the m(6)A methyltransferase complex (MTC) is a downstream effector of mTORC1 during autophagy in Drosophila and human cells. Furthermore, the Chaperonin Containing Tailless complex polypeptide 1 (CCT) complex, which facilitates protein folding, acts as a link between mTORC1 and MTC. The mTORC1 activates the chaperonin CCT complex to stabilize MTC, thereby increasing m(6)A levels on the messenger RNAs encoding autophagy-related genes, leading to their degradation and suppression of autophagy. Altogether, this study reveals an evolutionarily conserved mechanism linking mTORC1 signaling with m(6)A RNA methylation and demonstrates their roles in suppressing autophagy. Nagai, H., Tatara, H., Tanaka-Furuhashi, K., Kurata, S. and Yano, T. (2021). Homeostatic Regulation of ROS-Triggered Hippo-Yki Pathway via Autophagic Clearance of Ref(2)P/p62 in the Drosophila Intestine. Dev Cell 56(1): 81-94.e10. PubMed ID: 33400912
Homeostasis of intestinal epithelia is maintained by coordination of the proper rate of regeneration by stem cell division with the rate of cell loss. Regeneration of host epithelia is normally quiescent upon colonization of commensal bacteria; however, the epithelia often develop dysplasia in a context-dependent manner, the cause and underlying mechanism of which remain unclear. This study shows that in Drosophila intestine, autophagy lowers the sensitivity of differentiated enterocytes to reactive oxygen species (ROS) that are produced in response to commensal bacteria. Autophagy deficiency provokes ROS-dependent excessive regeneration and subsequent epithelial dysplasia and barrier dysfunction. Mechanistically, autophagic substrate Ref(2)P/p62, which co-localizes and physically interacts with Dachs, a Hippo signaling regulator, accumulates upon autophagy deficiency and thus inactivates Hippo signaling, resulting in stem cell over-proliferation non-cell autonomously. These findings uncover a mechanism whereby suppression of undesirable regeneration by autophagy maintains long-term homeostasis of intestinal epithelia.
Wednesday, May 5th - Adult Neural Development and Function
Smolic, T., Tavcar, P., Horvat, A., Cerne, U., Haluzan Vasle, A., Tratnjek, L., Kreft, M. E., Scholz, N., Matis, M., Petan, T., Zorec, R. and Vardjan, N. (2021). Astrocytes in stress accumulate lipid droplets. Glia. PubMed ID: 33609060
When the brain is in a pathological state, the content of lipid droplets (LDs), the lipid storage organelles, is increased, particularly in glial cells, but rarely in neurons. The biology and mechanisms leading to LD accumulation in astrocytes, glial cells with key homeostatic functions, are poorly understood. Fluorescently labeled LDs were imaged by microscopy in isolated and brain tissue rat astrocytes and in glia-like cells in Drosophila brain to determine the (sub)cellular localization, mobility, and content of LDs under various stress conditions characteristic for brain pathologies. LDs exhibited confined mobility proximal to mitochondria and endoplasmic reticulum that was attenuated by metabolic stress and by increased intracellular Ca(2+) , likely to enhance the LD-organelle interaction imaged by electron microscopy. When de novo biogenesis of LDs was attenuated by inhibition of DGAT1 and DGAT2 enzymes, the astrocyte cell number was reduced by ~40%, suggesting that in astrocytes LD turnover is important for cell survival and/or proliferative cycle. Exposure to noradrenaline, a brain stress response system neuromodulator, and metabolic and hypoxic stress strongly facilitated LD accumulation in astrocytes. The observed response of stressed astrocytes may be viewed as a support for energy provision, but also to be neuroprotective against the stress-induced lipotoxicity. Pagni, M., Haikala, V., Oberhauser, V., Meyer, P. B., Reiff, D. F. and Schnaitmann, C. (2021). Interaction of "chromatic" and "achromatic" circuits in Drosophila color opponent processing. Curr Biol. PubMed ID: 33636123
Color vision is an important sensory capability of humans and many animals. It relies on color opponent processing in visual circuits that gradually compare the signals of photoreceptors with different spectral sensitivities. In Drosophila, this comparison begins already in the presynaptic terminals of UV-sensitive R7 and longer wavelength-sensitive R8 inner photoreceptors that inhibit each other in the medulla. How downstream neurons process their signals is unknown. This study reports that the second order medulla interneuron Dm8 is inhibited when flies are stimulated with UV light and strongly excited in response to a broad range of longer wavelength (VIS) stimuli. Inhibition to UV light is mediated by histaminergic input from R7 and expression of the histamine receptor ort in Dm8, as previously suggested. However, two additional excitatory inputs antagonize the R7 input. First, activation of R8 leads to excitation of Dm8 by non-canonical photoreceptor signaling and cholinergic neurotransmission in the visual circuitry. Second, activation of outer photoreceptors R1-R6 with broad spectral sensitivity causes excitation in Dm8 through the cholinergic medulla interneuron Mi1, which is known for its major contribution to the detection of spatial luminance contrast and visual motion. In summary, Dm8 mediates a second step in UV/VIS color opponent processing in Drosophila by integrating input from all types of photoreceptors. These results demonstrate novel insights into the circuit integration of R1-R6 into color opponent processing and reveal that chromatic and achromatic circuitries of the fly visual system interact more extensively than previously thought.
Scaplen, K. M., Talay, M., Fisher, J. D., Cohn, R., Sorkac, A., Aso, Y., Barnea, G. and Kaun, K. R. (2021). Transsynaptic mapping of Drosophila mushroom body output neurons. Elife 10. PubMed ID: 33570489
The mushroom body (MB) is a well-characterized associative memory structure within the Drosophila brain. Analyzing MB connectivity using multiple approaches is critical for understanding the functional implications of this structure. Using the genetic anterograde transsynaptic tracing tool, trans-Tango, this study identified divergent projections across the brain and convergent downstream targets of the MB output neurons (MBONs). This analysis revealed at least three separate targets that receive convergent input from MBONs: other MBONs, the fan-shaped body (FSB), and the lateral accessory lobe (LAL). A multilayer circuit is described, both anatomically and functionally, in which inhibitory and excitatory MBONs converge on the same genetic subset of FSB and LAL neurons. This circuit architecture enables the brain to update and integrate information with previous experience before executing appropriate behavioral responses. This use of trans-Tango provides a genetically accessible anatomical framework for investigating the functional relevance of components within these complex and interconnected circuits. Quiquand, M., Rimesso, G., Qiao, N., Suo, S., Zhao, C., Slattery, M., White, K. P., Han, J. J. and Baker, N. E. (2021). New regulators of Drosophila eye development identified from temporal transcriptome changes. Genetics. PubMed ID: 33681970
In the last larval instar, uncommitted progenitor cells in the Drosophila eye primordium start to adopt individual retinal cell fates, arrest their growth and proliferation, and initiate terminal differentiation into photoreceptor neurons and other retinal cell types. To explore the regulation of these processes, mRNA-Seq studies of the larval eye and antennal primordial were performed at multiple developmental stages. A total of 10,893 fly genes were expressed during these stages and could be adaptively clustered into gene groups, some of whose expression increases or decreases in parallel with the cessation of proliferation and onset of differentiation. Using in situ hybridization of a sample of 98 genes to verify spatial and temporal expression patterns, it is estimated that 534 genes or more are transcriptionally upregulated during retinal differentiation, and 1367 or more downregulated as progenitor cells differentiate. Each group of co-expressed genes is enriched for regulatory motifs recognized by co-expressed transcription factors, suggesting that they represent coherent transcriptional regulatory programs. Using available mutant strains, novel roles are described for the transcription factors SoxNeuro (SoxN), H6-like homeobox (Hmx), CG10253, without children (woc), Structure specific recognition protein (Ssrp), and multisex combs (mxc).
Persico, V., Callaini, G. and Riparbelli, M. G. (2021). Sas-4 Colocalizes with the Ciliary Rootlets of the Drosophila Sensory Organs. J Dev Biol 9(1). PubMed ID: 33466292
The Drosophila eye displays peculiar sensory organs of unknown function, the mechanosensory bristles, that are intercalated among the adjacent ommatidia. Like the other Drosophila sensory organs, the mechanosensory bristles consist of a bipolar neuron and two tandemly aligned centrioles, the distal of which nucleates the ciliary axoneme and represents the starting point of the ciliary rootlets. This study reports that the centriole associated protein Sas-4 colocalizes with the short ciliary rootlets of the mechanosensory bristles and with the elongated rootlets of chordotonal and olfactory neurons. This finding suggests an unexpected cytoplasmic localization of Sas-4 protein and points to a new underscored role for this protein. Moreover, it was observed that the sheath cells associated with the sensory neurons also display two tandemly aligned centrioles but lacks ciliary axonemes, suggesting that the dendrites of the sensory neurons are dispensable for the assembly of aligned centrioles and rootlets. Sabandal, J. M., Berry, J. A. and Davis, R. L. (2021). Dopamine-based mechanism for transient forgetting. Nature. PubMed ID: 33473212
Active forgetting is an essential component of the memory management system of the brain. Forgetting can be permanent, in which prior memory is lost completely, or transient, in which memory exists in a temporary state of impaired retrieval. Temporary blocks on memory seem to be universal, and can disrupt an individual's plans, social interactions and ability to make rapid, flexible and appropriate choices. However, the neurobiological mechanisms that cause transient forgetting are unknown. This study identified a single dopamine neuron in Drosophila that mediates the memory suppression that results in transient forgetting. Artificially activating this neuron did not abolish the expression of long-term memory. Instead, it briefly suppressed memory retrieval, with the memory becoming accessible again over time. The dopamine neuron modulates memory retrieval by stimulating a unique dopamine receptor that is expressed in a restricted physical compartment of the axons of mushroom body neurons. This mechanism for transient forgetting is triggered by the presentation of interfering stimuli immediately before retrieval.
Tuesday, May 4th - RNA and Transposons
Phillips, J. B. and Ardell, D. H. (2021). Structural and Genetic Determinants of Convergence in the Drosophila tRNA Structure-Function Map. J Mol Evol 89(1-2): 103-116. PubMed ID: 33528599
The evolution of tRNA multigene families remains poorly understood, exhibiting unusual phenomena such as functional conversions of tRNA genes through anticodon shift substitutions. This study has improved FlyBase tRNA gene annotations from twelve Drosophila species, incorporating previously identified ortholog sets to compare substitution rates across tRNA bodies at single-site and base-pair resolution. All rapidly evolving sites fell within the same metal ion-binding pocket that lies at the interface of the two major stacked helical domains. A tRNA Structure-Function Mapper (tSFM) method was applied independently to each Drosophila species and one outgroup species Musca domestica; although predicted tRNA structure-function maps are generally highly conserved in flies, one tRNA Class-Informative Feature (CIF) within the rapidly evolving ion-binding pocket-Cytosine 17 (C17), ancestrally informative for lysylation identity, independently gained asparaginylation identity and substituted in parallel across tRNA(Asn) paralogs at least once, possibly multiple times, during evolution of the genus. In D. melanogaster, most tRNA(Lys) and tRNA(Asn) genes are co-arrayed in one large heterologous gene cluster, suggesting that heterologous gene conversion as well as structural similarities of tRNA-binding interfaces in the closely related asparaginyl-tRNA synthetase (AsnRS) and lysyl-tRNA synthetase (LysRS) proteins may have played a role in these changes. A previously identified Asn-to-Lys anticodon shift substitution in D. ananassae may have arisen to compensate for the convergent and parallel gains of C17 in tRNA(Asn) paralogs in that lineage. These results underscore the functional and evolutionary relevance of the tRNA structure-function map predictions and illuminate multiple genomic and structural factors contributing to rapid, parallel and compensatory evolution of tRNA multigene families. Sahu, R. K., Mutt, E. and Lakhotia, S. C. (2020). Conservation of gene architecture and domains amidst sequence divergence in the hsromega lncRNA gene across the Drosophila genus: an in silico analysis. J Genet 99. PubMed ID: 33622991
The developmentally active and cell-stress responsive hsrω; locus in Drosophila melanogaster carries two exons, one omega intron, one short translatable open reading frame (ORFω), long stretch of unique tandem repeats and an overlapping mir-4951 near its 3' end. It produces multiple long noncoding RNAs (lncRNAs) using two transcription start and four termination sites. Earlier cytogenetic studies revealed functional conservation of hsrω in several Drosophila species. However, sequence analysis in three species showed poor conservation for ORFω, tandem repeat and other regions while the 16 nt at 50 and 60 nt at 3' splice junctions of the omega intron, respectively, were found to be ultra-conserved. The present bioinformatic study using the splice-junction landmarks in D. melanogaster hsrω identified orthologues in publicly available 34 Drosophila species genomes. Each orthologue carries a short ORFω, ultra-conserved splice junctions of omega intron, repeat region, conserved 3' end located at mir-4951, and syntenic neighbours. Multiple copies of conserved nonamer motifs are seen in the tandem repeat region, despite a high variability in the repeat sequences. Intriguingly, only the omega intron sequences in different species show evolutionary relationships matching the general phylogenetic history in the genus. Search in other known insect genomes did not reveal sequence homology although a locus with similar functional properties is suggested in Chironomus and Ceratitis genera. Amidst the high sequence divergence, the conserved organization of exons, ORFω and omega intron in this gene's proximal part and tandem repeats in distal part across the Drosophila genus is remarkable and possibly reflects functional importance of higher order structure of hsrω lncRNAs and the small omega peptide.
Safieddine, A., Coleno, E., Salloum, S., Imbert, A., Traboulsi, A. M., Kwon, O. S., Lionneton, F., Georget, V., Robert, M. C., Gostan, T., Lecellier, C. H., Chouaib, R., Pichon, X., Le Hir, H., Zibara, K., Mueller, F., Walter, T., Peter, M. and Bertrand, E. (2021). A choreography of centrosomal mRNAs reveals a conserved localization mechanism involving active polysome transport. Nat Commun 12(1): 1352. PubMed ID: 33649340
Local translation allows for a spatial control of gene expression. This study used high-throughput smFISH to screen centrosomal protein-coding genes, and 8 human mRNAs were found to accumulate at centrosomes. These mRNAs localize at different stages during cell cycle with a remarkable choreography, indicating a finely regulated translational program at centrosomes. Interestingly, drug treatments and reporter analyses reveal a common translation-dependent localization mechanism requiring the nascent protein. Using ASPM (Drosophila homolog: abnormal spindle) and NUMA1 (Drosophila homolog: GRIP and coiled-coil domain containing 185 kDa) as models, single mRNA and polysome imaging reveals active movements of endogenous polysomes towards the centrosome at the onset of mitosis, when these mRNAs start localizing. ASPM polysomes associate with microtubules and localize by either motor-driven transport or microtubule pulling. Remarkably, the Drosophila orthologs of the human centrosomal mRNAs also localize to centrosomes and also require translation. These data identify a conserved family of centrosomal mRNAs that localize by active polysome transport mediated by nascent proteins. Li, R., Yao, X., Zhou, H., Jin, P. and Ma, F. (2021). The Drosophila miR-959-962 Cluster Members Repress Toll Signaling to Regulate Antibacterial Defense during Bacterial Infection. Int J Mol Sci 22(2). PubMed ID: 33477373
MicroRNAs (miRNAs) are a class of ~22 nt non-coding RNA molecules in metazoans capable of down-regulating target gene expression by binding to the complementary sites in the mRNA transcripts. Many individual miRNAs are implicated in a broad range of biological pathways, but functional characterization of miRNA clusters in concert is limited. This study reports that miR-959-962 cluster (miR-959/960/961/962) can weaken Drosophila immune response to bacterial infection evidenced by the reduced expression of antimicrobial peptide Drosomycin (Drs) and short survival within 24 h upon infection. Each of the four miRNA members is confirmed to contribute to the reduced Drs expression and survival rate of Drosophila. Mechanically, RT-qPCR and Dual-luciferase reporter assay verify that tube and dorsal (dl) mRNAs, key components of Toll pathway, can simultaneously be targeted by miR-959 and miR-960, miR-961, and miR-962, respectively. Furthermore, miR-962 can even directly target to the 3' untranslated region (UTR) of Toll. In addition, the dynamic expression pattern analysis in wild-type flies reveals that four miRNA members play important functions in Drosophila immune homeostasis restoration at the late stage of Micrococcus luteus (M. luteus) infection. Taken together, these results identify four miRNA members from miR-959-962 cluster as novel suppressors of Toll signaling and enrich the repertoire of immune-modulating miRNA in Drosophila.
Schnabl, J., Wang, J., Hohmann, U., Gehre, M., Batki, J., Andreev, V. I., Purkhauser, K., Fasching, N., Duchek, P., Novatchkova, M., Mechtler, K., Plaschka, C., Patel, D. J. and Brennecke, J. (2021). Molecular principles of Piwi-mediated cotranscriptional silencing through the dimeric SFiNX complex. Genes Dev 35(5-6): 392-409. PubMed ID: 33574069
Nuclear Argonaute proteins, guided by their bound small RNAs to nascent target transcripts, mediate cotranscriptional silencing of transposons and repetitive genomic loci through heterochromatin formation. The molecular mechanisms involved in this process are incompletely understood. This study shows that the SFiNX complex, a silencing mediator downstream from nuclear Piwi-piRNA complexes in Drosophila, facilitates cotranscriptional silencing as a homodimer. The dynein light chain protein Cut up/LC8 mediates SFiNX dimerization, and its function can be bypassed by a heterologous dimerization domain, arguing for a constitutive SFiNX dimer. Dimeric, but not monomeric SFiNX, is capable of forming molecular condensates in a nucleic acid-stimulated manner. Mutations that prevent SFiNX dimerization result in loss of condensate formation in vitro and the inability of Piwi to initiate heterochromatin formation and silence transposons in vivo. It is proposed that multivalent SFiNX-nucleic acid interactions are critical for heterochromatin establishment at piRNA target loci in a cotranscriptional manner. Proshkina, E., Yushkova, E., Koval, L., Zemskaya, N., Shchegoleva, E., Solovev, I., Yakovleva, D., Pakshina, N., Ulyasheva, N., Shaposhnikov, M. and Moskalev, A. (2021). Tissue-Specific Knockdown of Genes of the Argonaute Family Modulates Lifespan and Radioresistance in Drosophila Melanogaster. Int J Mol Sci 22(5). PubMed ID: 33673647
Small RNAs are essential to coordinate many cellular processes, including the regulation of gene expression patterns, the prevention of genomic instability, and the suppression of the mutagenic transposon activity. These processes determine the aging, longevity, and sensitivity of cells and an organism to stress factors (particularly, ionizing radiation). The biogenesis and activity of small RNAs are provided by proteins of the Argonaute family. These proteins participate in the processing of small RNA precursors and the formation of an RNA-induced silencing complex. However, the role of Argonaute proteins in regulating lifespan and radioresistance remains poorly explored. The effect of knockdown of Argonaute genes (AGO1, AGO2, AGO3, piwi) in various tissues on the Drosophila melanogaster lifespan and survival was studied after the γ-irradiation at a dose of 700 Gy. In most cases, these parameters are reduced or did not change significantly in flies with tissue-specific RNA interference. Surprisingly, piwi knockdown in both the fat body and the nervous system causes a lifespan increase. But changes in radioresistance depend on the tissue in which the gene was knocked out. In addition, analysis of changes in retrotransposon levels and expression of stress response genes allowed determination of associated molecular mechanisms.
Monday, May 3rd - Enhancers and Transcriptional Regulation
Brovkina, M. V., Duffie, R., Burtis, A. E. C. and Clowney, E. J. (2021). Fruitless decommissions regulatory elements to implement cell-type-specific neuronal masculinization. PLoS Genet 17(2): e1009338. PubMed ID: 33600447
In the fruit fly Drosophila melanogaster, male-specific splicing and translation of the Fruitless transcription factor (FruM) alters the presence, anatomy, and/or connectivity of >60 types of central brain neurons that interconnect to generate male-typical behaviors. While the indispensable function of FruM in sex-specific behavior has been understood for decades, the molecular mechanisms underlying its activity remain unknown. This study took a genome-wide, brain-wide approach to identifying regulatory elements whose activity depends on the presence of FruM. 436 high-confidence genomic regions were identified differentially accessible in male fruitless neurons, validate candidate regions as bona fide, differentially regulated enhancers, and the particular cell types in which these enhancers are active is described. It was found that individual enhancers are not activated universally but are dedicated to specific fru+ cell types. Aside from fru itself, genes are not dedicated to or common across the fru circuit; rather, FruM appears to masculinize each cell type differently, by tweaking expression of the same effector genes used in other circuits. Finally, this study found FruM motifs enriched among regulatory elements that are open in the female but closed in the male. Together, these results suggest that FruM acts cell-type-specifically to decommission regulatory elements in male fruitless neurons. Ruiz, L., Kaczmarska, Z., Gomes, T., Aragon, E., Torner, C., Freier, R., Baginski, B., Martin-Malpartida, P., de Martin Garrido, N., Marquez, J. A., Cordeiro, T. N., Pluta, R. and Macias, M. J. (2021). Unveiling the dimer/monomer propensities of Smad MH1-DNA complexes. Comput Struct Biotechnol J 19: 632-646. PubMed ID: 33510867
Smad transcription factors are the main downstream effectors of the Transforming growth factor β superfamily (TGFβ) signalling network. The DNA complexes determined here by X-ray crystallography for the Bone Morphogenetic Proteins (BMP) activated Smad5 (see Drosophila Mad) and Smad8 proteins reveal that all MH1 domains bind [GGC(GC)|(CG)] motifs similarly, although TGFβ-activated Smad2/3 (see Drosophila Smox) and Smad4 (see Drosophila Medea) MH1 domains bind as monomers whereas Smad1/5/8 form helix-swapped dimers. Dimers and monomers are also present in solution, as revealed by NMR. To decipher the characteristics that defined these dimers, chimeric MH1 domains were designed and characterized using X-ray crystallography. Swapping the loop1 between TGFβ- and BMP- activated MH1 domains switches the dimer/monomer propensities. When the distribution of Smad-bound motifs was scanned in ChIP-Seq peaks (Chromatin immunoprecipitation followed by high-throughput sequencing) in Smad-responsive genes, specific site clustering and spacing were observed depending on whether the peaks correspond to BMP- or TGFβ-responsive genes. Significant correlations were observed between site distribution and monomer or dimer propensities. It is proposed that the MH1 monomer or dimer propensity of Smads contributes to the distinct motif selection genome-wide and together with the MH2 domain association, help define the composition of R-Smad/Smad4 trimeric complexes.
Deliconstantinos, G., Kalodimou, K. and Delidakis, C. (2021). Translational Control of Serrate Expression in Drosophila Cells. In Vivo 35(2): 859-869. PubMed ID: 33622878
The DSL proteins, Serrate and Delta, which act as Notch receptor ligands, mediate signalling between adjacent cells, when a ligand-expressing cell binds to Notch on an adjacent receiving cell. Notch is ubiquitously expressed and DSL protein mis-expression can have devastating developmental consequences. Although transcriptional regulation of Delta and Serrate has been amply documented, this study examined whether they are also regulated at the level of translation. A series of deletions were generated to investigate the initiation codon usage for Serrate using Drosophila S2 cells. Serrate mRNA contains three putative ATG initiation codons spanning a 60-codon region upstream of its signal peptide; each one can act as an initiation codon, however, with a different translational efficiency. It is concluded that serrate expression is strictly regulated at the translational level. Postika, N., Schedl, P., Georgiev, P. and Kyrchanova, O.(2021). Mapping of functional elements of the Fab-6 boundary involved in the regulation of the Abd-B hox gene in Drosophila melanogaster. Sci Rep 11(1): 4156. PubMed ID: 33603202
The autonomy of segment-specific regulatory domains in the Bithorax complex: is conferred by boundary elements and associated Polycomb response elements (PREs). The Fab-6 boundary is located at the junction of the iab-5 and iab-6 domains. Previous studies mapped it to a nuclease hypersensitive region 1 (HS1), while the iab-6 PRE was mapped to a second hypersensitive region HS2 nearly 3 kb away. To analyze the role of HS1 and HS2 in boundary, deletions were generated of HS1 or HS1 + HS2 that have attP site for boundary replacement experiments. The 1389 bp HS1 deletion can be rescued by a 529 bp core Fab-6 sequence that includes two CTCF sites. However, Fab-6 HS1 cannot rescue the HS1 + HS2 deletion or substitute for another BX-C boundary - Fab-7. For this it must be combined with a PRE, either Fab-7 HS3, or Fab-6 HS2. These findings suggest that the boundary function of Fab-6 HS1 must be bolstered by a second element that has PRE activity.
Galouzis, C. C. and Prud'homme, B. (2021). Transvection regulates the sex-biased expression of a fly X-linked gene. Science 371(6527): 396-400. PubMed ID: 33479152
Sexual dimorphism in animals results from sex-biased gene expression patterns. These patterns are controlled by genetic sex determination hierarchies that establish the sex of an individual. This study shows that the male-biased wing expression pattern of the Drosophila biarmipes gene yellow, located on the X chromosome, is independent of the fly sex determination hierarchy. Instead, it was found that a regulatory interaction between yellow alleles on homologous chromosomes (a process known as transvection) silences the activity of a yellow enhancer functioning in the wing. Therefore, this enhancer can be active in males (XY) but not in females (XX). This transvection-dependent enhancer silencing requires the yellow intron and the chromatin architecture protein Mod(mdg4). These results suggest that transvection can contribute more generally to the sex-biased expression of X-linked genes. Rao, S., Ahmad, K. and Ramachandran, S. (2021). Cooperative binding between distant transcription factors is a hallmark of active enhancers. Mol Cell. PubMed ID: 33705711
Enhancers harbor binding motifs that recruit transcription factors (TFs) for gene activation. While cooperative binding of TFs at enhancers is known to be critical for transcriptional activation of a handful of developmental enhancers, the extent of TF cooperativity genome-wide is unknown. This study coupled high-resolution nuclease footprinting with single-molecule methylation profiling to characterize TF cooperativity at active enhancers in the Drosophila genome. Enrichment of short micrococcal nuclease (MNase)-protected DNA segments indicates that the majority of enhancers harbor two or more TF-binding sites, and this study uncovered protected fragments that correspond to co-bound sites in thousands of enhancers. From the analysis of co-binding, this study found that cooperativity dominates TF binding in vivo at the majority of active enhancers. Cooperativity is highest between sites spaced 50 bp apart, indicating that cooperativity occurs without apparent protein-protein interactions. These findings suggest nucleosomes promote cooperativity because co-binding may effectively clear nucleosomes and promote enhancer function.
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