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Current papers in developmental biology and gene function


Thursday, December 31st, 2020 - RNA and Transposons

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Cheng, L., Zhang, Y., Zhang, Y., Chen, T., Xu, Y. Z. and Rong, Y. S. (2020). Loss of the RNA trimethylguanosine cap is compatible with nuclear accumulation of spliceosomal snRNAs but not pre-mRNA splicing or snRNA processing during animal development. PLoS Genet 16(10): e1009098. PubMed ID: 33085660
The 2,2,7-trimethylguanosine (TMG) cap is one of the first identified modifications on eukaryotic RNAs. TMG, synthesized by the conserved Tgs1 enzyme, is abundantly present on snRNAs essential for pre-mRNA splicing. Results from ex vivo experiments in vertebrate cells suggested that TMG ensures nuclear localization of snRNAs. Functional studies of TMG using tgs1 mutations in unicellular organisms yield results inconsistent with TMG being indispensable for either nuclear import or splicing. Utilizing a hypomorphic Tgs1 mutation in Drosophila, this study shows that TMG reduction impairs germline development by disrupting the processing, particularly of introns with smaller sizes and weaker splice sites. Unexpectedly, loss of TMG does not disrupt snRNAs localization to the nucleus, disputing an essential role of TMG in snRNA transport. Tgs1 loss also leads to defective 3' processing of snRNAs. Remarkably, stronger Tgs1 mutations cause lethality without severely disrupting splicing, likely due to the preponderance of TMG-capped snRNPs. Tgs1, a predominantly nucleolar protein in Drosophila, likely carries out splicing-independent functions indispensable for animal development. Taken together, these results suggest that nuclear import is not a conserved function of TMG. As a distinctive structure on RNA, particularly non-coding RNA, it is suggested that TMG prevents spurious interactions detrimental to the function of RNAs that it modifies.
Laohakieat, K., Isasawin, S. and Thanaphum, S. (2020). The transformer-2 and fruitless characterisation with developmental expression profiles of sex-determining genes in Bactrocera dorsalis and B. correcta. Sci Rep 10(1): 17938. PubMed ID: 33087807
Sex determination in tephritid fruit flies involves a signaling cascade of alternatively spliced genes. The Transformer (TRA) and Transformer-2 (TRA-2) complex establishes an autoregulatory loop switching sex-specific splicing of tra pre-mRNA in females. The TRA/TRA-2 complex also regulates the sex-specific splicing of downstream effector genes, doublesex (dsx) and fruitless (fru). In Ceratitis capitata, a Maleness-on the-Y (MoY) gene modulates sex-specifically spliced Cctra pre-mRNA and results in the breakdown of the Cctra autoregulatory loop in males. In this study, the tra-2 and fru genes were characterised in two key pests, Bactrocera dorsalis and B. correcta. The tra-2 genes showed high degrees of conservation among tephritids. The complex gene organisation for each of Bdfru and Bcfru were identified. There are sex-specific and non sex-specific transcripts generated by alternative promoters as found in Drosophila melanogaster and other insects. RNAi knockdown of Bdtra transcripts showed that BdTRA controls the sex-specific splicing of Bddsx and Bdfru pre-mRNAs. Developmental expression analysis shows that multiple splice variants of Bdtra and Bctra RNAs are present before and during cellular blastoderm formation and that the mature sex-specific variants become fixed later in embryogenesis. Furthermore, the Bddsx(M) splice variants are found in early embryos at the beginning of gastulation, but Bdfru(M) does not appear until the larval stage. It is proposed that the zygotic tra loop is initiated in both female and male embryos before becoming automatised or abolished by MoY, respectively.
Hao, Y., Wang, D., Wu, S., Li, X., Shao, C., Zhang, P., Chen, J. Y., Lim, D. H., Fu, X. D., Chen, R. and He, S. (2020). Active retrotransposons help maintain pericentromeric heterochromatin required for faithful cell division. Genome Res 30(11): 1570-1582. PubMed ID: 33060173
Retrotransposons are populated in vertebrate genomes, and when active, are thought to cause genome instability with potential benefit to genome evolution. Retrotransposon-derived RNAs are also known to give rise to small endo-siRNAs to help maintain heterochromatin at their sites of transcription; however, as not all heterochromatic regions are equally active in transcription, it remains unclear how heterochromatin is maintained across the genome. This study addresses these problems by defining the origins of repeat-derived RNAs and their specific chromatin locations in Drosophila S2 cells. Repeat RNAs are predominantly derived from active gypsy elements and processed by Dcr-2 into small RNAs to help maintain pericentromeric heterochromatin. In cultured S2 cells that synthetic repeat-derived endo-siRNA mimics are sufficient to rescue Dcr-2-deficiency-induced defects in heterochromatin formation in interphase and chromosome segregation during mitosis, demonstrating that active retrotransposons are required for stable genetic inheritance.
Tatomer, D. C., Liang, D. and Wilusz, J. E. (2021). RNAi Screening to Identify Factors That Control Circular RNA Localization. Methods Mol Biol 2209: 321-332. PubMed ID: 33201478
Thousands of eukaryotic protein-coding genes are noncanonically spliced to generate circular RNAs that have covalently linked ends. In general, exonic circular RNAs accumulate in the cytoplasm. Recent work has revealed that circular RNAs are exported from the nucleus in a length-dependent manner and that the subcellular localization of these transcripts can be controlled by the DExH/D-box helicase Hel25E in Drosophila. This study describes how RNAi screening combined with subcellular fractionation and quantitative reverse transcription PCR (RT-qPCR) can be used to identify regulators of circular RNA localization in Drosophila cells. Long double-stranded RNAs (dsRNAs) that activate the RNA interference (RNAi) pathway are used to deplete factors of interest followed by biochemical fractionation to separate nuclear and cytoplasmic RNAs. RT-qPCR primers that amplify across the backsplicing junction of specific circular RNAs are then used to quantify the relative amounts of these transcripts in the nuclear and cytoplasmic compartments. In total, this approach can be broadly used to characterize circular RNA nuclear export and localization mechanisms, including to identify novel regulatory factors and their breadth of circular RNA targets.
Zhang, R., Du, J., Zhao, X., Wei, L. and Zhao, Z. (2020). Regulation of circadian behavioural output via clock-responsive miR-276b. Insect Mol Biol. PubMed ID: 33131172
Growing evidence indicates that microRNAs play numerous important roles. However, the roles of some microRNAs involved in regulation of circadian rhythm and sleep are still not well understood. This study shows that the miR-276b is essential for maintaining both sleep and circadian rhythm by targeting tim, NPFR and DopR1 genes, with miR-276b deleted mutant flies sleeping more, and vice versa in miR-276b overexpressing flies. Through analysing its promoter, mir-276b was found to be responsive to CLOCK and regulates circadian rhythm through the negative feedback loop of the CLK/CYC-TIM/PER. Furthermore, miR-276b is broadly expressed in the clock neurons and the central complexes such as the mushroom body and the fan-shape body of Drosophila brain, in which up-regulation of miR-276b in tim, npfr1 and DopR1 expressing tissues significantly causes sleep decreases. This study clarifies that the mir-276b is very important for participating in regulation of circadian rhythm and sleep.
Huang, D. Y., Xia, X. L., Huang, R., Li, S., Yuan, D. W. and Liu, S. N. (2020). The steroid-induced microRNA let-7 regulates developmental growth by targeting cdc7 in the Drosophila fat body. Insect Sci. PubMed ID: 33089948
In insects, 20-hydroxyecdysone (20E) limits systemic growth by triggering developmental transitions. Previous studies have shown that 20E-induced let-7 exhibits crosstalk with the cell cycle. This study examined the underlying molecular mechanisms and physiological functions of 20E-induced let-7 in the fat body, an organ for energy storage and nutrient mobilization which plays a critical role in the larval growth. First, the overexpression of let-7 decreased the body size and led to the reduction of both nucleolus and cell sizes in the larval fat body. In contrast, the overexpression of let-7-Sponge increased the nucleolus and cell sizes. Moreover, cdc7, encoding a conserved protein kinase that controls the endocycle, is a target of let-7. Notably, the mutation of cdc7 in the fat body resulted in growth defects. Overall, these findings revealed a novel role of let-7 in the control of endoreduplication-related growth during larval-prepupal transition in Drosophila.

Wednesday, December 30th - Adult neural development and function

Tsao, C. K., Huang, Y. F. and Sun, Y. H. (2020). Early lineage segregation of the retinal basal glia in the Drosophila eye disc. Sci Rep 10(1): 18522. PubMed ID: 33116242
The retinal basal glia (RBG) is a group of glia that migrates from the optic stalk into the third instar larval eye disc while the photoreceptor cells (PR) are differentiating. The RBGs are grouped into three major classes based on molecular and morphological characteristics: surface glia (SG), wrapping glia (WG) and carpet glia (CG). The SGs migrate and divide. The WGs are postmitotic and wraps PR axons. The CGs have giant nucleus and extensive membrane extension that each covers half of the eye disc. This study used lineage tracing methods to determine the lineage relationships among these glia subtypes and the temporal profile of the lineage decisions for RBG development. The CG lineage was found to segregate from the other RBG very early in the embryonic stage. It has been proposed that the SGs migrate under the CG membrane, which prevented SGs from contacting with the PR axons lying above the CG membrane. Upon passing the front of the CG membrane, which is slightly behind the morphogenetic furrow that marks the front of PR differentiation, the migrating SG contact the nascent PR axon, which in turn release FGF to induce SGs' differentiation into WG. Interestingly, it was found that SGs are equally distributed apical and basal to the CG membrane, so that the apical SGs are not prevented from contacting PR axons by CG membrane. Clonal analysis reveals that the apical and basal RBG are derived from distinct lineages determined before they enter the eye disc. Moreover, the basal SG lack the competence to respond to FGFR signaling, preventing its differentiation into WG. Thes findings suggest that this novel glia-to-glia differentiation is both dependent on early lineage decision and on a yet unidentified regulatory mechanism, which can provide spatiotemporal coordination of WG differentiation with the progressive differentiation of photoreceptor neurons.
Eschment, M., Franz, H. R., Gullu, N., Holscher, L. G., Huh, K. E. and Widmann, A. (2020). Insulin signaling represents a gating mechanism between different memory phases in Drosophila larvae. PLoS Genet 16(10): e1009064. PubMed ID: 33104728
Formation of short term memory is energetically costly and by the reason of restricted availability of food or fluctuations in energy expanses, efficient metabolic homeostasis modulating different needs like survival, growth, reproduction, or investment in longer lasting memories is crucial. Whilst equipped with cellular and molecular pre-requisites for formation of a protein synthesis dependent long-term memory (LTM), its existence in the larval stage of Drosophila remains elusive. Considering it from the viewpoint that larval brain structures are completely rebuilt during metamorphosis, and that this process depends completely on accumulated energy stores formed during the larval stage, investing in LTM represents an unnecessary expenditure. However, as an alternative, Drosophila larvae are equipped with the capacity to form a protein synthesis independent so-called larval anaesthesia resistant memory (lARM), which is consolidated in terms of being insensitive to cold-shock treatments. Motivated by the fact that LTM formation causes an increase in energy uptake in Drosophila adults, this study tested the question of whether an energy surplus can induce the formation of LTM in the larval stage. Surprisingly, increasing the metabolic state by feeding Drosophila larvae the disaccharide sucrose directly before aversive olfactory conditioning led to the formation of a protein synthesis dependent longer lasting memory. Moreover, formation of this memory component is accompanied by the suppression of lARM. It was ascertained that insulin receptors (InRs) expressed in the mushroom body Kenyon cells suppresses the formation of lARM and induces the formation of a protein synthesis dependent longer lasting memory in Drosophila larvae. Given the numerical simplicity of the larval nervous system this work offers a unique prospect to study the impact of insulin signaling on the formation of protein synthesis dependent memories on a molecular level.
Duhart, J. M., Baccini, V., Zhang, Y., Machado, D. R. and Koh, K. (2020). Modulation of sleep-courtship balance by nutritional status in Drosophila. Elife 9. PubMed ID: 33084567
Sleep is essential but incompatible with other behaviors, and thus sleep drive competes with other motivations. Previous work has shown Drosophila males balance sleep and courtship via octopaminergic neurons that act upstream of courtship-regulating P1 neurons. This study shows that nutrition modulates the sleep-courtship balance and identify sleep-regulatory neurons downstream of P1 neurons. Yeast-deprived males exhibited attenuated female-induced nighttime sleep loss yet normal daytime courtship, which suggests male flies consider nutritional status in deciding whether the potential benefit of pursuing female partners outweighs the cost of losing sleep. Trans-synaptic tracing and calcium imaging identified dopaminergic neurons projecting to the protocerebral bridge (DA-PB) as postsynaptic partners of P1 neurons. Activation of DA-PB neurons led to reduced sleep in normally fed but not yeast-deprived males. Additional PB-projecting neurons regulated male sleep, suggesting several groups of PB-projecting neurons act downstream of P1 neurons to mediate nutritional modulation of the sleep-courtship balance.
Cervantes-Sandoval, I., Davis, R. L. and Berry, J. A. (2020). Rac1 Impairs Forgetting-Induced Cellular Plasticity in Mushroom Body Output Neurons. Front Cell Neurosci 14: 258. PubMed ID: 33061890
Active memory forgetting is a well-regulated biological process thought to be adaptive and to allow proper cognitive functions. Recent efforts have elucidated several molecular players involved in the regulation of olfactory forgetting in Drosophila, including the small G protein Rac1, the dopamine receptor DAMB, and the scaffold protein Scribble. Similarly, recent work has reported that dopaminergic neurons mediate both learning- and forgetting-induced plasticity in the mushroom body output neuron MBON-γ2α'1. Two open questions remain: how does forgetting affect plasticity in other, highly plastic, mushroom body compartments and how do genes that regulate forgetting affect this cellular plasticity? This study shows that forgetting reverses short-term synaptic depression induced by aversive conditioning in the highly plastic mushroom body output neuron MBON-γ1pedc>α/β. In addition, the results indicate that genetic tampering with normal forgetting by inhibition of small G protein Rac1 impairs restoration of depressed odor responses to learned odor by intrinsic forgetting through time passing and forgetting induced acutely by shock stimulation after conditioning or reversal learning. These data further indicate that some forms of forgetting truly erase physiological changes generated by memory encoding.
Timaeus, L., Geid, L., Sancer, G., Wernet, M. F. and Hummel, T. (2020). Parallel Visual Pathways with Topographic versus Nontopographic Organization Connect the Drosophila Eyes to the Central Brain. iScience 23(10): 101590. PubMed ID: 33205011
One hallmark of the visual system is a strict retinotopic organization from the periphery toward the central brain, where functional imaging in Drosophila revealed a spatially accurate representation of visual cues in the central complex. This raised the question how, on a circuit level, the topographic features are implemented, as the majority of visual neurons enter the central brain converge in optic glomeruli. This study discovered a spatial segregation of topographic versus nontopographic projections of distinct classes of medullo-tubercular (MeTu) neurons into a specific visual glomerulus, the anterior optic tubercle (AOTU). These parallel channels synapse onto different tubercular-bulbar (TuBu) neurons, which in turn relay visual information onto specific central complex ring neurons in the bulb neuropil. Hence, these results provide the circuit basis for spatially accurate representation of visual information and highlight the AOTU's role as a prominent relay station for spatial information from the retina to the central brain.
Fendl, S., Vieira, R. M. and Borst, A. (2020). Parallel Visual Conditional protein tagging methods reveal highly specific subcellular distribution of ion channels in motion-sensing neurons. Elife 9. PubMed ID: 33079061

Neurotransmitter receptors and ion channels shape the biophysical properties of neurons, from the sign of the response mediated by neurotransmitter receptors to the dynamics shaped by voltage-gated ion channels. Therefore, knowing the localizations and types of receptors and channels present in neurons is fundamental to understanding of neural computation. This study developed two approaches to visualize the subcellular localization of specific proteins in Drosophila: The flippase-dependent expression of GFP-tagged receptor subunits in single neurons and 'FlpTag', a versatile new tool for the conditional labelling of endogenous proteins. Using these methods, the subcellular distribution of the receptors GluClα, Rdl, and Dα7 and the ion channels Para and Ih in motion-sensing T4/T5 neurons of the Drosophila visual system was investigated. A strictly segregated subcellular distribution of these proteins a sequential spatial arrangement of glutamate, acetylcholine, and GABA receptors was discovered along the dendrite that matched the previously reported EM-reconstructed synapse distributions.

Tuesday, December 29th - Gonads

Yuzawa, T., Matsuoka, M., Sumitani, M., Aoki, F., Sezutsu, H. and Suzuki, M. G. (2020). Transgenic and knockout analyses of Masculinizer and doublesex illuminated the unique functions of doublesex in germ cell sexual development of the silkworm, Bombyx mori. BMC Dev Biol 20(1): 19. PubMed ID: 32957956
Masculinizer (Masc) plays a pivotal role in male sex determination in the silkworm, Bombyx mori. Masc is required for male-specific splicing of B. mori doublesex (Bmdsx; see Drosophila Dsx) transcripts. The male isoform of Bmdsx (BmdsxM) induces male differentiation in somatic cells, while females express the female isoform of Bmdsx (BmdsxF), which promotes female differentiation in somatic cells. Previous findings suggest that Masc could direct the differentiation of genetically female (ZW) germ cells into sperms. However, it remains unclear whether Masc directly induces spermatogenesis or if it promotes male differentiation in germ cells indirectly by inducing the expression of BmdsxM. In this study, genetic analyses were performed using the transgenic line that expressed Masc, as well as various Bmdsx knockout lines. Masc-expressing females with a homozygous mutation in BmdsxM showed normal development in ovaries. The formation of testis-like tissues was abolished in these females. On the other hand, Masc-expressing females carrying a homozygous mutation in BmdsxF exhibited almost complete male-specific development in gonads and germ cells. These results suggest that BmdsxM has an ability to induce male development in germ cells as well as internal genital organs, while BmdsxF inhibits BmdsxM activity and represses male differentiation. To investigate whether MASC directly controls male-specific splicing of Bmdsx and identify RNAs that form complexes with MASC in testes, RNA immunoprecipitation (RIP) was performed using an anti-MASC antibody. MASC was found to form a complex with AS1 lncRNA, which is a testis-specific factor involved in the male-specific splicing of Bmdsx pre-mRNA. Taken together, these findings suggest that Masc induces male differentiation in germ cells by enhancing the production of BmdsxM. Physical interaction between MASC and AS1 lncRNA may be important for the BmdsxM expression in the testis. Unlike in the Drosophila dsx, BmdsxM was able to induce spermatogenesis in genetically female (ZW) germ cells. This is the first report that the role of dsx in germ cell sexual development is different between insect species.
Miscopein Saler, L., Hauser, V., Bartoletti, M., Mallart, C., Malartre, M., Lebrun, L., Pret, A. M., Theodore, L., Chalvet, F. and Netter, S. (2020). The Bric-a-Brac BTB/POZ transcription factors are necessary in niche cells for germline stem cells establishment and homeostasis through control of BMP/DPP signaling in the Drosophila melanogaster ovary. PLoS Genet 16(11): e1009128. PubMed ID: 33151937
How formation of a functional niche is initiated, including how stem cells within a niche are established, is less well understood. Adult Drosophila ovary Germline Stem Cell (GSC) niches are comprised of somatic cells forming a stack called a Terminal Filament (TF) and associated Cap and Escort Cells (CCs and ECs, respectively), which are in direct contact with GSCs. In the adult ovary, the transcription factor Engrailed is specifically expressed in niche cells where it directly controls expression of the decapentaplegic (dpp) gene encoding a member of the Bone Morphogenetic Protein (BMP) family of secreted signaling molecules, which are key factors for GSC maintenance. In larval ovaries, in response to BMP signaling from newly formed niches, adjacent primordial germ cells become GSCs. The bric-à-brac paralogs (bab1 and bab2) encode BTB/POZ domain-containing transcription factors that are expressed in developing niches of larval ovaries. This study shows that their functions are necessary specifically within precursor cells for TF formation during these stages. A new function was identified for Bab1 and Bab2 within developing niches for GSC establishment in the larval ovary and for robust GSC maintenance in the adult. Moreover, the presence of Bab proteins in niche cells was shown to be necessary for activation of transgenes reporting dpp expression as of larval stages in otherwise correctly specified Cap Cells, independently of Engrailed and its paralog Invected (En/Inv). Moreover, strong reduction of engrailed/invected expression during larval stages does not impair TF formation and only partially reduces GSC numbers. In the adult ovary, Bab proteins are also required for dpp reporter expression in CCs. Finally, when bab2 was overexpressed at this stage in somatic cells outside of the niche, there were no detectable levels of ectopic En/Inv, but ectopic expression of a dpp transgene was found in these cells and BMP signaling activation was induced in adjacent germ cells, which produced GSC-like tumors. Together, these results indicate that Bab transcription factors are positive regulators of BMP signaling in niche cells for establishment and homeostasis of GSCs in the Drosophila ovary.
Chen, W., Luan, X., Yan, Y., Wang, M., Zheng, Q., Chen, X., Yu, J. and Fang, J. (2020). CG8005 Mediates Transit-Amplifying Spermatogonial Divisions via Oxidative Stress in Drosophila Testes. Oxid Med Cell Longev 2020: 2846727. PubMed ID: 33193998
The generation of reactive oxygen species (ROS) widely occurs in metabolic reactions and affects stem cell activity by participating in stem cell self-renewal. However, the mechanisms of transit-amplifying (TA) spermatogonial divisions mediated by oxidative stress are not fully understood. Through genetic manipulation of Drosophila testes, this study demonstrated that CG8005 regulated TA spermatogonial divisions and redox homeostasis. Using in vitro approaches, it was shown that the knockdown of CG8005 increased ROS levels in S2 cells; the induced ROS generation was inhibited by NAC and exacerbated by H(2)O(2) pretreatments. Furthermore, the silencing of CG8005 increased the mRNA expression of oxidation-promoting factors Keap1, GstD1, and Mal-A6 and decreased the mRNA expression of antioxidant factors cnc, Gclm, maf-S, ND-42, and ND-75. The functions of the antioxidant factor cnc, a key factor in the Keap1-cnc signaling pathway was further investigated; cnc mimicked the phenotype of CG8005 in both Drosophila testes and S2 cells. These results indicated that CG8005, together with cnc, controlled TA spermatogonial divisions by regulating oxidative stress in Drosophila.
Delbare, S. Y. N., Ahmed-Braimah, Y. H., Wolfner, M. F. and Clark, A. G. (2020). Interactions between the microbiome and mating influence the female's transcriptional profile in Drosophila melanogaster. Sci Rep 10(1): 18168. PubMed ID: 33097776
Drosophila melanogaster females undergo a variety of post-mating changes that influence their activity, feeding behavior, metabolism, egg production and gene expression. These changes are induced either by mating itself or by sperm or seminal fluid proteins. In addition, studies have shown that axenic females-those lacking a microbiome-have altered fecundity compared to females with a microbiome, and that the microbiome of the female's mate can influence reproductive success. This study investigated fecundity and the post-mating transcript abundance profile of axenic or control females after mating with either axenic or control males. Interactions between the female's microbiome and her mating status were observed: transcripts of genes involved in reproduction and genes with neuronal functions were differentially abundant depending on the females' microbiome status, but only in mated females. In addition, immunity genes showed varied responses to either the microbiome, mating, or a combination of those two factors. It was further observed that the male's microbiome status influences the fecundity of both control and axenic females, while only influencing the transcriptional profile of axenic females. These results indicate that the microbiome plays a vital role in the post-mating switch of the female's transcriptome.
Dai, W., Guo, X., Cao, Y., Mondo, J. A., Campanale, J. P., Montell, B. J., Burrous, H., Streichan, S., Gov, N., Rappel, W. J. and Montell, D. J. (2020). Tissue topography steers migrating Drosophila border cells. Science 370(6519): 987-990. PubMed ID: 33214282
Moving cells can sense and respond to physical features of the microenvironment; however, in vivo, the significance of tissue topography is mostly unknown. This study used Drosophila border cells, an established model for in vivo cell migration, to study how chemical and physical information influences path selection. Although chemical cues were thought to be sufficient, live imaging, genetics, modeling, and simulations show that microtopography is also important. Chemoattractants promote predominantly posterior movement, whereas tissue architecture presents orthogonal information, a path of least resistance concentrated near the center of the egg chamber. E-cadherin supplies a permissive haptotactic cue. These results provide insight into how cells integrate and prioritize topographical, adhesive, and chemoattractant cues to choose one path among many.
Rust, K., Byrnes, L. E., Yu, K. S., Park, J. S., Sneddon, J. B., Tward, A. D. and Nystul, T. G. (2020). A single-cell atlas and lineage analysis of the adult Drosophila ovary. Nat Commun 11(1): 5628. PubMed ID: 33159074
The Drosophila ovary is a widely used model for germ cell and somatic tissue biology. This study used single-cell RNA-sequencing (scRNA-seq) to build a comprehensive cell atlas of the adult Drosophila ovary that contains transcriptional profiles for every major cell type in the ovary, including the germline stem cells and their niche cells, follicle stem cells, and previously undescribed subpopulations of escort cells. In addition, Gal4 lines were identified with specific expression patterns, and lineage tracing of subpopulations of escort cells and follicle cells was performed. A distinct subpopulation of escort cells was capable of converting to follicle stem cells in response to starvation or upon genetic manipulation, including knockdown of escargot, or overactivation of mTor or Toll signalling.

Monday December 28th - Synapse and Vesicles

Spinner, M. A., Pinter, K., Drerup, C. M. and Herman, T. G. (2020). A Conserved Role for Vezatin Proteins in Cargo-Specific Regulation of Retrograde Axonal Transport. Genetics. PubMed ID: 32788307
Active transport of organelles within axons is critical for neuronal health. Retrograde axonal transport, in particular, relays neurotrophic signals received by axon terminals to the nucleus and circulates new material among en passant synapses. A single motor protein complex, cytoplasmic dynein, is responsible for nearly all retrograde transport within axons: its linkage to and transport of diverse cargos is achieved by cargo-specific regulators. This study has identified Vezatin as a conserved regulator of retrograde axonal transport. Vertebrate Vezatin (Vezt) is required for the maturation and maintenance of cell-cell junctions and has not previously been implicated in axonal transport. However, a related fungal protein, VezA, has been shown to regulate retrograde transport of endosomes in hyphae. In a forward genetic screen,a loss-of-function mutation was identified in the Drosophila vezatin-like (vezl) gene. vezl loss prevents a subset of endosomes, including signaling endosomes containing activated BMP receptors, from initiating transport out of motor neuron terminal boutons. vezl loss also decreases the transport of endosomes and dense core vesicles (DCVs) but not mitochondria within axon shafts. vezl was disrupted in zebrafish; vezl loss specifically impairs the retrograde axonal movement of late endosomes, causing their accumulation in axon terminals. This work establishes a conserved, cargo-specific role for Vezatin proteins in retrograde axonal transport.
Karunanithi, S., Lin, Y. Q., Odierna, G. L., Menon, H., Gonzalez, J. M., Neely, G. G., Noakes, P. G., Lavidis, N. A., Moorhouse, A. J. and van Swinderen, B. (2020). Activity-dependent global downscaling of evoked neurotransmitter release across glutamatergic inputs in Drosophila. J Neurosci. PubMed ID: 32928887
Within mammalian brain circuits, activity-dependent synaptic adaptations such as synaptic scaling stabilise neuronal activity in the face of perturbations. It remains unclear whether activity-dependent uniform scaling also operates within peripheral circuits. This study tested for such scaling in a Drosophila larval neuromuscular circuit, where the muscle receives synaptic inputs from different motoneurons. Motoneuron-specific genetic manipulations were employed to increase the activity of only one motoneuron and recordings of postsynaptic currents from inputs formed by the different motoneurons. An adaptation was detected which caused uniform downscaling of evoked neurotransmitter release across all inputs through decreases in release probabilities. This 'presynaptic downscaling' maintained the relative differences in neurotransmitter release across all inputs around a homeostatic set point, caused a compensatory decrease in synaptic drive to the muscle affording robust and stable muscle activity, and was induced within hours. Presynaptic downscaling was associated with an activity-dependent increase in Drosophila vesicular glutamate transporter (DVGLUT) expression. Activity-dependent uniform scaling can therefore manifest also on the presynaptic side to produce robust and stable circuit outputs. Within brain circuits, uniform downscaling on the postsynaptic side is implicated in sleep- and memory-related processes. These results suggest that evaluation of such processes might be broadened to include uniform downscaling on the presynaptic side.
Wan, B., Poirie, M. and Gatti, J. L. (2020). Parasitoid wasp venom vesicles (venosomes) enter Drosophila melanogaster lamellocytes through a flotillin/lipid raft-dependent endocytic pathway. Virulence 11(1): 1512-1521. PubMed ID: 33135553
Venosomes are extracellular vesicles found in the venom of Leptopilina endoparasitoids wasps, which transport and target virulence factors to impair the parasitoid egg encapsulation by the lamellocytes of their Drosophila melanogaster host larva. Using the co-immunolocalization of fluorescent L. boulardi venosomes and one of the putative-transported virulence factors, LbGAP, with known markers of cellular endocytosis, this study showed that venosomes endocytosis by lamellocytes is not a process dependent on clathrin or macropinocytosis and internalization seems to bypass the early endosomal compartment Rab5. After internalization, LbGAP colocalizes strongly with flotillin-1 and the GPI-anchored protein Atilla/L1 (a lamellocyte surface marker) suggesting that entry occurs via a flotillin/lipid raft-dependent pathway. Once internalized, venosomes reach all intracellular compartments, including late and recycling endosomes, lysosomes, and the endoplasmic reticulum network. Venosomes therefore enter their target cells by a specific mechanism and the virulence factors are widely distributed in the lamellocytes' compartments to impair their functions.
Damulewicz, M., Woznicka, O., Jasinska, M. and Pyza, E. (2020). CRY-dependent plasticity of tetrad presynaptic sites in the visual system of Drosophila at the morning peak of activity and sleep. Sci Rep 10(1): 18161. PubMed ID: 33097794
Tetrad synapses are formed between the retina photoreceptor terminals and postsynaptic cells in the first optic neuropil (lamina) of Drosophila. They are remodelled in the course of the day and show distinct functional changes during activity and sleep. These changes result from fast degradation of the presynaptic scaffolding protein Bruchpilot (BRP) by Cryptochrome (CRY) in the morning and depend on BRP-170, one of two BRP isoforms. This process also affects the number of synaptic vesicles, both clear and dense-core, delivered to the presynaptic elements. In cry01 mutants lacking CRY and in brpΔ170, the number of synaptic vesicles is lower in the morning peak of activity than during night-sleep while in wild-type flies the number of synaptic vesicles is similar at these two time points. CRY may also set phase of the circadian rhythm in plasticity of synapses. The process of synapse remodelling stimulates the formation of clear synaptic vesicles in the morning. They carry histamine, a neurotransmitter in tetrad synapses and seem to be formed from glial capitate projections inside the photoreceptor terminals. In turn dense-core vesicles probably carry synaptic proteins building the tetrad presynaptic element.
Greenhalgh, A., Istas, O. and Cooper, R. L. (2020). Bacterial endotoxin lipopolysaccharide enhances synaptic transmission at low-output glutamatergic synapse. Neurosci Res. PubMed ID: 32987087
The endotoxin lipopolysaccharides (LPS), secreted from gram-negative bacteria, has direct effects on synaptic transmission independent of systemic secondary cytokine responses. High concentration of LPS (500 microg/ml) from Serratia marcescens increased synaptic efficacy at glutamatergic low-output synapses more than for high-output synapses. Over an hour of exposure was not toxic to the preparation and continued to enhance synaptic transmission. A small but significant rapid hyperpolarization of the post-synaptic cells occurred, in addition to a slower enhancement of in the amplitude of evoked excitatory junction potentials. LPS may promote reserve pool vesicles to the readily releasable pool for low-output synapses. The action of LPS at the glutamatergic synapses of the crayfish neuromuscular junction is unique in promoting synaptic transmission as compared to other glutamatergic synapses in Drosophila and mammals, where synaptic transmission is depressed.
Revaitis, N. T., Niepielko, M. G., Marmion, R. A., Klein, E. A., Piccoli, B. and Yakoby, N. (2020). Quantitative analyses of EGFR localization and trafficking dynamics in the follicular epithelium. Development. PubMed ID: 32680934
To bridge the gap between qualitative and quantitative analyses of the epidermal growth factor receptor (EGFR) in tissues, this study generated a sfGFP-tagged EGF receptor (EGFR-sfGFP) in Drosophila. The homozygous fly appears wild type with EGFR expression and activation patterns that are consistent with previous reports in the ovary, early embryo, and imaginal discs. Using ELISA, an average of 1100, 6200, and 2500 receptors was quantified per follicle cell (FC) at stages 8/9, 10, and ≥11 of oogenesis, respectively. Interestingly, the spatial localization of the EGFR to the apical side of the FCs at early stages depended on the TGF-alpha-like ligand Gurken. At later stages, EGFR localized to basolateral positions of the FCs. Finally, the endosomal localization of EGFR was followed in the FCs. The EGFR co-localized with the late endosome, but no significant co-localization of the receptor was found with the early endosome. The EGFR-sfGFP fly is an exciting new resource to study cellular localization and regulation of EGFR in tissues.

Thursday, December 24th - Signaling

Wong, K. K. L., Liao, J. Z., Shih, C. R. Y., Harden, N. and Verheyen, E. M. (2020). Hyperpolarized mitochondria accumulate in Drosophila Hipk-overexpressing cells to drive tumor-like growth. J Cell Sci. PubMed ID: 33199523
Both functional and dysfunctional mitochondria are known to underlie tumor progression. This study established use of the proto-oncogene Drosophila Homeodomain-interacting protein kinase (Hipk) as a new tool to address this paradox. In Hipk-overexpressing tumor-like cells, it was found that mitochondria accumulate and switch from fragmented to highly fused interconnected morphologies. Moreover, elevated Hipk promotes mitochondrial membrane hyperpolarization. These mitochondrial changes are at least in part driven by the upregulation of Myc. Furthermore, the altered mitochondrial energetics, but not morphology, was shown to be required for Hipk tumor-like growth as knockdown of pdsw (NDUFB10 in mammals; a Complex I subunit) abrogates the growth. Knockdown of ATPsynβ (a Complex V subunit), which produces higher levels of reactive oxygen species (ROS) than pdsw knockdown, instead synergizes with Hipk to potentiate JNK activation and the downstream induction of Matrix metalloproteinases. Accordingly, ATPsynβ knockdown suppresses Hipk tumor-like growth only when ROS scavengers are co-expressed. Altogether, this work presents an in vivo tumor model featuring the accumulation of hyperfused and hyperpolarized mitochondria, and reveals respiratory Complex subunit-dependent, opposing effects on tumorigenic outcomes.
Budnar, P., Singh, N. P. and Rao, C. M. (2020). HSPB5 (αB-crystallin) confers protection against paraquat-induced oxidative stress at the organismal level in a tissue-dependent manner. Cell Stress Chaperones. PubMed ID: 33078332
Oxidative stress is one of the major and continuous stresses, an organism encounters during its lifetime. Tissues such as the brain, liver and muscles are more prone to damage by oxidative stress due to their metabolic activity, differences in physiological and adaptive processes. One of the defence mechanisms against continuous oxidative stress is a set of small heat shock proteins. αB-Crystallin/HSPB5 (see Drosophila l(2)efl), a small heat shock protein, gets upregulated under stress and acts as a molecular chaperone. In addition to acting as a molecular chaperone, HSPB5 is shown to have a role in other cytoprotective functions such as inhibition of apoptosis, prevention of oxidative stress and stabilisation of cytoskeletal system. Such protection in vivo, at the organism level, particularly in a tissue-dependent manner, has not been investigated. This study expressed HSPB5 in fat body (liver), neurons and specifically in dopaminergic and motor neurons in Drosophila and investigated its protective effect against paraquat-induced oxidative stress. Expression of HSPB5 in neurons and fat body confers protection against paraquat-induced oxidative stress. Expression in dopaminergic neurons showed a higher protective effect. These results clearly establish the protective ability of HSPB5 in vivo; the extent of protection, however, varies depending on the tissue in which it is expressed. Interestingly, neuronal expression of HSPB5 resulted in an improvement in negative geotropic behaviour, whereas specific expression in muscle tissue did not show such a beneficial effect.
Sun, G., Ding, X. A., Argaw, Y., Guo, X. and Montell, D. J. (2020). Akt1 and dCIZ1 promote cell survival from apoptotic caspase activation during regeneration and oncogenic overgrowth. Nat Commun 11(1): 5726. PubMed ID: 33184261
Apoptosis is an ancient and evolutionarily conserved cell suicide program. During apoptosis, executioner caspase enzyme activation has been considered a point of no return. However, emerging evidence suggests that some cells can survive caspase activation following exposure to apoptosis-inducing stresses, raising questions as to the physiological significance and underlying molecular mechanisms of this unexpected phenomenon. This study shows that, following severe tissue injury, Drosophila wing disc cells that survive executioner caspase activation contribute to tissue regeneration. Through RNAi screening, this study identified akt1 and a previously uncharacterized Drosophila gene CG8108, which is homologous to the human gene CIZ1, as essential for survival from the executioner caspase activation. It was also shown that cells expressing activated oncogenes experience apoptotic caspase activation, and that Akt1 and dCIZ1 are required for their survival and overgrowth. Thus, survival following executioner caspase activation is a normal tissue repair mechanism usurped to promote oncogene-driven overgrowth.
Ai, X., Wang, D., Zhang, J. and Shen, J. (2020). Hippo signaling promotes Ets21c-dependent apical cell extrusion in the Drosophila wing disc. Development 147(22). PubMed ID: 33028612
Cell extrusion is a crucial regulator of epithelial tissue development and homeostasis. Epithelial cells undergoing apoptosis, bearing pathological mutations or possessing developmental defects are actively extruded toward elimination. However, the molecular mechanisms of Drosophila epithelial cell extrusion are not fully understood. This study reports that activation of the conserved Hippo (Hpo) signaling pathway induces both apical and basal cell extrusion in the Drosophila wing disc epithelia. Canonical Yorkie targets Diap1, Myc and Cyclin E are not required for either apical or basal cell extrusion induced by activation of this pathway. Another target gene, bantam, is only involved in basal cell extrusion, suggesting novel Hpo-regulated apical cell extrusion mechanisms. Using RNA-seq analysis, it was found that JNK signaling is activated in the extruding cells. Genetic evidence is provided that JNK signaling activation is both sufficient and necessary for Hpo-regulated cell extrusion. Furthermore, it was demonstrate that the ETS-domain transcription factor Ets21c, an ortholog of proto-oncogenes FLI1 and ERG, acts downstream of JNK signaling to mediate apical cell extrusion. These findings reveal a novel molecular link between Hpo signaling and cell extrusion.
Xu, J., Zhao, H. and Wang, T. (2020). Suppression of retinal degeneration by two novel ERAD ubiquitin E3 ligases SORDD1/2 in Drosophila. PLoS Genet 16(11): e1009172. PubMed ID: 33137101
Mutations in the gene rhodopsin are one of the major causes of autosomal dominant retinitis pigmentosa (adRP). Mutant forms of Rhodopsin frequently accumulate in the endoplasmic reticulum (ER), cause ER stress, and trigger photoreceptor cell degeneration. This study performed a genome-wide screen to identify suppressors of retinal degeneration in a Drosophila model of adRP, carrying a point mutation in the major rhodopsin, Rh1 (Rh1G69D). Two novel E3 ubiquitin ligases, SORDD1 (CG8974) and SORDD2 (CG32581), were identified that effectively suppressed Rh1G69D-induced photoreceptor dysfunction and retinal degeneration. SORDD1/2 promoted the ubiquitination and degradation of Rh1G69D through VCP (valosin containing protein) and independent of processes reliant on the HRD1 (HMG-CoA reductase degradation protein 1)/HRD3 complex. This study further demonstrated that SORDD1/2 and HRD1 function in parallel and in a redundant fashion to maintain rhodopsin homeostasis and integrity of photoreceptor cells. These findings identify a new ER-associated protein degradation (ERAD) pathway and suggest that facilitating SORDD1/2 function may be a therapeutic strategy to treat adRP.
Cheng, Y., Cai, J., Fu, Y., Feng, C., Hao, Y. and Wei, Y. (2020). Royal jelly attenuates metabolic defects in a Drosophila mutant with elevated TORC1 activity. Biol Open 9(11). PubMed ID: 33037015
Target of rapamycin complex 1 (TORC1) is a master regulator of cell metabolism, and its dysregulation has been linked to an array of pathologies, including cancer and age-related diseases. Nprl3, a component of GTPase-activating protein towards Rags complex 1 (GATOR1), inhibits TORC1 activity under nutrient scarcity status. The nprl3 mutant exhibits some metabolic defects due to hyper TORC1 activity in Drosophila. Royal jelly (RJ) is a honeybee-secreted product and plays an essential role in caste differentiation that requires TORC1 activity. RJ is also used as a health-benefit food for its potential roles on antioxidant and anti-aging. In this study, nprl3-mutant flies were used to measure the effect of RJ on metabolic modulation. Interestingly, RJ feeding significantly increased survival and decreased TORC1 activity in the nprl3 mutant. RJ feeding also ameliorated the abnormal reactive oxygen species (ROS) levels and energy status in the nprl3 mutant. The proteins in RJ were characterized to be the essential components in increasing nprl3 mutant viability. These findings suggest that RJ modulates some metabolic defects associated with elevated TORC1 activity and that the nprl3-mutant fly might be a useful tool for investigating the bioactive components of RJ in vivo.

Wednesday December 23rd - Apoptosis and Autophagy

Sheel, A., Shao, R., Brown, C., Johnson, J., Hamilton, A., Sun, D., Oppenheimer, J., Smith, W., Visconti, P. E., Markstein, M., Bigelow, C. and Schwartz, L. M. (2020). Acheron/Larp6 Is a Survival Protein That Protects Skeletal Muscle From Programmed Cell Death During Development. Front Cell Dev Biol 8: 622. PubMed ID: 32850788
programmed cell death (PCD) requires de novo gene expression. Using the ISMs from the tobacco hawkmoth Manduca sexta, this study has found that Acheron/LARP6 mRNA is induced ∼1,000-fold on the day the muscles become committed to die. Acheron functions as a survival protein that protects cells until cell death is initiated at eclosion (emergence), at which point it becomes phosphorylated and degraded in response to the peptide Eclosion Hormone (EH). Acheron binds to a novel BH3-only protein that was named BBH1 (BAD/BNIP3 homology 1/CG5059). BBH1 accumulates on the day the ISMs become committed to die and is presumably liberated when Acheron is degraded. This is correlated with the release and rapid degradation of cytochrome c and the subsequent demise of the cell. RNAi experiments in the fruit fly Drosophila confirmed that loss of Acheron results in precocious ecdysial muscle death while targeting BBH1 prevents death altogether. Acheron is highly expressed in neurons and muscles in humans and drives metastatic processes in some cancers, suggesting that it may represent a novel survival protein that protects terminally differentiated cells and some cancers from death.
Brown, J., Bush, I., Bozon, J. and Su, T. T. (2020). Cells with loss-of-heterozygosity after exposure to ionizing radiation in Drosophila are culled by p53-dependent and p53-independent mechanisms. PLoS Genet 16(10): e1009056. PubMed ID: 33075096
Loss of Heterozygosity (LOH) typically refers to a phenomenon in which diploid cells that are heterozygous for a mutant allele lose their wild type allele through mutations. LOH is implicated in oncogenesis when it affects the remaining wild type copy of a tumor suppressor. Drosophila has been a useful model to identify genes that regulate the incidence of LOH, but most of these studies use adult phenotypic markers such as multiple wing hair (mwh). This study described a cell-autonomous fluorescence-based system that relies on the the QF/QS transcriptional module to detect LOH, which may be used in larval, pupal and adult stages and in conjunction with the GAL4/UAS system.. Using the QF/QS system, it was possible to detect the induction of cells with LOH by X-rays in a dose-dependent manner in the larval wing discs, and to monitor their fate through subsequent development in pupa and adult stages. The genetic requirement was tested for changes in LOH, using both classical mutants and GAL4/UAS-mediated RNAi. The results identify two distinct culling phases that eliminate cells with LOH, one in late larval stages and another in the pupa. The two culling phases are genetically separable, showing differential requirement for pro-apoptotic genes of the H99 locus and transcription factor Srp. A direct comparison of mwh LOH and QF/QS LOH suggests that cells with different LOH events are distinguished from each other in a p53-dependent manner and are retained to different degrees in the final adult structure. These studies reveal previously unknown mechanisms for the elimination of cells with chromosome aberrations.
Allen, E. A., Amato, C., Fortier, T. M., Velentzas, P., Wood, W. and Baehrecke, E. H. (2020). A conserved myotubularin-related phosphatase regulates autophagy by maintaining autophagic flux. J Cell Biol 219(11). PubMed ID: 32915229
Macroautophagy (autophagy) targets cytoplasmic cargoes to the lysosome for degradation. Like all vesicle trafficking, autophagy relies on phosphoinositide identity, concentration, and localization to execute multiple steps in this catabolic process. This study screened for phosphoinositide phosphatases that influence autophagy in Drosophila and identified CG3530. CG3530 is homologous to the human MTMR6 subfamily of myotubularin-related 3-phosphatases, and therefore, was named dMtmr6. dMtmr6, which is required for development and viability in Drosophila, functions as a regulator of autophagic flux in multiple Drosophila cell types. The MTMR6 family member MTMR8 has a similar function in autophagy of higher animal cells. Decreased dMtmr6 and MTMR8 function results in autophagic vesicle accumulation and influences endolysosomal homeostasis.
Jacomin, A. C., Petridi, S., Di Monaco, M., Bhujabal, Z., Jain, A., Mulakkal, N. C., Palara, A., Powell, E. L., Chung, B., Zampronio, C., Jones, A., Cameron, A., Johansen, T. and Nezis, I. P. (2020). Regulation of Expression of Autophagy Genes by Atg8a-Interacting Partners Sequoia, YL-1, and Sir2 in Drosophila. Cell Rep 31(8): 107695. PubMed ID: 32460019
Autophagy is the degradation of cytoplasmic material through the lysosomal pathway. One of the most studied autophagy-related proteins is LC3. Despite growing evidence that LC3 is enriched in the nucleus, its nuclear role is poorly understood. This study shows that Drosophila Atg8a protein, homologous to mammalian LC3, interacts with the transcription factor Sequoia in a LIR motif-dependent manner. Sequoia depletion induces autophagy in nutrient-rich conditions through the enhanced expression of autophagy genes. Atg8a interacts with YL-1, a component of a nuclear acetyltransferase complex, and it is acetylated in nutrient-rich conditions. Atg8a interacts with the deacetylase Sir2, which deacetylates Atg8a during starvation to activate autophagy. These results suggest a mechanism of regulation of the expression of autophagy genes by Atg8a, which is linked to its acetylation status and its interaction with Sequoia, YL-1, and Sir2.
Ahmad, V., Vadla, G. P. and Chabu, C. Y. (2020). Syd/JIP3 controls tissue size by regulating Diap1 protein turnover downstream of Yorkie/YAP. Dev Biol 469: 37-45. PubMed ID: 33022230
How organisms control organ size is not fully understood. This study found that Syd/JIP3 is required for proper wing size in Drosophila. JIP3 mutations are associated with organ size defects in mammals. The underlying mechanisms are not well understood. Syd/JIP3 inhibition was found to result in a downregulation of the inhibitor of apoptosis protein 1 (Diap1) in the Drosophila wing. Correspondingly, Syd/JIP3 deficient tissues exhibit ectopic cell death and yield smaller wings. Syd/JIP3 inhibition generated similar effects in mammalian cells, indicating a conserved mechanism. Yorkie/YAP stimulates Syd/JIP3 in Drosophila and mammalian cells. Notably, Syd/JIP3 is required for the full effect of Yorkie-mediated tissue growth. Thus Syd/JIP3 regulation of Diap1 functions downstream of Yorkie/YAP to control growth. This study provides mechanistic insights into the recent and perplexing link between JIP3 mutations and organ size defects in mammals, including in humans where de novo JIP3 variants are associated with microcephaly.
Pop, S., Chen, C. L., Sproston, C. J., Kondo, S., Ramdya, P. and Williams, D. W. (2020). Extensive and diverse patterns of cell death sculpt neural networks in insects. Elife 9. PubMed ID: 32894223l
Changes to the structure and function of neural networks are thought to underlie the evolutionary adaptation of animal behaviours. Among the many developmental phenomena that generate change programmed cell death appears to play a key role. This study shows that cell death occurs continuously throughout insect neurogenesis and happens soon after neurons are born. Mimicking an evolutionary role for increasing cell numbers, programmed cell death was artificially blocked in the medial neuroblast lineage in Drosophila melanogaster, which results in the production of 'undead' neurons with complex arborisations and distinct neurotransmitter identities. Activation of these 'undead' neurons and recordings of neural activity in behaving animals demonstrate that they are functional. Focusing on two dipterans which have lost flight during evolution this study revealed that reductions in populations of flight interneurons are likely caused by increased cell death during development. These findings suggest that the evolutionary modulation of death-based patterning could generate novel network configurations.

Tuesday, December 22 - Embryonic neural development

Palavalli, A., Tizon-Escamilla, N., Rupprecht, J. F. and Lecuit, T. (2020). Deterministic and Stochastic Rules of Branching Govern Dendrite Morphogenesis of Sensory Neurons. Curr Biol. PubMed ID: 33212017
Dendrite morphology is necessary for the correct integration of inputs that neurons receive. The branching mechanisms allowing neurons to acquire their type-specific morphology remain unclear. Classically, axon and dendrite patterns were shown to be guided by molecules, providing deterministic cues. However, the extent to which deterministic and stochastic mechanisms, based upon purely statistical bias, contribute to the emergence of dendrite shape is largely unknown. This issue was addressed using the Drosophila class I vpda multi-dendritic neurons. Detailed quantitative analysis of vpda dendrite morphogenesis indicates that the primary branch grows very robustly in a fixed direction, though secondary branch numbers and lengths showed fluctuations characteristic of stochastic systems. Live-tracking dendrites and computational modeling revealed how neuron shape emerges from few local statistical parameters of branch dynamics. This study reports key opposing aspects of how tree architecture feedbacks on the local probability of branch shrinkage. Child branches promote stabilization of parent branches, although self-repulsion promotes shrinkage. Finally, it was shown that self-repulsion, mediated by the adhesion molecule Dscam1, indirectly patterns the growth of secondary branches by spatially restricting their direction of stable growth perpendicular to the primary branch. Thus, the stochastic nature of secondary branch dynamics and the existence of geometric feedback emphasize the importance of self-organization in neuronal dendrite morphogenesis.
Brown, H. E. and Evans, T. A. (2020). Minimal structural elements required for midline repulsive signaling and regulation of Drosophila Robo1. PLoS One 15(10): e0241150. PubMed ID: 33091076
The Roundabout (Robo) family of axon guidance receptors has a conserved ectodomain arrangement of five immunoglobulin-like (Ig) domains plus three fibronectin type III (Fn) repeats. Based on the strong evolutionary conservation of this domain structure among Robo receptors, as well as in vitro structural and domain-domain interaction studies of Robo family members, this ectodomain arrangement is predicted to be important for Robo receptor signaling in response to Slit ligands. This study defined the minimal ectodomain structure required for Slit binding and midline repulsive signaling in vivo by Drosophila Robo1. The majority of the Robo1 ectodomain is dispensable for both Slit binding and repulsive signaling. A significant level of midline repulsive signaling activity is retained when all Robo1 ectodomain elements apart from Ig1 are deleted, and the combination of Ig1 plus one additional ectodomain element (Ig2, Ig5, or Fn3) is sufficient to restore midline repulsion to wild type levels. Further, deleting four out of five Robo1 Ig domains (ΔIg2-5) does not affect negative regulation of Robo1 by Commissureless (Comm) or Robo2, while variants lacking all three fibronectin repeats (ΔFn1-3 and ΔIg2-Fn3) are insensitive to regulation by both Comm and Robo2, signifying a novel regulatory role for Robo1's Fn repeats. These results provide an in vivo perspective on the importance of the conserved 5+3 ectodomain structure of Robo receptors, and suggest that specific biochemical properties and/or ectodomain structural conformations observed in vitro for domains other than Ig1 may have limited significance for in vivo signaling in the context of midline repulsion.
Terzi, A., Roeder, H., Weaver, C. J. and Suter, D. M. (2020). Neuronal NADPH oxidase 2 regulates growth cone guidance downstream of slit2/robo2. Dev Neurobiol. PubMed ID: 33191581
NADPH oxidases (Nox; see Drosophila Nox) are membrane-bound multi-subunit protein complexes producing reactive oxygen species (ROS) that regulate many cellular processes. Emerging evidence suggests that Nox-derived ROS also control neuronal development and axonal outgrowth. However, whether Nox act downstream of receptors for axonal growth and guidance cues is presently unknown. To answer this question, retinal ganglion cells (RGCs) derived from zebrafish embryos were cultured and these neurons were exposed to netrin-1, slit2 (see Drosophila Slit), and brain-derived neurotrophic factor (BDNF). To test the role of Nox in cue-mediated growth and guidance, Nox was either pharmacologically inhibited or neurons from mutant fish were investigated that are deficient in Nox2. Slit2-mediated growth cone collapse, and axonal retraction was eliminated by Nox inhibition. Though no effect of either BDNF or netrin-1 was seen on growth rates, growth in the presence of netrin-1 (see Drosophila Netrins) was reduced by Nox inhibition. Furthermore, attractive and repulsive growth cone turning in response to gradients of BDNF, netrin-1, and slit2, respectively, were eliminated when Nox was inhibited in vitro. ROS biosensor imaging showed that slit2 treatment increased growth cone hydrogen peroxide levels via mechanisms involving Nox2 activation. The possible relationship between Nox2 and slit2/Robo2 signaling was investigated in vivo. astray/nox2 double heterozygote larvae exhibited decreased area of tectal innervation as compared to individual heterozygotes, suggesting both Nox2 and Robo2 are required for establishment of retinotectal connections. These results provide evidence that Nox2 acts downstream of slit2/robo2 by mediating growth and guidance of developing zebrafish RGC neurons.
Gaziova, I., Gazi, M., Mar, J. and Bhat, K. M. (2020). Restriction on self-renewing asymmetric division is coupled to terminal asymmetric division in the Drosophila CNS. PLoS Genet 16(9): e1009011. PubMed ID: 32986715
Neuronal precursor cells undergo self-renewing and non-self-renewing asymmetric divisions to generate a large number of neurons of distinct identities. In Drosophila, primary precursor neuroblasts undergo a varying number of self-renewing asymmetric divisions, with one known exception, the MP2 lineage, which undergoes just one terminal asymmetric division similar to the secondary precursor cells. The mechanism and the genes that regulate the transition from self-renewing to non-self-renewing asymmetric division or the number of times a precursor divides is unknown. This study shows that the T-box transcription factor, Midline (Mid), couples these events. In mid loss of function mutants, MP2 undergoes additional self-renewing asymmetric divisions, the identity of progeny neurons generated dependent upon Numb localization in the parent MP2. MP2 expresses Mid transiently and an over-expression of mid in MP2 can block its division. The mechanism which directs the self-renewing asymmetric division of MP2 in mid involves an upregulation of Cyclin E. The results indicate that Mid inhibits cyclin E gene expression by binding to a variant Mid-binding site in the cyclin E promoter and represses its expression without entirely abolishing it. Consistent with this, over-expression of cyclin E in MP2 causes its multiple self-renewing asymmetric division. These results reveal a Mid-regulated pathway that restricts the self-renewing asymmetric division potential of cells via inhibiting cyclin E and facilitating their exit from cell cycle.
Rui, M., Bu, S., Chew, L. Y., Wang, Q. and Yu, F. (2020). The membrane protein Raw regulates dendrite pruning via the secretory pathway. Development. PubMed ID: 32928906
Neuronal pruning is essential for proper wiring of the nervous systems in invertebrates and vertebrates. Drosophila ddaC sensory neurons selectively prune their larval dendrites to sculpt the nervous system during early metamorphosis. However, the molecular mechanisms underlying ddaC dendrite pruning remain elusive. Here, this study has identified an important and cell-autonomous role of the membrane protein Raw in dendrite pruning of ddaC neurons. Raw appears to regulate dendrite pruning via a novel mechanism, which is independent of JNK signaling. Importantly, Raw was shown to promote endocytosis and downregulation of the conserved L1-type cell-adhesion molecule Neuroglian (Nrg) prior to dendrite pruning. Moreover, Raw is required to modulate the secretory pathway by regulating the integrity of secretory organelles and efficient protein secretion. Mechanistically, Raw facilitates Nrg downregulation and dendrite pruning in part through regulation of the secretory pathway. Thus, this study reveals a JNK-independent role of Raw in regulating the secretory pathway and thereby promoting dendrite pruning.
Matta, S. M., Moore, Z., Walker, F. R., Hill-Yardin, E. L. and Crack, P. J. (2020). An altered glial phenotype in the NL3(R451C) mouse model of autism. Sci Rep 10(1): 14492. PubMed ID: 32879325
Autism Spectrum Disorder (ASD; autism) is a neurodevelopmental disorder characterised by deficits in social communication, and restricted and/or repetitive behaviours. Increasing evidence supports a role for dysregulated neuroinflammation in the brain with potential effects on synapse function. Characteristics of microglia and astrocytes were studied in the Neuroligin-3 (NL3(R451C); see Drosophila Nlg) mouse model of autism. Increased microglial density was observed in the dentate gyrus (DG) of NL3(R451C) mice without morphological differences. In contrast, WT and NL3(R451C) mice had similar astrocyte density but astrocyte branch length, the number of branch points, as well as cell radius and area were reduced in the DG of NL3(R451C) mice. Because retraction of astrocytic processes has been linked to altered synaptic transmission and dendrite formation, regional changes in pre- and postsynaptic protein expression were assessed in the cortex, striatum and cerebellum in NL3(R451C) mice. NL3(R451C) mice showed increased striatal postsynaptic density 95 (PSD-95; see Drosophila Discs large) protein levels and decreased cortical expression of synaptosomal-associated protein 25 (SNAP-25; see Drosophila Snap-25). These changes could contribute to dysregulated neurotransmission and cognition deficits previously reported in these mice.

Monday, December 21st - Chromatin

Belyi, A., Argyridou, E. and Parsch, J. (2020). The influence of chromosomal environment on X-linked gene expression in Drosophila melanogaster. Nature. PubMed ID: 33208948
Sex chromosomes often differ from autosomes with respect to their gene expression and regulation. In Drosophila melanogaster, X-linked genes are dosage compensated by having their expression up-regulated in the male soma, a process mediated by the X chromosome-specific binding of the dosage compensation complex (DCC). Previous studies of X-linked gene expression found a negative correlation between a gene's male-to-female expression ratio and its distance to the nearest DCC binding site in somatic tissues, including head and brain, which suggests that dosage compensation influences sex-biased gene expression. A limitation of the previous studies, however, was that they focused on endogenous X-linked genes and, thus, could not disentangle the effects of chromosomal position from those of gene-specific regulation. To overcome this limitation, this study examined the expression of an exogenous reporter gene inserted at many locations spanning the X chromosome. A negative correlation was observed between the male-to-female expression ratio of the reporter gene and its distance to the nearest DCC binding site in somatic tissues, but not in gonads. A reporter gene's location relative to a DCC binding site had greater influence on its expression than the local regulatory elements of neighboring endogenous genes, suggesting that intra-chromosomal variation in the strength of dosage compensation is a major determinant of sex-biased gene expression. Average levels of sex-biased expression did not differ between head and brain, but there was greater positional effect variation in the brain, which may explain the observed excess of endogenous sex-biased genes located on the X chromosome in this tissue.
Hsu, S. J., Stow, E. C., Simmons, J. R., Wallace, H. A., Lopez, A. M., Stroud, S. and Labrador, M. (2020). Mutations in the insulator protein Suppressor of Hairy wing induce genome instability. Chromosoma 129(3-4): 255-274. PubMed ID: 33140220
Insulator proteins orchestrate the three-dimensional organization of the genome. Insulators function by facilitating communications between regulatory sequences and gene promoters, allowing accurate gene transcription regulation during embryo development and cell differentiation. However, the role of insulator proteins beyond genome organization and transcription regulation remains unclear. Suppressor of Hairy wing [Su(Hw)] is a Drosophila insulator protein that plays an important function in female oogenesis. This study found that su(Hw) has an unsuspected role in genome stability during cell differentiation. su(Hw) mutant developing egg chambers have poorly formed microtubule organization centers (MTOCs) in the germarium and display mislocalization of the anterior/posterior axis specification factor gurken in later oogenesis stages. Additionally, eggshells from partially rescued su(Hw) mutant female germline exhibit dorsoventral patterning defects. These phenotypes are very similar to phenotypes found in the important class of spindle mutants or in piRNA pathway mutants in Drosophila, in which defects generally result from the failure of germ cells to repair DNA damage. Similarities between mutations in su(Hw) and spindle and piRNA mutants are further supported by an excess of DNA damage in nurse cells, and because Gurken localization defects are partially rescued by mutations in the ATR (mei-41) and Chk1 (grapes) DNA damage response genes. Finally, this study also showed that su(Hw) mutants produce an elevated number of chromosome breaks in dividing neuroblasts from larval brains. Together, these findings suggest that Su(Hw) is necessary for the maintenance of genome integrity during Drosophila development, in both germline and dividing somatic cells.
Valsecchi, C. I. K., Basilicata, M. F., Georgiev, P., Gaub, A., Seyfferth, J., Kulkarni, T., Panhale, A., Semplicio, G., Manjunath, V., Holz, H., Dasmeh, P. and Akhtar, A. (2020). RNA nucleation by MSL2 induces selective X chromosome compartmentalization. Nature . PubMed ID: 33208948
Confinement of the X chromosome to a territory for dosage compensation is a prime example of how subnuclear compartmentalization is used to regulate transcription at the megabase scale. In Drosophila melanogaster, two sex-specific non-coding RNAs (roX1 and roX2) are transcribed from the X chromosome. They associate with the male-specific lethal (MSL) complex, which acetylates histone H4 lysine 16 and thereby induces an approximately twofold increase in expression of male X-linked genes. Current models suggest that X-over-autosome specificity is achieved by the recognition of cis-regulatory DNA high-affinity sites (HAS) by the MSL2 subunit. However, HAS motifs are also found on autosomes, indicating that additional factors must stabilize the association of the MSL complex with the X chromosome. This study shows that the low-complexity C-terminal domain (CTD) of MSL2 renders its recruitment to the X chromosome sensitive to roX non-coding RNAs. roX non-coding RNAs and the MSL2 CTD form a stably condensed state, and functional analyses in Drosophila and mammalian cells show that their interactions are crucial for dosage compensation in vivo. Replacing the CTD of mammalian MSL2 with that from Drosophila and expressing roX in cis is sufficient to nucleate ectopic dosage compensation in mammalian cells. Thus, the condensing nature of roX-MSL2(CTD) is the primary determinant for specific compartmentalization of the X chromosome in Drosophila.
Beaver, M., Bhatnagar, A., Panikker, P., Zhang, H., Snook, R., Parmar, V., Vijayakumar, G., Betini, N., Akhter, S. and Elefant, F. (2020). Disruption of Tip60 HAT mediated neural histone acetylation homeostasis is an early common event in neurodegenerative diseases. Sci Rep 10(1): 18265. PubMed ID: 33106538
Epigenetic dysregulation is a common mechanism shared by molecularly and clinically heterogenous neurodegenerative diseases (NDs). Histone acetylation homeostasis, maintained by the antagonistic activity of histone acetyltransferases (HATs) and histone deacetylases (HDACs), is necessary for appropriate gene expression and neuronal function. Disruption of neural acetylation homeostasis has been implicated in multiple types of NDs including Alzheimer's disease (AD), yet mechanisms underlying alterations remain unclear. This study shows that like AD, disruption of Tip60 HAT/HDAC2 balance with concomitantm epigenetic repression of common Tip60 target neuroplasticity genes occurs early in multiple types of Drosophila ND models such as Parkinson's Disease (PD), Huntington's Disease (HD) and Amyotrophic Lateral Sclerosis (ALS). Repressed neuroplasticity genes show reduced enrichment of Tip60 and epigentic acetylation signatures at all gene loci examined with certain genes showing inappropriate HDAC2 repressor enrichment. Functional neuronal consequences for these disease conditions are reminiscent of human pathology and include locomotion, synapse morphology, and short-term memory deficits. Increasing Tip60 HAT levels specifically in the mushroom body learning and meory center in the Drosophila brain protects against locomotion and short-term memory function deficits in multiple NDs. Together, these results support a model by which Tip60 protects against neurological impairments in different NDs via similar modes of action.
Finogenova, K., Bonnet, J., Poepsel, S., Schäfer, I. B., Finkl, K., Schmid, K., Litz, C., Strauss, M., Benda, C. and Muller, J. (2020). Structural basis for PRC2 decoding of active histone methylation marks H3K36me2/3. Elife 9. PubMed ID: 33211010
Repression of genes by Polycomb requires that PRC2 modifies their chromatin by trimethylating lysine 27 on histone H3 (H3K27me3; see Drosophila Histone H3). At transcriptionally active genes, di- and trimethylated H3K36 inhibit PRC2. In this study, the cryo-EM structure of PRC2 on dinucleosomes reveals how binding of its catalytic subunit EZH2 (see Drosophila Enhancer of zeste) to nucleosomal DNA orients the H3 N-terminus via an extended network of interactions to place H3K27 into the active site. Unmodified H3K36 occupies a critical position in the EZH2-DNA interface. Mutation of H3K36 to arginine or alanine inhibits H3K27 methylation by PRC2 on nucleosomes in vitro. Accordingly, Drosophila H3K36A and H3K36R mutants show reduced levels of H3K27me3 and defective Polycomb repression of HOX genes. The relay of interactions between EZH2, the nucleosomal DNA and the H3 N-terminus therefore creates the geometry that permits allosteric inhibition of PRC2 by methylated H3K36 in transcriptionally active chromatin.
Vaziri, A., Khabiri, M., Genaw, B. T., May, C. E., Freddolino, P. L. and Dus, M. (2020). Persistent epigenetic reprogramming of sweet taste by diet. Sci Adv 6(46). PubMed ID: 33177090
Diets rich in sugar, salt, and fat alter taste perception and food preference, contributing to obesity and metabolic disorders, but the molecular mechanisms through which this occurs are unknown. This study shows that in response to a high sugar diet, the epigenetic regulator Polycomb Repressive Complex 2.1 (PRC2.1) persistently reprograms the sensory neurons of Drosophila melanogaster flies to reduce sweet sensation and promote obesity. In animals fed high sugar, the binding of PRC2.1 to the chromatin of the sweet gustatory neurons is redistributed to repress a developmental transcriptional network that modulates the responsiveness of these cells to sweet stimuli, reducing sweet sensation. Half of these transcriptional changes persist despite returning the animals to a control diet, causing a permanent decrease in sweet taste. These results uncover a new epigenetic mechanism that, in response to the dietary environment, regulates neural plasticity and feeding behavior to promote obesity.

Friday December 18th - Disease Models

Zhang, H., Karisetty, B. C., Bhatnagar, A., Armour, E. M., Beaver, M., Roach, T. V., Mortazavi, S., Mandloi, S. and Elefant, F. (2020). Tip60 protects against amyloid-beta-induced transcriptomic alterations via different modes of action in early versus late stages of neurodegeneration. Mol Cell Neurosci 109: 103570. PubMed ID: 33160016
Alzheimer's disease (AD) is an age-related neurodegenerative disorder hallmarked by amyloid-β (Aβ) plaque accumulation, neuronal cell death, and cognitive deficits that worsen during disease progression. Histone acetylation dysregulation, caused by an imbalance between reduced histone acetyltransferases (HAT) Tip60 and increased histone deacetylase 2 (HDAC2) levels, can directly contribute to AD pathology. However, whether such AD-associated neuroepigenetic alterations occur in response to Aβ peptide production and can be protected against by increasing Tip60 levels over the course of neurodegenerative progression remains unknown. This study profiled Tip60 HAT/HDAC2 dynamics and transcriptome-wide changes across early and late stage AD pathology in the Drosophila brain produced solely by human amyloid-β(42). Early Aβ(42) induction leads to disruption of Tip60 HAT/HDAC2 balance during early neurodegenerative stages preceding Aβ plaque accumulation that persists into late AD stages. Correlative transcriptome-wide studies reveal alterations in biological processes were classified as transient (early-stage only), late-onset (late-stage only), and constant (both). Increasing Tip60 HAT levels in the Aβ(42) fly brain protects against AD functional pathologies that include Aβ plaque accumulation, neural cell death, cognitive deficits, and shorter life-span. Strikingly, Tip60 protects against Aβ(42)-induced transcriptomic alterations via distinct mechanisms during early and late stages of neurodegeneration. These findings reveal distinct modes of neuroepigenetic gene changes and Tip60 neuroprotection in early versus late stages in AD that can serve as early biomarkers for AD, and support the therapeutic potential of Tip60 over the course of AD progression.
Auxerre-Plantie, E., Nielsen, T., Grunert, M., Olejniczak, O., Perrot, A., Ozcelik, C., Harries, D., Matinmehr, F., Dos Remedios, C., Muhlfeld, C., Kraft, T., Bodmer, R., Vogler, G. and Sperling, S. R. (2020). Identification of MYOM2 as a candidate gene in hypertrophic cardiomyopathy and tetralogy of fallot and its functional evaluation in the Drosophila heart. Dis Model Mech. PubMed ID: 33033063
The causal genetic underpinnings of congenital heart diseases, which are often complex and with multigenic background, are still far from understood. Moreover, there are also predominantly monogenic heart defects, such as cardiomyopathies, with known disease genes for the majority of cases. This study identified mutations in myomesin 2 (MYOM2) in patients with Tetralogy of Fallot (TOF), the most common cyanotic heart malformation, as well as in patients with hypertrophic cardiomyopathy (HCM), who do not exhibit any mutations in the known disease genes. MYOM2 is a major component of the myofibrillar M-band of the sarcomere and a hub gene within interactions of sarcomere genes. This study shows that patient-derived cardiomyocytes exhibit myofibrillar disarray and reduced passive force with increasing sarcomere lengths. Moreover, a comprehensive functional analyses in the Drosophila animal model reveal that the so far uncharacterized fly gene CG14964 may be an ortholog of MYOM2, as well as other myosin binding proteins (henceforth named as Drosophila Myomesin and Myosin Binding protein (dMnM)). Its partial loss-of-function or moderate cardiac knockdown results in cardiac dilation, whereas more severely reduced function causes a constricted phenotype and an increase in sarcomere myosin protein. Moreover, compound heterozygous combinations of CG14964 and the sarcomere gene Mhc (MYH6/7) exhibited synergistic genetic interactions. In summary, these results suggest that MYOM2 not only plays a critical role in maintaining robust heart function but may also be a candidate gene for heart diseases such as HCM and TOF, as it is clearly involved in the development of the heart.
Scholes, H. M., Cryar, A., Kerr, F., Sutherland, D., Gethings, L. A., Vissers, J. P. C., Lees, J. G., Orengo, C. A., Partridge, L. and Thalassinos, K. (2020). Dynamic changes in the brain protein interaction network correlates with progression of Abeta42 pathology in Drosophila. Sci Rep 10(1): 18517. PubMed ID: 33116184
Alzheimer's disease (AD), the most prevalent form of dementia, is a progressive and devastating neurodegenerative condition for which there are no effective treatments. Understanding the molecular pathology of AD during disease progression may identify new ways to reduce neuronal damage. This paper presents a longitudinal study tracking dynamic proteomic alterations in the brains of an inducible Drosophila melanogaster model of AD expressing the Arctic mutant Aβ42 gene. 3093 proteins from flies that were induced to express Aβ42 and age-matched healthy controls were identified using label-free quantitative ion-mobility data independent analysis mass spectrometry. Of these, 228 proteins were significantly altered by Aβ42 accumulation and were enriched for AD-associated processes. Network analyses further revealed that these proteins have distinct hub and bottleneck properties in the brain protein interaction network, suggesting that several may have significant effects on brain function. This unbiased analysis provides useful insights into the key processes governing the progression of amyloid toxicity and forms a basis for further functional analyses in model organisms and translation to mammalian systems.
Rodriguez, L. R., Calap-Quintana, P., Lapena-Luzon, T., Pallardo, F. V., Schneuwly, S., Navarro, J. A. and Gonzalez-Cabo, P. (2020). Oxidative stress modulates rearrangement of endoplasmic reticulum-mitochondria contacts and calcium dysregulation in a Friedreich's ataxia model. Redox Biol 37: 101762. PubMed ID: 33128998
Friedreich ataxia (FRDA) is a neurodegenerative disorder characterized by neuromuscular and neurological manifestations. It is caused by mutations in the FXN gene, which results in loss of the mitochondrial protein frataxin. Endoplasmic Reticulum-mitochondria associated membranes (MAMs) are inter-organelle structures involved in the regulation of essential cellular processes, including lipid metabolism and calcium signaling. This study has analyzed in both, unicellular and multicellular models of FRDA, calcium management and integrity of MAMs. Function of MAMs was observed to be compromised in the cellular model of FRDA, which was improved upon treatment with antioxidants. In agreement, promoting mitochondrial calcium uptake was sufficient to restore several defects caused by frataxin deficiency in Drosophila melanogaster. Remarkably, the findings describe for the first time frataxin as a member of the protein network of MAMs, where interacts with two of the main proteins implicated in endoplasmic reticulum-mitochondria communication. These results suggest a new role of frataxin, indicate that FRDA goes beyond mitochondrial defects and highlight MAMs as novel therapeutic candidates to improve patient's conditions.
Takai, A., Chiyonobu, T., Ueoka, I., Tanaka, R., Tozawa, T., Yoshida, H., Morimoto, M., Hosoi, H. and Yamaguchi, M. (2020). A novel Drosophila model for neurodevelopmental disorders associated with Shwachman-Diamond syndrome. Neurosci Lett 739: 135449. PubMed ID: 33115644
Genetic defects in ribosome biogenesis result in a group of diseases called ribosomopathies. Patients with ribosomopathies manifest multiorgan phenotypes, including neurological impairments. A well-characterized ribosomopathy, Shwachman-Diamond syndrome (SDS), is mainly associated with loss-of-function mutations in the causal gene SBDS. Children with SDS have neurodevelopmental disorders; however, the neurological consequences of SBDS dysfunction remain poorly defined. This study investigated the phenotype of Drosophila melanogaster following knockdown of CG8549, the Drosophila ortholog of human SBDS, to provide evidence for the neurological consequences of reduction in physiological SBDS functions. The pan-neuron-specific knockdown of CG8549 was associated with locomotive disabilities, mechanically induced seizures, hyperactivity, learning impairments, and anatomical defects in presynaptic terminals. These results provide the first evidence of a direct link between a reduction in physiological SBDS function and neurological impairments.
Rigon, L., Kucharowski, N., Eckardt, F. and Bauer, R. (2020). Modeling Mucopolysaccharidosis Type II in the Fruit Fly by Using the RNA Interference Approach. Life (Basel) 10(11). PubMed ID: 33142967
Mucopolysaccharidosis type II (MPS II) is a lysosomal storage disorder that occurs due to the deficit of the lysosomal enzyme iduronate 2-sulfatase (IDS) that leads to the storage of the glycosaminoglycan heparan- and dermatan-sulfate in all organs and tissues. It is characterized by important clinical features and the severe form presents with a heavy neurological involvement. However, almost nothing is known about the neuropathogenesis of MPS II. To address this issue, a ubiquitous, neuronal, and glial-specific knockdown model was developed in Drosophila melanogaster by using the RNA interference (RNAi) approach. Knockdown of the Ids/CG12014 gene resulted in a significant reduction of the Ids gene expression and enzymatic activity. However, glycosaminoglycan storage, survival, molecular markers (Atg8a, Lamp1, Rab11), and locomotion behavior were not affected. Even strongly reduced, IDS-activity was enough to prevent a pathological phenotype in a MPS II RNAi fruit fly. Thus, a Drosophila MPS II model requires complete abolishment of the enzymatic activity.

Thursday, December 17th - Behavior

Zhao, F., Zeng, Y., Guo, A., Su, H. and Xu, B. (2020). A neural algorithm for Drosophila linear and nonlinear decision-making. Sci Rep 10(1): 18660. PubMed ID: 33122701
It has been evidenced that vision-based decision-making in Drosophila consists of both simple perceptual (linear) decision and value-based (non-linear) decision. This paper proposes a general computational spiking neural network (SNN) model to explore how different brain areas are connected contributing to Drosophila linear and nonlinear decision-making behavior. First, the SNN model could successfully describe all the experimental findings in fly visual reinforcement learning and action selection among multiple conflicting choices as well. Second, the computational modeling shows that dopaminergic neuron-GABAergic neuron-mushroom body (DA-GABA-MB) works in a recurrent loop providing a key circuit for gain and gating mechanism of nonlinear decision making. Compared with existing models, this model shows more biologically plausible on the network design and working mechanism, and could amplify the small differences between two conflicting cues more clearly. Finally, based on the proposed model, the unmanned aerial vehicle (UAV) could quickly learn to make clear-cut decisions among multiple visual choices and flexible reversal learning resembling to real fly. Compared with linear and uniform decision-making methods, the DA-GABA-MB mechanism helps UAV complete the decision-making task with fewer steps.
Qiu, S., Li, C., Cao, G. and Xiao, C. (2020). Mating experience modifies locomotor performance and promotes episodic motor activity in Drosophila melanogaster. Zoology (Jena) 144: 125854. PubMed ID: 33186862
Sexual behavior is a routine among animal species. Sexual experience has several behavioral consequences in insects, but its physiological basis is less well-understood. The episodic motor activity with a periodicity around 19 s was unintentionally observed in the wildtype Canton-S flies and was greatly reduced in the white-eyed mutant w(1118) flies. Episodic motor activity co-exists with several consistent locomotor performances in Canton-S flies whereas reduced episodic motor activity is accompanied by neural or behavioral abnormalities in w(1118) flies. The improvements of both episodic motor activity and locomotor performance are co-inducible by a pulsed light illumination in w(1118). This study shows that mating experience of w(1118) males promoted fast and consistent locomotor activities and increased the power of episodic motor activities. Compared with virgin males, mated ones showed significant increases of boundary preference, travel distance over 60 s, and increased path increments per 0.2 s. In contrast, mated males of Canton-S showed decreased boundary preference, increased travel distance over 60 s, and increased path increments per 0.2 s. Additionally, mated males of w(1118) displayed increased power amplitude of periodic motor activities at 0.03-0.1 Hz. These data indicated that mating experience promoted fast and consistent locomotion and improved episodic motor activities in w(1118) male flies.
Demir, M., Kadakia, N., Anderson, H. D., Clark, D. A. and Emonet, T. (2020). Walking Drosophila navigate complex plumes using stochastic decisions biased by the timing of odor encounters. Elife 9. PubMed ID: 33140723
How insects navigate complex odor plumes, where the location and timing of odor packets are uncertain, remains unclear. This study imaged complex odor plumes simultaneously with freely-walking flies, quantifying how behavior is shaped by encounters with individual odor packets. Navigation was stochastic and did not rely on the continuous modulation of speed or orientation. Instead, flies turned stochastically with stereotyped saccades, whose direction was biased upwind by the timing of prior odor encounters, while the magnitude and rate of saccades remained constant. Further, flies used the timing of odor encounters to modulate the transition rates between walks and stops. In more regular environments, flies continuously modulate speed and orientation, even though encounters can still occur randomly due to animal motion. In less predictable environments, where encounters are random in both space and time, walking flies navigate with random walks biased by encounter timing.
Filice, D. C. S., Bhargava, R. and Dukas, R. (2020). Female mating experience and genetic background independently influence male mating success in fruit flies. J Evol Biol. PubMed ID: 33128417
When the reproductive interests of males and females conflict, males can evolve traits that are harmful to females, and females can coevolve traits to resist this harm. This study tested how the genetic background of a female and her previous mating experience interact to affect the mating success of focal males. In the experience phase, females from 28 distinct genetic backgrounds were placed individually either with a single male (low conflict) or with three males (high conflict) for 48 hr. In the subsequent test phase, the mating and post-mating fertilization success was measured of focal males paired individually with each female. focal males paired with females from the high-conflict treatment were less successful at mating, took longer to mate when they were successful, and had a lower proportion of paternity share. Furthermore, significant female genetic variation associated with male mating success was identified. These results indicate that female experience, along with intrinsic genetic factors, can independently influence different fitness components of her subsequent mates and has implications for understanding of plastic female mating strategies and the evolution of sexually antagonistic traits in males and females.
Park, A., Tran, T., Scheuermann, E. A., Smith, D. P. and Atkinson, N. S. (2020). Alcohol potentiates a pheromone signal in flies. Elife 9. PubMed ID: 33141025
For decades, numerous researchers have documented the presence of the fruit fly or Drosophila melanogaster on alcohol-containing food sources. Although fruit flies are a common laboratory model organism of choice, there is relatively little understood about the ethological relationship between flies and ethanol. This study finds that when male flies inhabit ethanol-containing food substrates they become more aggressive. A possible mechanism was identified for this behavior. The odor of ethanol potentiates the activity of sensory neurons in response to an aggression-promoting pheromone. Finally, it was observed that the odor of ethanol also promotes attraction to a food-related citrus odor. Understanding how flies interact with the complex natural environment they inhabit can provide valuable insight into how different natural stimuli are integrated to promote fundamental behaviors.
Hu, S. W., Yang, Y. T., Sun, Y., Zhan, Y. P. and Zhu, Y. (2020). Serotonin Signals Overcome Loser Mentality in Drosophila. iScience 23(11): 101651. PubMed ID: 33117967
Traumatic experiences generate stressful neurological effects in the exposed persons and animals. Previous studies have demonstrated that in many species, including Drosophila, the defeated animal has a higher probability of losing subsequent fights. However, the neural basis of this "loser effect" is largely unknown. This study reports that elevated serotonin (5-HT) signaling helps a loser to overcome suppressive neurological states. Coerced activation of 5-HT neurons increases aggression in males and promotes losers to both vigorously re-engage in fights and even defeat the previous winners and regain mating motivation. P1 neurons act upstream and 5-HT1B neurons in the ellipsoid body act downstream of 5-HT neurons to arouse losers. These results demonstrate an ancient neural mechanism of regulating depressive behavioral states after distressing events.

Wednesday, December 16th - RNA and Transposons

Weilguny, L., Vlachos, C., Selvaraju, D. and Kofler, R. (2020). Reconstructing the Invasion Route of the P-Element in Drosophila melanogaster Using Extant Population Samples. Genome Biol Evol 12(11): 2139-2152. PubMed ID: 33210145
The P-element, one of the best understood eukaryotic transposable elements, spread in natural Drosophila melanogaster populations in the last century. It invaded American populations first and later spread to the Old World. Inferring this invasion route was made possible by a unique resource available in D. melanogaster: Many strains sampled from different locations over the course of the last century. This study tested the hypothesis that the invasion route of the P-element may be reconstructed from extant population samples using internal deletions (IDs) as markers. These IDs arise at a high rate when DNA transposons, such as the P-element, are active. It is suggested that inferring invasion routes is possible as: 1) the fraction of IDs increases in successively invaded populations, which also explains the striking differences in the ID content between American and European populations, and 2) successively invaded populations end up with similar sets of IDs. This approach allowed reconstruction of the invasion route of the P-element with reasonable accuracy. The approach also sheds light on the unknown timing of the invasion in African populations: it is suggested that African populations were invaded after American but before European populations. Simulations of TE invasions in spatially distributed populations confirm that IDs may allow inference ot invasion routes. This approach might be applicable to other DNA transposons in different host species.
Xie, W., Sowemimo, I., Hayashi, R., Wang, J., Burkard, T. R., Brennecke, J., Ameres, S. L. and Patel, D. J. (2020). Structure-function analysis of microRNA 3'-end trimming by Nibbler. Proc Natl Acad Sci U S A. PubMed ID: 33199607
Nibbler (Nbr) is a 3'-to-5' exoribonuclease whose catalytic 3'-end trimming activity impacts microRNA (miRNA) and PIWI-interacting RNA (piRNA) biogenesis. This study reports on structural and functional studies to decipher the contributions of Nbr's N-terminal domain (NTD) and exonucleolytic domain (EXO) in miRNA 3'-end trimming. This study has solved the crystal structures of the NTD core and EXO domains of Nbr, both in the apo-state. The NTD-core domain of Aedes aegypti Nbr adopts a HEAT-like repeat scaffold with basic patches constituting an RNA-binding surface exhibiting a preference for binding double-strand RNA (dsRNA) over single-strand RNA (ssRNA). Structure-guided functional assays in Drosophila S2 cells confirmed a principal role of the NTD in exonucleolytic miRNA trimming, which depends on basic surface patches. Gain-of-function experiments revealed a potential role of the NTD in recruiting Nbr to Argonaute-bound small RNA substrates. The EXO domain of A. aegypti and Drosophila melanogaster Nbr adopt a mixed α/β-scaffold with a deep pocket lined by a DEDDy catalytic cleavage motif. Nbr's EXO domain exhibits Mn(2+)-dependent ssRNA-specific 3'-to-5' exoribonuclease activity. Modeling of a 3' terminal Uridine into the catalytic pocket of Nbr EXO indicates that 2'-O-methylation of the 3'-U would result in a steric clash with a tryptophan side chain, suggesting that 2'-O-methylation protects small RNAs from Nbr-mediated trimming. Overall, thee data establish that Nbr requires its NTD as a substrate recruitment platform to execute exonucleolytic miRNA maturation, catalyzed by the ribonuclease EXO domain.
Iki, T., Takami, M. and Kai, T. (2020). Modulation of Ago2 Loading by Cyclophilin 40 Endows a Unique Repertoire of Functional miRNAs during Sperm Maturation in Drosophila. Cell Rep 33(6): 108380. PubMed ID: 33176138
In gene silencing, Hsp90 chaperone machinery assists Argonaute (Ago) binding and unwinding of silencing small RNA (sRNA) duplexes. This enables the formation of effector RNA-induced silencing complex (RISC) that often displays cargo preferences. Hence, in Drosophila, microRNAs (miRNAs) and small-interfering RNAs (siRNAs) are differentially sorted into Ago1-RISC and Ago2-RISC, respectively. This study identified fly Cyclophilin 40 (Cyp40) as a testis-specialized Hsp90 co-chaperone essential for spermatogenesis and for modulating Ago2-RISC formation. Testis-distinctive Ago-sorting and strand-selection mechanisms accumulate a unique set of miRNAs on Ago2. Cyp40 interacts with duplex-incorporating Ago2 through Hsp90 in vitro and selectively promotes the build-up of Ago2-bound miRNAs, but not endogenous siRNAs, in vivo. Moreover, one of Cyp40-dependent Ago2-sorted miRNAs is required for late spermatogenesis, unraveling the physiological relevance of the unconventional yet conserved Drosophila miRNA-Ago2 sorting pathway. Collectively, these results identify RISC-regulatory roles for Hsp90 machinery and, more generally, highlight the tissue-specific adaptation of sRNA pathways through chaperone diversification.
Molla-Herman, A., Angelova, M. T., Ginestet, M., Carre, C., Antoniewski, C. and Huynh, J. R. (2020). tRNA Fragments Populations Analysis in Mutants Affecting tRNAs Processing and tRNA Methylation. Front Genet 11: 518949. PubMed ID: 33193603
tRNA fragments (tRFs) are a class of small non-coding RNAs (sncRNAs) derived from tRNAs. tRFs are highly abundant in many cell types including stem cells and cancer cells, and are found in all domains of life. Beyond translation control, tRFs have several functions ranging from transposon silencing to cell proliferation control. However, the analysis of tRFs presents specific challenges and their biogenesis is not well understood. They are very heterogeneous and highly modified by numerous post-transcriptional modifications. This study describes a bioinformatic pipeline (tRFs-Galaxy) to study tRFs populations and shed light onto tRNA fragments biogenesis in Drosophila melanogaster. Indeed, small RNAs Illumina sequencing datasets were used that were extracted from wild type and mutant ovaries affecting two different highly conserved steps of tRNA biogenesis: 5'pre-tRNA processing (RNase-P subunit Rpp30) and tRNA 2'-O-methylation (dTrm7_34 and dTrm7_32). Using this pipeline, it was shown how defects in tRNA biogenesis affect nuclear and mitochondrial tRFs populations and other small non-coding RNAs biogenesis, such as small nucleolar RNAs (snoRNAs). This tRF analysis workflow will advance the current understanding of tRFs biogenesis, which is crucial to better comprehend tRFs roles and their implication in human pathology.
Tindell, S. J., Rouchka, E. C. and Arkov, A. L. (2020). Glial granules contain germline proteins in the Drosophila brain, which regulate brain transcriptome. Commun Biol 3(1): 699. PubMed ID: 33219296
Membraneless RNA-protein granules play important roles in many different cell types and organisms. In particular, granules found in germ cells have been used as a paradigm to study large and dynamic granules. These germ granules contain RNA and proteins required for germline development. This study unexpectedly identified large granules in specific subtypes of glial cells ("glial granules") of the adult Drosophila brain which contain polypeptides with previously characterized roles in germ cells including scaffold Tudor, Vasa, Polar granule component and Piwi family proteins. Interestingly, super-resolution microscopy analysis shows that in the glial granules, these proteins form distinct partially overlapping clusters. Furthermore, it was shown that glial granule scaffold protein Tudor functions in silencing of transposable elements and in small regulatory piRNA biogenesis. Remarkably, the data indicate that the adult brain contains a small population of cells, which express both neuroblast and germ cell proteins. These distinct cells are evolutionarily conserved and expand during aging suggesting the existence of age-dependent signaling. This work uncovers previously unknown glial granules and indicates the involvement of their components in the regulation of brain transcriptome.
Wei, K. H., Gibilisco, L. and Bachtrog, D. (2020). Epigenetic conflict on a degenerating Y chromosome increases mutational burden in Drosophila males. Nat Commun 11(1): 5537. PubMed ID: 33139741
Large portions of eukaryotic genomes consist of transposable elements (TEs), and the establishment of transcription-repressing heterochromatin during early development safeguards genome integrity in Drosophila. Repeat-rich Y chromosomes can act as reservoirs for TEs ('toxic' Y effect), and incomplete epigenomic defenses during early development can lead to deleterious TE mobilization. This study contrasted the dynamics of early TE activation in two Drosophila species with vastly different Y chromosomes of different ages. Zygotic TE expression is elevated in male embryos relative to females in both species, mostly due to expression of Y-linked TEs. Interestingly, male-biased TE expression diminishes across development in D. pseudoobscura, but remains elevated in D. miranda, the species with the younger and larger Y chromosome. The repeat-rich Y of D. miranda still contains many actively transcribed genes, which compromise the formation of silencing heterochromatin. Elevated TE expression results in more de novo insertions of repeats in males compared to females. This lends support to the idea that the 'toxic' Y chromosome can create a mutational burden in males when genome-wide defense mechanisms are compromised, and suggests a previously unappreciated epigenetic conflict on evolving Y chromosomes between transcription of essential genes and silencing of selfish DNA.

Tuesday, December 15th - Evolution

Zhou, F., Cao, G., Dai, S., Li, G., Li, H., Ding, Z., Hou, S., Xu, B., You, W., Wiseglass, G., Shi, F., Yang, X., Rubinstein, R. and Jin, Y. (2020). Chelicerata sDscam isoforms combine homophilic specificities to define unique cell recognition. Proc Natl Acad Sci U S A. PubMed ID: 32963097
Thousands of Down syndrome cell adhesion molecule (Dscam1) isoforms and ∼60 clustered protocadhrein (cPcdh) proteins are required for establishing neural circuits in insects and vertebrates, respectively. The strict homophilic specificity exhibited by these proteins has been extensively studied and is thought to be critical for their function in neuronal self-avoidance. In contrast, significantly less is known about the Dscam1-related family of ∼100 shortened Dscam (sDscam) proteins in Chelicerata. Chelicerata sDscamα and some sDscamβ protein trans interactions are strictly homophilic, and the trans interaction is meditated via the first Ig domain through an antiparallel interface. Additionally, different sDscam isoforms interact promiscuously in cis via membrane proximate fibronectin-type III domains. Cell-cell interactions depend on the combined identity of all sDscam isoforms expressed. A single mismatched sDscam isoform can interfere with the interactions of cells that otherwise express an identical set of isoforms. Thus, these data support a model by which sDscam association in cis and trans generates a vast repertoire of combinatorial homophilic recognition specificities. It is proposed that in Chelicerata, sDscam combinatorial specificity is sufficient to provide each neuron with a unique identity for self-nonself discrimination. Surprisingly, while sDscams are related to Drosophila Dscam1, the results mirror the findings reported for the structurally unrelated vertebrate cPcdh. Thus, these findings suggest a remarkable example of convergent evolution for the process of neuronal self-avoidance and provide insight into the basic principles and evolution of metazoan self-avoidance and self-nonself discrimination.
Erickson, P. A., Weller, C. A., Song, D. Y., Bangerter, A. S., Schmidt, P. and Bergland, A. O. (2020). Unique genetic signatures of local adaptation over space and time for diapause, an ecologically relevant complex trait, in Drosophila melanogaster. PLoS Genet 16(11): e1009110. PubMed ID: 33216740
Local adapation can result in variation in seasonal responses, but the genetic basis and evolutionary history of this variation remains elusive. Many insects, including Drosophila melanogaster, are able to undergo an arrest of reproductive development (diapause) in response to unfavorable conditions. In D. melanogaster, the ability to diapause is more common in high latitude populations, where flies endure harsher winters, and in the spring, reflecting differential survivorship of overwintering populations. Using a novel hybrid swarm-based genome wide association study, this study examined the genetic basis and evolutionary history of ovarian diapause. Outbred females were exposed to different temperatures and day lengths, ovarian development was characterized for over 2800 flies, and their complete, phased genomes were reconstructed. Diapause, scored at two different developmental cutoffs, was found to be modest heritability, and hundreds of SNPs associated with each of the two phenotypes were identified. Alleles associated with one of the diapause phenotypes tend to be more common at higher latitudes, but these alleles do not show predictable seasonal variation. The collective signal of many small-effect, clinally varying SNPs can plausibly explain latitudinal variation in diapause seen in North America. Alleles associated with diapause are segregating in Zambia, suggesting that variation in diapause relies on ancestral polymorphisms, and both pro- and anti-diapause alleles have experienced selection in North America. Finally, outdoor mesocosms were used to track diapause under natural conditions. Hybrid swarms reared outdoors were found to evolve increased propensity for diapause in late fall, whereas indoor control populations experienced no such change. These results indicate that diapause is a complex, quantitative trait with different evolutionary patterns across time and space.
Singh, N. P., De Kumar, B., Paulson, A., Parrish, M. E., Zhang, Y., Florens, L., Conaway, J. W., Si, K. and Krumlauf, R. (2020). A six-amino-acid motif is a major determinant in functional evolution of HOX1 proteins. Genes Dev. PubMed ID: 33184220
Gene duplication and divergence is a major driver in the emergence of evolutionary novelties. How variations in amino acid sequences lead to loss of ancestral activity and functional diversification of proteins is poorly understood. This study used cross-species functional analysis of Drosophila Labial and its mouse HOX1 orthologs (HOXA1, HOXB1, and HOXD1) as a paradigm to address this issue. Mouse HOX1 proteins display low (30%) sequence similarity with Drosophila Labial. However, substituting endogenous Labial with the mouse proteins revealed that HOXA1 has retained essential ancestral functions of Labial, while HOXB1 and HOXD1 have diverged. Genome-wide analysis demonstrated similar DNA-binding patterns of HOXA1 and Labial in mouse cells, while HOXB1 binds to distinct targets. Compared with HOXB1, HOXA1 shows an enrichment in co-occupancy with PBX (see Drosophila Extradenticle) proteins on target sites and exists in the same complex with PBX on chromatin. Functional analysis of HOXA1-HOXB1 chimeric proteins uncovered a novel six-amino-acid C-terminal motif (CTM) flanking the homeodomain that serves as a major determinant of ancestral activity. In vitro DNA-binding experiments and structural prediction show that CTM provides an important domain for interaction of HOXA1 proteins with PBX. These findings show that small changes outside of highly conserved DNA-binding regions can lead to profound changes in protein function.
Videlier, M., Careau, V., Wilson, A. J. and Rundle, H. D. (2020). Quantifying selection on standard metabolic rate and body mass in Drosophila melanogaster. Evolution. PubMed ID: 33196104
Standard metabolic rate (SMR), defined as the minimal energy expenditure required for self-maintenance, is a key physiological trait. Few studies have estimated its relationship with fitness, most notably in insects. This is presumably due to the difficulty of measuring SMR in a large number of very small individuals. Using high-throughput flow-through respirometry and a Drosophila melanogaster laboratory population adapted to a life-cycle that facilitates fitness measures, SMR, body mass, and fitness were quantified in 515 female and 522 male adults. A novel multivariate approach was used to estimate linear and non-linear selection differentials and gradients from the variance-covariance matrix of fitness, SMR, and body mass, allowing traits specific covariates to be accommodated within a single model. In males, linear selection differentials for mass and SMR were positive and individually significant. Selection gradients were also positive but, despite substantial sample sizes, were non-significant due to increased uncertainty given strong SMR-mass collinearity. In females, only nonlinear selection was detected and it appeared to act primarily on body size, although the individual gradients were again non-significant. Selection did not differ significantly between sexes although differences in the fitness surfaces suggest sex-specific selection as an important topic for further study.
Banerjee, T. D. and Monteiro, A. (2020). Molecular mechanisms underlying simplification of venation patterns in holometabolous insects. Development. PubMed ID: 33144394
How mechanisms of pattern formation evolve has remained a central research theme in the field of evolutionary and developmental biology. The mechanism of wing vein differentiation in Drosophila is a classic text-book example of pattern formation using a system of positional-information, yet very little is known about how species with a different number of veins pattern their wings, and how insect venation patterns evolved. This study examine the expression pattern of genes previously implicated in vein differentiation in Drosophila in two butterfly species with more complex venation Bicyclus anynana and Pieris canidia. The function of some of these genes was tested in B. anynana. Both conserved as well as new domains of decapentaplegic, engrailed, invected, spalt, optix, wingless, armadillo, blistered, and rhomboid gene expression in butterflies were identified, and a proposal is made about how the simplified venation in Drosophila might have evolved via loss of decapentaplegic, spalt and optix gene expression domains, along with silencing of vein inducing programs at Spalt-expression boundaries, and changes in gene expression of vein maintenance genes.
Kasinathan, B., Colmenares, S. U., McConnell, H., Young, J. M., Karpen, G. H. and Malik, H. S. (2020). Innovation of heterochromatin functions drives rapid evolution of essential ZAD-ZNF genes in Drosophila. Elife 9. PubMed ID: 33169670
Contrary to dogma, evolutionarily young and dynamic genes can encode essential functions. Rvolutionarily dynamic ZAD-ZNF genes, which encode the most abundant class of insect transcription factors, are more likely to encode essential functions in Drosophila melanogaster than ancient, conserved ZAD-ZNF genes. This study focused on the Nicknack ZAD-ZNF gene, which is evolutionarily young, poorly retained in Drosophila species, and evolves under strong positive selection. Yet it was found that it is necessary for larval development in D. melanogaster. Nicknack encodes a heterochromatin-localizing protein like its paralog Oddjob, also an evolutionarily dynamic yet essential ZAD-ZNF gene. The divergent D. simulans Nicknack protein can still localize to D. melanogaster heterochromatin and rescue viability of female but not male Nicknack-null D. melanogaster. These findings suggest that innovation for rapidly changing heterochromatin functions might generally explain the essentiality of many evolutionarily dynamic ZAD-ZNF genes in insects.

Monday, December 14th - Adult neural development and function

Privman Champaloux, E., Donelson, N., Pyakurel, P., Wolin, D., Ostendorf, L., Denno, M., Borman, R., Burke, C., Short-Miller, J. C., Yoder, M. R., Copeland, J. M., Sanyal, S. and Jill Venton, B. (2020). Ring Finger Protein 11 (RNF11) Modulates Dopamine Release in Drosophila. Neuroscience. PubMed ID: 33176188
Recent work indicates a role for RING finger protein 11 (RNF11) in Parkinson disease (PD) pathology, which involves the loss of dopaminergic neurons. However, the role of RNF11 in regulating dopamine neurotransmission has not been studied. This work tested the effect of RNF11 RNAi knockdown or overexpression on stimulated dopamine release in the larval Drosophila central nervous system. Dopamine release was stimulated using optogenetics and monitored in real-time using fast-scan cyclic voltammetry at an electrode implanted in an isolated ventral nerve cord. RNF11 knockdown doubled dopamine release, but there was no decrease in dopamine from RNF11 overexpression. RNF11 knockdown did not significantly increase stimulated serotonin or octopamine release, indicating the effect is dopamine specific. Dopamine clearance was also changed, as RNF11 RNAi flies had a higher V(max) and RNF11 overexpressing flies had a lower V(max) than control flies. RNF11 RNAi flies had increased mRNA levels of dopamine transporter (DAT) in RNF11, confirming changes in DAT. In RNF11 RNAi flies, release was maintained better for stimulations repeated at short intervals, indicating increases in the recycled releasable pool of dopamine. Nisoxetine, a DAT inhibitor, and flupenthixol, a D2 antagonist, did not affect RNF11 RNAi or overexpressing flies differently than control. Thus, RNF11 knockdown causes early changes in dopamine neurotransmission, and this is the first work to demonstrate that RNF11 affects both dopamine release and uptake. RNF11 expression decreases in human dopaminergic neurons during PD, and that decrease may be protective by increasing dopamine neurotransmission in the surviving dopaminergic neurons.
Perry, R. J., Saunders, C. J., Nelson, J. M., Rizzo, M. J., Braco, J. T. and Johnson, E. C. (2020). Regulation of Metabolism by an Ensemble of Different Ion Channel Types: Excitation-Secretion Coupling Mechanisms of Adipokinetic Hormone Producing Cells in Drosophila. Front Physiol 11: 580618. PubMed ID: 33192586
Adipokinetic Hormone (AKH) is the primary insect hormone that mobilizes stored energy and is functional equivalent to mammalian glucagon. Relatively little is known about how AKH secretion is regulated. The AKH cell transcriptome was assessed, and the data set was mined for for specific insight into the identities of different ion channels expressed in this cell lineage. Significant signals were found for 39 of the either known or suspected ion channel genes within the Drosophila genome. A targeted RNAi screen was performed that aimed to identify the functional contribution of these different ion channels that may participate in excitation-secretion coupling in AKH producing cells (APCs). Starvation survival was assessed, because changes in AKH signaling have previously been shown to impact starvation sensitivity. Genetic knockdown of three genes (Ca-Beta, Sur, and sei), in AKH producing cells caused highly significant changes in both male and female lifespan, and knockdown of six other genes (Shaw, cac, Ih, NaCP60E, stj, and TASK6) caused significant changes in only female lifespan. Specifically, the genetic knockdown of Ca-Beta and Sur led to increases in starvation lifespan, whereas the knockdown of sei decreased starvation survivorship. Focusing on these three strongest candidates from the behavioral screen, other AKH-dependent phenotypes were assessed. The AKH hormone is required for starvation-induced hyperactivity, and these three ion channel gene knockdowns were found to change activity profiles, and a modulatory role of these channels in AKH release was further suggested. The possibility that these genetic elements caused AKH cell lethality was eliminated, and using independent methods, expression of these genes was verified in AKH cells. Collectively, these results suggest a model of AKH-cell excitability and establish an experimental framework for evaluating intrinsic mechanisms of AKH release.
Shieh, B. H., Nuzum, L. and Kristaponyte, I. (2020). Exploring Excitotoxicity and Regulation of a Constitutively Active TRP Ca(2+) Channel in Drosophila. Fly (Austin). PubMed ID: 33200658
Unregulated Ca(2+) influx affects intracellular Ca(2+) homeostasis, which may lead to neuronal death. In Drosophila, following the activation of rhodopsin the TRP Ca(2+) channel is open to mediate the light-dependent depolarization. A constitutively active TRP channel triggers the degeneration of Trp(P365)/+ photoreceptors. To explore retinal degeneration, a multidisciplinary approach was employed including live imaging using GFP tagged actin and arrestin 2. Importantly, it was demonstrated that the major rhodopsin (Rh1) was greatly reduced before the onset of rhabdomere degeneration; a great reduction of Rh1 affects the maintenance of rhabdomere leading to degeneration of photoreceptors. Trp(P365)/+ also led to the up-regulation of CaMKII, which is beneficial as suppression of CaMKII accelerated retinal degeneration. This study explored the regulation of TRP by investigating the genetic interaction between Trp(P365)/+ and mutants affecting the turnover of diacylglycerol (DAG). A loss of phospholipase C in norpA(P24) was shown to exhibit a great reduction of the DAG content delayed degeneration of Trp(P365)/+ photoreceptors. In contrast, knockdown or mutations in DAG lipase (InaE) that is accompanied by slightly reduced levels of most DAG but an increased level of DAG 34:1, exacerbated retinal degeneration of Trp(P365) /+. Together, these findings support the notion that DAG plays a role in regulating TRP. Interestingly, DAG lipase is likely required during photoreceptor development as Trp(P365)/+; inaE(N125) double mutants contained severely degenerated rhabdomeres.
Tai, C. Y., Chin, A. L. and Chiang, A. S. (2020). A Comprehensive Map of Visual Projection Neurons for Processing Ultraviolet Information in the Drosophila Brain. J Comp Neurol. PubMed ID: 33174208
The brain perceives visual information and controls behavior depending on its underlying neural circuits. How UV information is represented and processed in the brain remains poorly understood. In Drosophila melanogaster, UV light is detected by the R7 photoreceptor that project exclusively into the medulla layer 6 (M(6)). This study imaged 28,768 single neurons and identified 238 visual projection neurons linking M(6) to the central brain. Based on morphology and connectivity, these visual projection neurons were systematically classified into 94 cell types belonging to 12 families. Three tracts connected M(6) in each optic lobe to the central brain: One dorsal tract linking to the ipsilateral lateral anterior optic tubercle (L-AOTU) and two medial tracts linking to the ipsilateral ventral medial protocerebrum (VMP) and the contralateral VMP. The M(6) information was primarily represented in the L-AOTU. Each L-AOTU consisted of four columns that each contained three glomeruli. Each L-AOTU glomerulus received inputs from M(6) subdomains and gave outputs to a glomerulus within the ellipsoid body dendritic region, suggesting specific processing of spatial information through the dorsal pathway. Furthermore, the middle columns of the L-AOTUs of both hemispheres were connected via the intertubercle tract, suggesting information integration between the two eyes. In contrast, an ascending neuron linked each VMP to all glomeruli in the bulb and the L-AOTU, bilaterally, suggesting general processing of information through the ventral pathway. Altogether, these diverse morphologies of the visual projection neurons suggested multi-dimensional processing of UV information through parallel and bilateral circuits in the Drosophila brain.
Schretter, C. E., Aso, Y., Robie, A. A., Dreher, M., Dolan, M. J., Chen, N., Ito, M., Yang, T., Parekh, R., Branson, K. M. and Rubin, G. M. (2020). Cell types and neuronal circuitry underlying female aggression in Drosophila. Elife 9. PubMed ID: 33141021
Aggressive social interactions are used to compete for limited resources and are regulated by complex sensory cues and the organism's internal state. While both sexes exhibit aggression, its neuronal underpinnings are understudied in females. This study identified a population of sexually dimorphic aIPg neurons in the adult Drosophila melanogaster central brain whose optogenetic activation increased, and genetic inactivation reduced, female aggression. Analysis of GAL4 lines identified in an unbiased screen for increased female chasing behavior revealed the involvement of another sexually dimorphic neuron, pC1d, and implicated aIPg and pC1d neurons as core nodes regulating female aggression. Connectomic analysis demonstrated that aIPg neurons and pC1d are interconnected and suggest that aIPg neurons may exert part of their effect by gating the flow of visual information to descending neurons. This work reveals important regulatory components of the neuronal circuitry that underlies female aggressive social interactions and provides tools for their manipulation.
Sharma, A. and Hasan, G. (2020). Modulation of flight and feeding behaviours requires presynaptic IP(3)Rs in dopaminergic neurons. Elife 9. PubMed ID: 33155978
Innate behaviours, although robust and hard wired, rely on modulation of neuronal circuits, for eliciting an appropriate response according to internal states and external cues. Drosophila flight is one such innate behaviour that is modulated by intracellular calcium release through inositol 1,4,5-trisphosphate receptors (IP(3)Rs). Cellular mechanism(s) by which IP(3)Rs modulate neuronal function for specific behaviours remain speculative, in vertebrates and invertebrates. To address this, an inducible dominant negative form of the IP(3)R (IP(3)R(DN)) was generated. Flies with neuronal expression of IP(3)R(DN) exhibit flight deficits. Expression of IP(3)R(DN) helped identify key flight-modulating dopaminergic neurons with axonal projections in the mushroom body. Flies with attenuated IP(3)Rs in these presynaptic dopaminergic neurons exhibit shortened flight bouts and a disinterest in seeking food, accompanied by reduced excitability and dopamine release upon cholinergic stimulation. These findings suggest that the same neural circuit modulates the drive for food search and for undertaking longer flight bouts.

Friday, December 11th - Cell Cycle

Gallaud, E., Ramdas Nair, A., Horsley, N., Monnard, A., Singh, P., Pham, T. T., Salvador Garcia, D., Ferrand, A. and Cabernard, C. (2020). Dynamic centriolar localization of Polo and Centrobin in early mitosis primes centrosome asymmetry. PLoS Biol 18(8): e3000762. PubMed ID: 32760088
Centrosomes, the main microtubule organizing centers (MTOCs) of metazoan cells, contain an older "mother" and a younger "daughter" centriole. Stem cells either inherit the mother or daughter-centriole-containing centrosome, providing a possible mechanism for biased delivery of cell fate determinants. However, the mechanisms regulating centrosome asymmetry and biased centrosome segregation are unclear. Using 3D-structured illumination microscopy (3D-SIM) and live-cell imaging, this study shows in fly neural stem cells (neuroblasts) that the mitotic kinase Polo and its centriolar protein substrate Centrobin (Cnb) accumulate on the daughter centriole during mitosis, thereby generating molecularly distinct mother and daughter centrioles before interphase. Cnb's asymmetric localization, potentially involving a direct relocalization mechanism, is regulated by Polo-mediated phosphorylation, whereas Polo's daughter centriole enrichment requires both Wdr62 and Cnb. Based on optogenetic protein mislocalization experiments, it is proposed that the establishment of centriole asymmetry in mitosis primes biased interphase MTOC activity, necessary for correct spindle orientation.
Garrido, D., Bourouh, M., Bonneil, É., Thibault, P., Swan, A. and Archambault, V. (2020). Cyclin B3 activates the Anaphase-Promoting Complex/Cyclosome in meiosis and mitosis. PLoS Genet 16(11): e1009184. PubMed ID: 33137813
In mitosis and meiosis, chromosome segregation is triggered by the Anaphase-Promoting Complex/Cyclosome (APC/C), a multi-subunit ubiquitin ligase that targets proteins for degradation, leading to the separation of chromatids. APC/C activation requires phosphorylation of its APC3 and APC1 subunits, which allows the APC/C to bind its co-activator Cdc20. The identity of the kinase(s) responsible for APC/C activation in vivo is unclear. Cyclin B3 (CycB3) is an activator of the Cyclin-Dependent Kinase 1 (Cdk1) that is required for meiotic anaphase. Using Drosophila, this study found that mutations in CycB3 genetically enhance mutations in tws, which encodes the B55 regulatory subunit of Protein Phosphatase 2A (PP2A) known to promote mitotic exit. Females heterozygous for CycB3 and tws loss-of-function alleles lay embryos that arrest in mitotic metaphase in a maternal effect, indicating that CycB3 promotes anaphase in mitosis in addition to meiosis. This metaphase arrest is not due to the Spindle Assembly Checkpoint (SAC) because mutation of mad2 that inactivates the SAC does not rescue the development of embryos from CycB3-/+, tws-/+ females. Moreover, CycB3 was found to promote APC/C activity and anaphase in cells in culture. CycB3 physically associates with the APC/C, is required for phosphorylation of APC3, and promotes APC/C association with its Cdc20 co-activators Fizzy and Cortex. These results strongly suggest that CycB3-Cdk1 directly activates the APC/C to promote anaphase in both meiosis and mitosis.
Torras-Llort, M., Medina-Giro, S., Escudero-Ferruz, P., Lipinszki, Z., Moreno-Moreno, O., Karman, Z., Przewloka, M. R. and Azorin, F. (2020). A fraction of barrier-to-autointegration factor (BAF) associates with centromeres and controls mitosis progression. Commun Biol 3(1): 454. PubMed ID: 32814801
Barrier-to-Autointegration Factor (BAF) is a conserved nuclear envelope (NE) component that binds chromatin and helps its anchoring to the NE. Cycles of phosphorylation and dephosphorylation control BAF function. Entering mitosis, phosphorylation releases BAF from chromatin and facilitates NE-disassembly. At mitotic exit, PP2A-mediated dephosphorylation restores chromatin binding and nucleates NE-reassembly. This study shows that in Drosophila a small fraction of BAF (cenBAF) associates with centromeres. PP4 phosphatase, which is recruited to centromeres by CENP-C, prevents phosphorylation and release of cenBAF during mitosis. cenBAF is necessary for proper centromere assembly and accurate chromosome segregation, being critical for mitosis progression. Disrupting cenBAF localization prevents PP2A inactivation in mitosis compromising global BAF phosphorylation, which in turn leads to its persistent association with chromatin, delays anaphase onset and causes NE defects. These results suggest that, together with PP4 and CENP-C, cenBAF forms a centromere-based mechanism that controls chromosome segregation and mitosis progression.
Unnikannan, C. P., Reuveny, A., Grunberg, D. and Volk, T. (2020). Recruitment of BAF to the nuclear envelope couples the LINC complex to endoreplication. Development. PubMed ID: 33168584
DNA endoreplication has been implicated as a cell strategy to grow in size and in tissue injury. This study demonstrates that barrier to autointegration factor (BAF), represses endoreplication in Drosophila myofibers. This study shows that BAF localization at the nuclear envelope was eliminated either in mutants of the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex, in which the LEM-domain protein Otefin was similarly excluded, or after disruption of the nucleus-sarcomere connections. Furthermore, BAF localization at the nuclear envelope required the activity of the BAF kinase VRK1/Ball, and consistently non-phosphorytable BAF-GFP was excluded from the nuclear envelope. Importantly, removal of BAF from the nuclear envelope correlated with increased DNA content in the myonuclei. E2F1, a key regulator of endoreplication was found to overlap BAF localization at the myonuclear envelope, and BAF removal from the nuclear envelope resulted with increased E2F1 levels in the nucleoplasm, and subsequent elevated DNA content. It is suggested that LINC-dependent, and phospho-sensitive attachment of BAF to the nuclear envelope, through its binding to Otefin, tethers E2F1 to the nuclear envelope thus inhibiting its accumulation at the nucleoplasm.
Ryder, P. V., Fang, J. and Lerit, D. A. (2020). centrocortin RNA localization to centrosomes is regulated by FMRP and facilitates error-free mitosis. J Cell Biol 219(12). PubMed ID: 33196763
Centrosomes are microtubule-organizing centers required for error-free mitosis and embryonic development. The microtubule-nucleating activity of centrosomes is conferred by the pericentriolar material (PCM), a composite of numerous proteins subject to cell cycle-dependent oscillations in levels and organization. In diverse cell types, mRNAs localize to centrosomes and may contribute to changes in PCM abundance. This study investigated the regulation of mRNA localization to centrosomes in the rapidly cycling Drosophila melanogaster embryo. RNA localization to centrosomes was found to be regulated during the cell cycle and developmentally. A novel role for the fragile-X mental retardation protein was identified in the posttranscriptional regulation of a model centrosomal mRNA, centrocortin (cen). Further, mistargeting cen mRNA is sufficient to alter cognate protein localization to centrosomes and impair spindle morphogenesis and genome stability.
Wang, X. F., Liu, J. X., Ma, Z. Y., Shen, Y., Zhang, H. R., Zhou, Z. Z., Suzuki, E., Liu, Q. X. and Hirose, S. (2020). Evolutionarily Conserved Roles for Apontic in Induction and Subsequent Decline of Cyclin E Expression. iScience 23(8): 101369. PubMed ID: 32736066
Cyclin E is a key factor for S phase entry, and deregulation of Cyclin E results in developmental defects and tumors. Therefore, proper cycling of Cyclin E is crucial for normal growth. This study found that transcription factors Apontic (Apt) and E2f1 cooperate to induce cyclin E in Drosophila. Functional binding motifs of Apt and E2f1 are clustered in the first intron of Drosophila cyclin E and directly contribute to the cyclin E transcription. Knockout of apt and e2f1 together abolished Cyclin E expression. Furthermore, Apt up-regulates Retinoblastoma family protein 1 (Rbf1) for proper chromatin compaction, which is known to repress cyclin E. Notably, Apt-dependent up-regulation of Cyclin E and Rbf1 is evolutionarily conserved in mammalian cells. These findings reveal a unique mechanism underlying the induction and subsequent decline of Cyclin E expression.

Thursday, December 10th - Enhancers and gene regulation

Huang, W., Carbone, M. A., Lyman, R. F., Anholt, R. R. H. and Mackay, T. F. C. (2020). Genotype by environment interaction for gene expression in Drosophila melanogaster. Nat Commun 11(1): 5451. PubMed ID: 33116142
The genetics of phenotypic responses to changing environments remains elusive. Using whole-genome quantitative gene expression as a model, this study examined how the genetic architecture of regulatory variation in gene expression changed in a population of fully sequenced inbred Drosophila melanogaster strains when flies developed in different environments (25 °C and 18 °C). A substantial fraction of the transcriptome exhibited genotype by environment interaction, implicating environmentally plastic genetic architecture of gene expression. Genetic variance in expression increases at 18 °C relative to 25 °C for most genes that have a change in genetic variance. Although the majority of expression quantitative trait loci (eQTLs) for the gene expression traits in the two environments are shared and have similar effects, analysis of the environment-specific eQTLs reveals enrichment of binding sites for two transcription factors. Finally, although genotype by environment interaction in gene expression could potentially disrupt genetic networks, the co-expression networks are highly conserved across environments. Genes with higher network connectivity are under stronger stabilizing selection, suggesting that stabilizing selection on expression plays an important role in promoting network robustness.
Nakamura, S., Hira, S., Kojima, M., Kondo, A. and Mukai, M. (2020). Expression of the core promoter factors TBP and TRF2 in Drosophila germ cells and their distinct functions in germline development. Dev Growth Differ. PubMed ID: 33219538
In Drosophila, the expression of germline genes is initiated in primordial germ cells (PGCs) and its expression is known to be associated with germline establishment. However, the transcriptional regulation of germline genes remains elusive. Previous studies found that the BTB/POZ-Zn-finger protein, Mamo, is necessary for the expression of the germline gene, vasa, in PGCs. Moreover, the truncated Mamo lacking the BTB/POZ domain (MamoAF) is a potent vasa activator. This study investigated the genetic interaction between MamoAF and specific transcriptional regulators to gain insight into the transcriptional regulation of germline development. A general transcription factor, TATA box binding protein (TBP)-associated factor 3 (TAF3/BIP2), and a member of the TBP-like proteins, TBP-related factor 2 (TRF2), were found as new genetic modifiers of MamoAF. In contrast to TRF2, TBP was found to show no genetic interaction with MamoAF, suggesting that Trf2 has a selective function. Therefore, this study focused on Trf2 expression and investigated its function in germ cells. The Trf2 mRNA, rather than the Tbp mRNA, was found to be preferentially expressed in PGCs during embryogenesis. The depletion of TRF2 in PGCs resulted in decreased mRNA expression of vasa. RNA interference-mediated knockdown showed that while Trf2 is required for the maintenance of germ cells, Tbp is needed for their differentiation during oogenesis. Therefore, these results suggest that Trf2 and Tbp expression is differentially regulated in germ cells, and that these factors have distinct functions in Drosophila germline development.
Vasudevan, D., Neuman, S. D., Yang, A., Lough, L., Brown, B., Bashirullah, A., Cardozo, T. and Ryoo, H. D. (2020). Translational induction of ATF4 during integrated stress response requires noncanonical initiation factors eIF2D and DENR. Nat Commun 11(1): 4677. PubMed ID: 32938929
The Integrated Stress Response (ISR) helps metazoan cells adapt to cellular stress by limiting the availability of initiator methionyl-tRNA for translation. Such limiting conditions paradoxically stimulate the translation of ATF4 mRNA through a regulatory 5' leader sequence with multiple upstream Open Reading Frames (uORFs), thereby activating stress-responsive gene expression. This study reports the identification of two critical regulators of such ATF4 induction, the noncanonical initiation factors eIF2D and DENR. Loss of eIF2D and DENR in Drosophila results in increased vulnerability to amino acid deprivation, susceptibility to retinal degeneration caused by endoplasmic reticulum (ER) stress, and developmental defects similar to ATF4 mutants. eIF2D requires its RNA-binding motif for regulation of 5' leader-mediated ATF4 translation. Consistently, eIF2D and DENR deficient human cells show impaired ATF4 protein induction in response to ER stress. Altogether, these findings indicate that eIF2D and DENR are critical mediators of ATF4 translational induction and stress responses in vivo.
Berndt, A. J., Othonos, K. M., Lian, T., Flibotte, S., Miao, M., Bhuiyan, S. A., Cho, R. Y., Fong, J. S., Hur, S. A., Pavlidis, P. and Allan, D. W. (2020). A low affinity cis-regulatory BMP response element restricts target gene activation to subsets of Drosophila neurons. Elife 9. PubMed ID: 33124981
Retrograde BMP signaling and canonical pMad/Medea-mediated transcription regulate diverse target genes across subsets of Drosophila efferent neurons, to differentiate neuropeptidergic neurons and promote motor neuron terminal maturation. How a common BMP signal regulates diverse target genes across many neuronal subsets remains largely unresolved, although available evidence implicates subset-specific transcription factor codes rather than differences in BMP signaling. This study examined the cis-regulatory mechanisms restricting BMP-induced FMRFa neuropeptide expression to Tv4-neurons. pMad/Medea bind at an atypical, low affinity motif in the FMRFa enhancer. Converting this motif to high affinity caused ectopic enhancer activity and eliminated Tv4-neuron expression. In silico searches identified additional motif instances functional in other efferent neurons, implicating broader functions for this motif in BMP-dependent enhancer activity. Thus, differential interpretation of a common BMP signal, conferred by low affinity pMad/Medea binding motifs, can contribute to the specification of BMP target genes in efferent neuron subsets.
Schnepf, M., von Reutern, M., Ludwig, C., Jung, C. and Gaul, U. (2020). Transcription Factor Binding Affinities and DNA Shape Readout. iScience 23(11): 101694. PubMed ID: 33163946
An essential event in gene regulation is the binding of a transcription factor (TF) to its target DNA. Models considering the interactions between the TF and the DNA geometry proved to be successful approaches to describe this binding event, while conserving data interpretability. However, a direct characterization of the DNA shape contribution to binding is still missing due to the lack of accurate and large-scale binding affinity data. This study use a recently established binding assay to measure with high sensitivity the binding specificities of 13 Drosophila TFs, including dinucleotide dependencies to capture non-independent amino acid-base interactions. Correlating the binding affinities with all DNA shape features, this study found that shape readout is widely used by these factors. A shape readout/TF-DNA complex structure analysis validates this approach while providing biological insights such as positively charged or highly polar amino acids often contact nucleotides that exhibit strong shape readout.
Reddington, J. P., Garfield, D. A., Sigalova, O. M., Karabacak Calviello, A., Marco-Ferreres, R., Girardot, C., Viales, R. R., Degner, J. F., Ohler, U. and Furlong, E. E. M. (2020). Lineage-Resolved Enhancer and Promoter Usage during a Time Course of Embryogenesis. Dev Cell. PubMed ID: 33171098
Enhancers are essential drivers of cell states, yet the relationship between accessibility, regulatory activity, and in vivo lineage commitment during embryogenesis remains poorly understood. This study measured chromatin accessibility in isolated neural and mesodermal lineages across a time course of Drosophila embryogenesis. Promoters, including tissue-specific genes, are often constitutively open, even in contexts where the gene is not expressed. In contrast, the majority of distal elements have dynamic, tissue-specific accessibility. Enhancer priming appears rarely within a lineage, perhaps reflecting the speed of Drosophila embryogenesis. However, many tissue-specific enhancers are accessible in other lineages early on and become progressively closed as embryogenesis proceeds. This study demonstrates the usefulness of this tissue- and time-resolved resource to definitively identify single-cell clusters, to uncover predictive motifs, and to identify many regulators of tissue development. For one such predicted neural regulator, l(3)neo38, a loss-of-function mutant was generated and an essential role for neuromuscular junction and brain development was uncovered.

Wednesday, December 9th - Signal transduction

Maddison, D. C., Alfonso-Nunez, M., Swaih, A. M., Breda, C., Campesan, S., Allcock, N., Straatman-Iwanowska, A., Kyriacou, C. P. and Giorgini, F. (2020). A novel role for kynurenine 3-monooxygenase in mitochondrial dynamics. PLoS Genet 16(11): e1009129. PubMed ID: 33170836
The enzyme kynurenine 3-monooxygenase (KMO) operates at a critical branch-point in the kynurenine pathway (KP), the major route of tryptophan metabolism. As the KP has been implicated in the pathogenesis of several human diseases, KMO and other enzymes that control metabolic flux through the pathway are potential therapeutic targets for these disorders. While KMO is localized to the outer mitochondrial membrane in eukaryotic organisms, no mitochondrial role for KMO has been described. In this study, KMO (cinnabar) deficient Drosophila melanogaster were investigated for mitochondrial phenotypes in vitro and in vivo. A loss of function allele or RNAi knockdown of the Drosophila KMO ortholog (cinnabar) causes a range of morphological and functional alterations to mitochondria, which are independent of changes to levels of KP metabolites. Notably, cinnabar genetically interacts with the Parkinson's disease associated genes Pink1 and parkin, as well as the mitochondrial fission gene Drp1, implicating KMO in mitochondrial dynamics and mitophagy, mechanisms which govern the maintenance of a healthy mitochondrial network. Overexpression of human KMO in mammalian cells finds that KMO plays a role in the post-translational regulation of DRP1. These findings reveal a novel mitochondrial role for KMO, independent from its enzymatic role in the kynurenine pathway.
Pelham, J. F., Dunlap, J. C. and Hurley, J. M. (2020). Intrinsic disorder is an essential characteristic of components in the conserved circadian circuit. Cell Commun Signal 18(1): 181. PubMed ID: 33176800
The circadian circuit, a roughly 24 h molecular feedback loop, or clock, is conserved from bacteria to animals and allows for enhanced organismal survival by facilitating the anticipation of the day/night cycle. Recent research has demonstrated that proteins comprising the circadian clock network display a significant amount of intrinsic disorder. This work focussed on the extent of intrinsic disorder in the circadian clock and its potential mechanistic role in circadian timing. The conservation of disorder was highlighted by quantifying the extent of computationally-predicted protein disorder in the core clock of the key eukaryotic circadian model organisms Drosophila melanogaster, Neurospora crassa, and Mus musculus. Previously published work, as well as feature novel experimental evidence, was examined, demonstrating that the core negative arm circadian period drivers FREQUENCY (Neurospora crassa) and PERIOD-2 (PER2) (Mus musculus), possess biochemical characteristics of intrinsically disordered proteins. Finally, the potential contributions are discussed of the inherent biophysical principals of intrinsically disordered proteins that may explain the vital mechanistic roles they play in the clock to drive their broad evolutionary conservation in circadian timekeeping. It is concluded that the pervasive conservation of disorder amongst the clock in the crown eukaryotes suggests that disorder is essential for optimal circadian timing from fungi to animals, providing vital homeostatic cellular maintenance and coordinating organismal physiology across phylogenetic kingdoms.
Maier, D. (2020). Membrane-Anchored Hairless Protein Restrains Notch Signaling Activity. Genes (Basel) 11(11). PubMed ID: 33171957
The Notch signaling pathway governs cell-to-cell communication in higher eukaryotes. In Drosophila, after cleavage of the transmembrane receptor Notch, the intracellular domain of Notch (ICN) binds to the transducer Suppressor of Hairless (Su(H)) and shuttles into the nucleus to activate Notch target genes. Similarly, the Notch antagonist Hairless transfers Su(H) into the nucleus to repress Notch target genes. With the aim to prevent Su(H) nuclear translocation, Hairless was fused to a transmembrane domain to anchor the protein at membranes. Indeed, endogenous Su(H) co-localized with membrane-anchored Hairless, demonstrating their binding in the cytoplasm. Moreover, adult phenotypes uncovered a loss of Notch activity, in support of membrane-anchored Hairless sequestering Su(H) in the cytosol. A combined overexpression of membrane-anchored Hairless with Su(H) lead to tissue proliferation, which is in contrast to the observed apoptosis after ectopic co-overexpression of the wild-type genes, indicating a shift to a gain of Notch activity. A mixed response, general de-repression of Notch signaling output, plus inhibition at places of highest Notch activity, perhaps reflects Su(H)'s role as activator and repressor, supported by results obtained with the Hairless-binding deficient Su(H)(LLL) mutant, inducing activation only. Overall, the results strengthen the idea of Su(H) and Hairless complex formation within the cytosolic compartment.
Verboon, J. M., Nakamura, M., Davidson, K. A., Decker, J. R., Nandakumar, V. and Parkhurst, S. M. (2020). Drosophila Wash and the Wash regulatory complex function in nuclear envelope budding. J Cell Sci 133(13). PubMed ID: 32503943
Nuclear envelope (NE) budding is a recently described phenomenon wherein large macromolecular complexes are packaged inside the nucleus and extruded through the nuclear membranes. Although a general outline of the cellular events occurring during NE budding is now in place, little is yet known about the molecular machinery and mechanisms underlying the physical aspects of NE bud formation. Using a multidisciplinary approach, this study identified Wash, its regulatory complex (SHRC), capping protein and Arp2/3 as new molecular components involved in the physical aspects of NE bud formation in a Drosophila model system. Interestingly, Wash affects NE budding in two ways: indirectly through general nuclear lamina disruption via an SHRC-independent interaction with Lamin B leading to inefficient NE bud formation, and directly by blocking NE bud formation along with its SHRC, capping protein and Arp2/3. In addition to NE budding emerging as an important cellular process, it shares many similarities with herpesvirus nuclear egress mechanisms, suggesting new avenues for exploration in both normal and disease biology.
Park, J., Jun, K., Choi, Y., Yoon, E., Kim, W., Jang, Y. G. and Chung, J. (2020). CORO7 functions as a scaffold protein for the core kinase complex assembly of the Hippo pathway. J Biol Chem. PubMed ID: 33162394
The Hippo pathway controls organ size and tissue homeostasis through the regulation of cell proliferation and apoptosis. However, the exact molecular mechanisms underpinning Hippo pathway regulation is not fully understood. This study identified a new component of the Hippo pathway: CORO7, a coronin protein family member that is involved in organization of the actin cytoskeleton. pod1, the Drosophila orthologue of CORO7, genetically interacts with key Hippo pathway genes in Drosophila. In mammalian cells, CORO7 is required for the activation of the Hippo pathway in response to cell-cell contact, serum deprivation, and cytoskeleton damage. CORO7 forms a complex with the core components of the pathway and functions as a scaffold for the Hippo core kinase complex. Collectively, these results demonstrate that CORO7 is a key scaffold controlling the Hippo pathway via modulating protein-protein interactions.
Luo, Y., Fefelova, E., Ninova, M., Chen, Y. A. and Aravin, A. A. (2020). Repression of interrupted and intact rDNA by the SUMO pathway in Drosophila melanogaster. Elife 9. PubMed ID: 33164748
Ribosomal RNAs (rRNAs) are essential components of the ribosome and are among the most abundant macromolecules in the cell. To ensure high rRNA level, eukaryotic genomes contain dozens to hundreds of rDNA genes, however, only a fraction of the rRNA genes seems to be active, while others are transcriptionally silent. This study found that individual rDNA genes have high level of cell-to-cell heterogeneity in their expression in Drosophila melanogaster. Insertion of heterologous sequences into rDNA leads to repression associated with reduced expression in individual cells and decreased number of cells expressing rDNA with insertions. SUMO (Small Ubiquitin-like Modifier) and SUMO ligase Ubc9 were shown to be required for efficient repression of interrupted rDNA units and variable expression of intact rDNA. Disruption of the SUMO pathway abolishes discrimination of interrupted and intact rDNAs and removes cell-to-cell heterogeneity leading to uniformly high expression of individual rDNA in single cells. These results suggest that the SUMO pathway is responsible for both repression of interrupted units and control of intact rDNA expression.

Tuesday, December 8th - Larval and adult neural development and function

Ozel, M. N., Simon, F., Jafari, S., Holguera, I., Chen, Y. C., Benhra, N., El-Danaf, R. N., Kapuralin, K., Malin, J. A., Konstantinides, N. and Desplan, C. (2020). Neuronal diversity and convergence in a visual system developmental atlas. Nature. PubMed ID: 33149298
Deciphering how neuronal diversity is established and maintained requires a detailed knowledge of neuronal gene expression throughout development. In contrast to mammalian brains, the large neuronal diversity of the Drosophila optic lobe and its connectome are almost completely characterized. However, a molecular characterization of this neuronal diversity, particularly during development, has been lacking. Thhis study presents insights into brain development through a nearly complete description of the transcriptomic diversity of the optic lobes of Drosophila. The transcriptome of 275,000 single cells was acquired at adult and at five pupal stages, and a machine-learning framework was built to assign them to almost 200 cell types at all time points during development. Two large neuronal populations were discovered that wrap neuropils during development but die just before adulthood, as well as neuronal subtypes that partition dorsal and ventral visual circuits by differential Wnt signalling throughout development. Moreover, it was shown that the transcriptomes of neurons that are of the same type but are produced days apart become synchronized shortly after their production. During synaptogenesis this study also resolved neuronal subtypes that, although differing greatly in morphology and connectivity, converge to indistinguishable transcriptomic profiles in adults. These datasets almost completely account for the known neuronal diversity of the Drosophila optic lobes, and serve as a paradigm to understand brain development across species.
Omamiuda-Ishikawa, N., Sakai, M. and Emoto, K. (2020). A pair of ascending neurons in the subesophageal zone mediates aversive sensory inputs-evoked backward locomotion in Drosophila larvae. PLoS Genet 16(11): e1009120. PubMed ID: 33137117
Animals typically avoid unwanted situations with stereotyped escape behavior. For instance, Drosophila larvae often escape from aversive stimuli to the head, such as mechanical stimuli and blue light irradiation, by backward locomotion. Responses to these aversive stimuli are mediated by a variety of sensory neurons including mechanosensory class III da (C3da) sensory neurons and blue-light responsive class IV da (C4da) sensory neurons and Bolwig's organ (BO). How these distinct sensory pathways evoke backward locomotion at the circuit level is still incompletely understood. This study shows that a pair of cholinergic neurons in the subesophageal zone, designated AMBs, evoke robust backward locomotion upon optogenetic activation. Anatomical and functional analysis shows that AMBs act upstream of MDNs, the command-like neurons for backward locomotion. Further functional analysis indicates that AMBs preferentially convey aversive blue light information from C4da neurons to MDNs to elicit backward locomotion, whereas aversive information from BO converges on MDNs through AMB-independent pathways. This study also found that, unlike in adult flies, MDNs are dispensable for the dead end-evoked backward locomotion in larvae. These findings thus reveal the neural circuits by which two distinct blue light-sensing pathways converge on the command-like neurons to evoke robust backward locomotion, and suggest that distinct but partially redundant neural circuits including the command-like neurons might be utilized to drive backward locomotion in response to different sensory stimuli as well as in adults and larvae.
Hadjieconomou, D., King, G., Gaspar, P., Mineo, A., Blackie, L., Ameku, T., Studd, C., de Mendoza, A., Diao, F., White, B. H., Brown, A. E. X., Placais, P. Y., Preat, T. and Miguel-Aliaga, I. (2020). Enteric neurons increase maternal food intake during reproduction. Nature 587(7834): 455-459. PubMed ID: 33116314
Reproduction induces increased food intake across females of many animal species, providing a physiologically relevant paradigm for the exploration of appetite regulation. By examining the diversity of enteric neurons in Drosophila melanogaster, this study identified a key role for gut-innervating neurons with sex- and reproductive state-specific activity in sustaining the increased food intake of mothers during reproduction. Steroid and enteroendocrine hormones functionally remodel these neurons, which leads to the release of their neuropeptide onto the muscles of the crop-a stomach-like organ-after mating. Neuropeptide release changes the dynamics of crop enlargement, resulting in increased food intake, and preventing the post-mating remodelling of enteric neurons reduces both reproductive hyperphagia and reproductive fitness. The plasticity of enteric neurons is therefore key to reproductive success. These findings provide a mechanism to attain the positive energy balance that sustains gestation, dysregulation of which could contribute to infertility or weight gain.
Han, X., Wang, M., Liu, C., Trush, O., Takayama, R., Akiyama, T., Naito, T., Tomomizu, T., Imamura, K. and Sato, M. (2020). DWnt4 and DWnt10 Regulate Morphogenesis and Arrangement of Columnar Units via Fz2/PCP Signaling in the Drosophila Brain. Cell Rep 33(4): 108305. PubMed ID: 33113378
Columns are structural and functional units of the brain. However, the mechanism of column formation remains unclear. The medulla of the fly visual center shares features with the mammalian cerebral cortex, such as columnar and layered structures, and provides a good opportunity to study the mechanisms of column formation. Column formation is initiated by three core neurons in the medulla, namely, Mi1, R8, and R7. The proper orientation of neurons is required for the orientation and arrangement of multiple columns. Their orientations may be under the control of planar cell polarity (PCP) signaling, because it is known to regulate the orientation of cells in two-dimensional tissue structures. This study demonstrates that the ligands DWnt4 and DWnt10 expressed specifically in the ventral medulla and dorsal medulla, respectively, globally regulate the columnar arrangement and orientation of Mi1 and R8 terminals through Fz2/PCP signaling in a three-dimensional space.
Kurmangaliyev, Y. Z., Yoo, J., Valdes-Aleman, J., Sanfilippo, P. and Zipursky, S. L. (2020). Transcriptional Programs of Circuit Assembly in the Drosophila Visual System. Neuron. PubMed ID: 33125872
Precise patterns of synaptic connections between neurons are encoded in their genetic programs. This study used single-cell RNA sequencing to profile neuronal transcriptomes at multiple stages in the developing Drosophila visual system. An efficient strategy was devised for profiling neurons at multiple time points in a single pool, thereby minimizing batch effects and maximizing the reliability of time-course data. A transcriptional atlas spanning multiple stages is generated, including more than 150 distinct neuronal populations; of these, 88 are followed through synaptogenesis. This analysis reveals a common (pan-neuronal) program unfolding in highly coordinated fashion in all neurons, including genes encoding proteins comprising the core synaptic machinery and membrane excitability. This program is overlaid by cell-type-specific programs with diverse cell recognition molecules expressed in different combinations and at different times. It is proposed that a pan-neuronal program endows neurons with the competence to form synapses and that cell-type-specific programs control synaptic specificity.
Morimoto, M. M., Nern, A., Zhao, A., Rogers, E. M., Wong, A., Isaacson, M. D., Bock, D., Rubin, G. M. and Reiser, M. B. (2020). Spatial readout of visual looming in the central brain of Drosophila. Elife 9. PubMed ID: 33205753
Visual systems can exploit spatial correlations in the visual scene by using retinotopy. However, retinotopy is often lost, such as when visual pathways are integrated with other sensory modalities. How is spatial information processed outside of strictly visual brain areas? This study focused on visual looming responsive LC6 cells in Drosophila, a population whose dendrites collectively cover the visual field, but whose axons form a single glomerulus-a structure without obvious retinotopic organization-in the central brain. Multiple cell types downstream of LC6 were identified in the glomerulus and they were found to more strongly respond to looming in different portions of the visual field, unexpectedly preserving spatial information. Through EM reconstruction of all LC6 synaptic inputs to the glomerulus, it was found that LC6 and downstream cell types form circuits within the glomerulus that enable spatial readout of visual features and contralateral suppression-mechanisms that transform visual information for behavioral control.

Monday, December 7th - Disease models

Montagna, A., Vajente, N., Pendin, D. and Daga, A. (2020). In vivo Analysis of CRISPR/Cas9 Induced Atlastin Pathological Mutations in Drosophila. Front Neurosci 14: 547746. PubMed ID: 33177972
The endoplasmic reticulum (ER) is a highly dynamic network whose shape is thought to be actively regulated by membrane resident proteins. Mutation of several such morphology regulators cause the neurological disorder Hereditary Spastic Paraplegia (HSP), suggesting a critical role of ER shape maintenance in neuronal activity and function. Human Atlastin-1 mutations are responsible for SPG3A, the earliest onset and one of the more severe forms of dominant HSP. Atlastin has been initially identified in Drosophila as the GTPase responsible for the homotypic fusion of ER membrane. The majority of SPG3A-linked Atlastin-1 mutations map to the GTPase domain, potentially interfering with atlastin GTPase activity, and to the three-helix-bundle (3HB) domain, a region critical for homo-oligomerization. This study examined the in vivo effects of four pathogenetic missense mutations (two mapping to the GTPase domain and two to the 3HB domain) using two complementary approaches: CRISPR/Cas9 editing to introduce such variants in the endogenous atlastin gene and transgenesis to generate lines overexpressing atlastin carrying the same pathogenic variants. Sll pathological mutations examined reduce atlastin activity in vivo although to different degrees of severity. Moreover, overexpression of the pathogenic variants in a wild type atlastin background does not give rise to the loss of function phenotypes expected for dominant negative mutations. These results indicate that the four pathological mutations investigated act through a loss of function mechanism.
Bakalov, V., Reyes-Uribe, L., Deshpande, R., Maloy, A. L., Shapiro, S. D., Angus, D. C., Chang, C. H., Le Moyec, L., Wendell, S. G. and Kaynar, A. M. (2020). Dichloroacetate-induced metabolic reprogramming improves lifespan in a Drosophila model of surviving sepsis. PLoS One 15(11): e0241122. PubMed ID: 33151963
Sepsis is the leading cause of death in hospitalized patients and beyond the hospital stay and these long-term sequelae are due in part to unresolved inflammation. Metabolic shift from oxidative phosphorylation to aerobic glycolysis links metabolism to inflammation and such a shift is commonly observed in sepsis under normoxic conditions. By shifting the metabolic state from aerobic glycolysis to oxidative phosphorylation, it was hypothesized that the shift would reverse unresolved inflammation and subsequently improve outcome. This study proposes that a shift from aerobic glycolysis to oxidative phosphorylation as a sepsis therapy by targeting the pathways involved in the conversion of pyruvate into acetyl-CoA via pyruvate dehydrogenase (PDH). Chemical manipulation of PDH using dichloroacetic acid (DCA) will promote oxidative phosphorylation over glycolysis and decrease inflammation. This hypothesis was tested in a Drosophila melanogaster model of surviving sepsis infected with Staphylococcus aureus. Drosophila were divided into 3 groups: unmanipulated, sham and sepsis survivors, all treated with linezolid; each group was either treated or not with DCA for one week following sepsis. Lifespan, measured gene expression of Toll, defensin, cecropin A, and drosomycin, and levels of lactate, pyruvate, acetyl-CoA was studied as well as TCA metabolites. In this model, metabolic effects of sepsis are modified by DCA with normalized lactate, TCA metabolites, and was associated with improved lifespan of sepsis survivors, yet had no lifespan effects on unmanipulated and sham flies. While Drosomycin and cecropin A expression increased in sepsis survivors, DCA treatment decreased both and selectively increased defensin.
Hartwig, C., Mendez, G. M., Bhattacharjee, S., Vrailas-Mortimer, A. D., Zlatic, S. A., Freeman, A. A. H., Gokhale, A., Concilli, M., Werner, E., Sapp Savas, C., Rudin-Rush, S., Palmer, L., Shearing, N., Margewich, L., McArthy, J., Taylor, S., Roberts, B., Lupashin, V., Polishchuk, R. S., Cox, D. N., Jorquera, R. A. and Faundez, V. (2020). Golgi-Dependent Copper Homeostasis Sustains Synaptic Development and Mitochondrial Content. J Neurosci. PubMed ID: 33208468
Rare genetic diseases preponderantly affect the nervous system causing neurodegeneration to neurodevelopmental disorders. This is the case for both Menkes and Wilson disease, arising from mutations in ATP7A and ATP7B, respectively. The ATP7A and ATP7B proteins localize to the Golgi and regulate copper homeostasis. Genetic and biochemical interactions were demonstrated between ATP7 paralogs with the Conserved Oligomeric Golgi complex, or COG complex, a Golgi apparatus vesicular tether. Disruption of Drosophila copper homeostasis by ATP7 tissue-specific transgenic expression caused alterations in epidermis, aminergic, sensory, and motor neurons. Prominent among neuronal phenotypes was a decreased mitochondrial content at synapses, a phenotype that paralleled with alterations of synaptic morphology, transmission, and plasticity. These neuronal and synaptic phenotypes caused by transgenic expression of ATP7 were rescued by downregulation of COG complex subunits. It is concluded that the integrity of Golgi-dependent copper homeostasis mechanisms, requiring ATP7 and COG, are necessary to maintain mitochondria functional integrity and localization to synapses.
Keramidis, I., Vourkou, E., Papanikolopoulou, K. and Skoulakis, E. M. C. (2020). Functional Interactions of Tau Phosphorylation Sites That Mediate Toxicity and Deficient Learning in Drosophila melanogaster. Front Mol Neurosci 13: 569520. PubMed ID: 33192295
Hyperphosphorylated Tau protein is the main component of the neurofibrillary tangles, characterizing degenerating neurons in Alzheimer's disease and other Tauopathies. Expression of human Tau protein (see Drosophila Tau) in Drosophila CNS results in increased toxicity, premature mortality and learning and memory deficits. This study used novel transgenic lines to investigate the contribution of specific phosphorylation sites previously implicated in Tau toxicity. These three different sites, Ser(238), Thr(245), and Ser(262) were tested either by blocking their phosphorylation, by Ser/Thr to Ala substitution, or pseudophosphorylation, by changing Ser/Thr to Glu. The hypothesis was validated that phosphorylation at Ser(262) is necessary for Tau-dependent learning deficits and a "facilitatory gatekeeper" to Ser(238) occupation, which is linked to Tau toxicity. Importantly this study reveals that phosphorylation at Thr(245) acts as a "suppressive gatekeeper", preventing phosphorylation of many sites including Ser(262) and consequently of Ser(238). Therefore, this study elucidates novel interactions among phosphosites central to Tau mediated neuronal dysfunction and toxicity, likely driven by phosphorylation-dependent conformational plasticity.
Li, L., Ding, Z., Pang, T. L., Zhang, B., Li, C. H., Liang, A. M., Wang, Y. R., Zhou, Y., Fan, Y. J. and Xu, Y. Z. (2020). Defective minor spliceosomes induce SMA-associated phenotypes through sensitive intron-containing neural genes in Drosophila. Nat Commun 11(1): 5608. PubMed ID: 33154379
The minor spliceosome is evolutionarily conserved in higher eukaryotes, but its biological significance remains poorly understood. By precise CRISPR/Cas9-mediated disruption of the U12 and U6atac snRNAs, this study reports that a defective minor spliceosome is responsible for spinal muscular atrophy (SMA) associated phenotypes in Drosophila. Using a newly developed bioinformatic approach, a large set of minor spliceosome-sensitive splicing events was identified and it was demonstrated that three sensitive intron-containing neural genes, Pcyt2, Zmynd10, and Fas3, directly contribute to disease development as evidenced by the ability of their cDNAs to rescue the SMA-associated phenotypes in muscle development, neuromuscular junctions, and locomotion. Interestingly, many splice sites in sensitive introns are recognizable by both minor and major spliceosomes, suggesting a new mechanism of splicing regulation through competition between minor and major spliceosomes. These findings reveal a vital contribution of the minor spliceosome to SMA and to regulated splicing in animals.
Lee, P. T., Lievens, J. C., Wang, S. M., Chuang, J. Y., Khalil, B., Wu, H. E., Chang, W. C., Maurice, T. and Su, T. P. (2020). Sigma-1 receptor chaperones rescue nucleocytoplasmic transport deficit seen in cellular and Drosophila ALS/FTD models. Nat Commun 11(1): 5580. PubMed ID: 33149115
In a subgroup of patients with amyotrophic lateral sclerosis (ALS)/Frontotemporal dementia (FTD), the (G4C2)-RNA repeat expansion from C9orf72 chromosome binds to the Ran-activating protein (RanGAP) at the nuclear pore, resulting in nucleocytoplasmic transport deficit and accumulation of Ran in the cytosol. This study found that the sigma-1 receptor (Sig-1R), a molecular chaperone, reverses the pathological effects of (G4C2)-RNA repeats in cell lines and in Drosophila. The Sig-1R colocalizes with RanGAP and nuclear pore proteins (Nups) and stabilizes the latter. Interestingly, Sig-1Rs directly bind (G4C2)-RNA repeats. Overexpression of Sig-1Rs rescues, whereas the Sig-1R knockout exacerbates, the (G4C2)-RNA repeats-induced aberrant cytoplasmic accumulation of Ran. In Drosophila, Sig-1R (but not the Sig-1R-E102Q mutant) overexpression reverses eye necrosis, climbing deficit, and firing discharge caused by (G4C2)-RNA repeats. These results on a molecular chaperone at the nuclear pore suggest that Sig-1Rs may benefit patients with C9orf72 ALS/FTD by chaperoning the nuclear pore assembly and sponging away deleterious (G4C2)-RNA repeats.

Friday, December 4th - Methods

Xu, X. S., Bulger, E. A., Gantz, V. M., Klanseck, C., Heimler, S. R., Auradkar, A., Bennett, J. B., Miller, L. A., Leahy, S., Juste, S. S., Buchman, A., Akbari, O. S., Marshall, J. M. and Bier, E. (2020). Active Genetic Neutralizing Elements for Halting or Deleting Gene Drives. Mol Cell. PubMed ID: 32949493
CRISPR-Cas9-based gene drive systems possess the inherent capacity to spread progressively throughout target populations. This study describes two self-copying (or active) guide RNA-only genetic elements, called e-CHACRs and ERACRs. These elements use Cas9 produced in trans by a gene drive either to inactivate the cas9 transgene (e-CHACRs) or to delete and replace the gene drive (ERACRs). e-CHACRs can be inserted at various genomic locations and carry two or more gRNAs, the first copying the e-CHACR and the second mutating and inactivating the cas9 transgene. Alternatively, ERACRs are inserted at the same genomic location as a gene drive, carrying two gRNAs that cut on either side of the gene drive to excise it. e-CHACRs efficiently inactivate Cas9 and can drive to completion in cage experiments. Similarly, ERACRs, particularly those carrying a recoded cDNA-restoring endogenous gene activity, can drive reliably to fully replace a gene drive. The strengths of these two systems are compared.
Liu, H., Pizzano, S., Li, R., Zhao, W., Veling, M. W., Hu, Y., Yang, L. and Ye, B. (2020). isoTarget: A Genetic Method for Analyzing the Functional Diversity of Splicing Isoforms In Vivo. Cell Rep 33(6): 108361. PubMed ID: 33176150
Protein isoforms generated by alternative splicing contribute to proteome diversity. Because of the lack of effective techniques, the isoform-specific function, expression, localization, and signaling of endogenous proteins are unknown for most genes. This paper reports a genetic method, isoTarget, for multi-purpose studies of targeted isoforms in select cells. Applying isoTarget to two isoforms of Drosophila Dscam, Dscam[TM1] and [TM2], it was found that, in neurons, endogenous Dscam[TM1] is in dendrites, whereas Dscam[TM2] is in both dendrites and axons. The difference in subcellular localization, rather than biochemical properties, leads to the two isoforms' functional differences. Moreover, the subcellular enrichment of functional partners was shown to result in a DLK/Wallenda-Dscam[TM2]-Dock signaling cascade in axons. IsoTarget was further applied to study two isoforms of a GABA receptor to demonstrate its general applicability. isoTarget is an effective technique for studying how alternative splicing enhances proteome complexity.
Xia, B., Amador, G., Viswanatha, R., Zirin, J., Mohr, S. E. and Perrimon, N. (2020). CRISPR-based engineering of gene knockout cells by homology-directed insertion in polyploid Drosophila S2R+ cells. Nat Protoc 15(10): 3478-3498. PubMed ID: 32958931
Precise and efficient genome modifications provide powerful tools for biological studies. Previous CRISPR gene knockout methods in cell lines have relied on frameshifts caused by stochastic insertion/deletion in all alleles. However, this method is inefficient for genes with high copy number due to polyploidy or gene amplification because frameshifts in all alleles can be difficult to generate and detect. This paper describes a homology-directed insertion method to knockout genes in the polyploid Drosophila S2R+ cell line. This protocol allows generation of homozygous mutant cell lines using an insertion cassette which autocatalytically generates insertion mutations in all alleles. Knockout cells generated using this method can be directly identified by PCR without a need for DNA sequencing. This protocol takes 2-3 months and can be applied to other polyploid cell lines or high-copy-number genes.
Ceolin, S., Hanf, M., Bozek, M., Storti, A. E., Gompel, N., Unnerstall, U., Jung, C. and Gaul, U. (2020). A sensitive mNeonGreen reporter system to measure transcriptional dynamics in Drosophila development. Commun Biol 3(1): 663. PubMed ID: 33184447
The gene regulatory network governing anterior-posterior axis formation in Drosophila is a well-established paradigm to study transcription in developmental biology. The rapid temporal dynamics of gene expression during early stages of development, however, are difficult to track with standard techniques. This study optimized the bright and fast-maturing fluorescent protein mNeonGreen as a real-time, quantitative reporter of enhancer expression. Enhancer activity is derived from the reporter fluorescence dynamics with high spatial and temporal resolution, using a robust reconstruction algorithm. By comparing these results with data obtained with the established MS2-MCP system, the higher detection sensitivity of this reporter is demonstrated. The reporter activity was used to quantify the activity of variants of a simple synthetic enhancer, and observe increased activity upon reduction of enhancer-promoter distance or addition of binding sites for the pioneer transcription factor Zelda. This reporter system constitutes a powerful tool to study spatio-temporal gene expression dynamics in live embryos.
Huynh, N., Depner, N., Larson, R. and King-Jones, K. (2020). A versatile toolkit for CRISPR-Cas13-based RNA manipulation in Drosophila. Genome Biol 21(1): 279. PubMed ID: 33203452
Advances in CRISPR technology have immensely improved the ability to manipulate nucleic acids, and the recent discovery of the RNA-targeting endonuclease Cas13 adds even further functionality. This study shows that Cas13 works efficiently in Drosophila, both ex vivo and in vivo. 44 different Cas13 variants were tested to identify enzymes with the best overall performance; Cas13 was able to target endogenous Drosophila transcripts in vivo with high efficiency and specificity. Cas13 applications were developed to edit mRNAs and target mitochondrial transcripts. This vector collection represents a versatile tool collection to manipulate gene expression at the post-transcriptional level.
Cachero, S., Gkantia, M., Bates, A. S., Frechter, S., Blackie, L., McCarthy, A., Sutcliffe, B., Strano, A., Aso, Y. and Jefferis, G. (2020). BAcTrace, a tool for retrograde tracing of neuronal circuits in Drosophila. Nat Methods. PubMed ID: 33139893
Animal behavior is encoded in neuronal circuits in the brain. To elucidate the function of these circuits, it is necessary to identify, record from and manipulate networks of connected neurons. This paper presents BAcTrace (Botulinum-Activated Tracer), a genetically encoded, retrograde, transsynaptic labeling system. BAcTrace is based on Clostridium botulinum neurotoxin A, Botox, which was engineered to travel retrogradely between neurons to activate an otherwise silent transcription factor. BAcTrace was validated at three neuronal connections in the Drosophila olfactory system. BAcTrace-mediated labeling allows electrophysiological recording of connected neurons. Finally, in a challenging circuit with highly divergent connections, BAcTrace correctly identified 12 of 16 connections that were previously observed by electron microscopy.

Thursday, December 3rd - Adult Physiology

Liao, S., Amcoff, M. and Nassel, D. R. (2020). Impact of high-fat diet on lifespan, metabolism, fecundity and behavioral senescence in Drosophila. Insect Biochem Mol Biol: 103495. PubMed ID: 33171202
Excess consumption of high-fat diet (HFD) is likely to result in obesity and increases the predisposition to associated health disorders. Drosophila melanogaster has emerged as an important model to study the effects of HFD on metabolism, gut function, behavior, and ageing. In this study, the effects of HFD on physiology and behavior of female flies was investigated at different time-points over several weeks. HFD was found to decrease lifespan, and also with age leads to accelerated decline of climbing ability in both virgins and mated flies. In virgins HFD also increased sleep fragmentation with age. Furthermore, long-term exposure to HFD results in elevated adipokinetic hormone (AKH) transcript levels and an enlarged crop with increased lipid stores. No long-term effects of HFD were detected on body mass, or levels of triacylglycerides (TAG), glycogen or glucose, although fecundity was diminished. However, one week of HFD resulted in decreased body mass and elevated TAG levels in mated flies. Finally, this study investigated the role of AKH in regulating effects of HFD during aging. Both with normal diet (ND) and HFD, Akh mutant flies displayed increased longevity compared to control flies. However, both mutants and controls showed shortened lifespan on HFD compared to ND. In flies exposed to ND, fecundity is decreased in Akh mutants compared to controls after one week, but increased after three weeks. However, HFD leads to a similar decrease in fecundity in both genotypes after both exposure times. Thus, long-term exposure to HFD increases AKH signaling, impairs lifespan and fecundity and augments age-related behavioral senescence.
Langmuller, A. M., Nolte, V., Galagedara, R., Poupardin, R., Dolezal, M. and Schlotterer, C. (2020). Fitness effects for Ace insecticide resistance mutations are determined by ambient temperature. BMC Biol 18(1): 157. PubMed ID: 33121485
Insect pest control programs often use periods of insecticide treatment with intermittent breaks, to prevent fixing of mutations conferring insecticide resistance. Such mutations are typically costly in an insecticide-free environment, and their frequency is determined by the balance between insecticide treatment and cost of resistance. Ace, a key gene in neuronal signaling, is a prominent target of many insecticides and across several species, three amino acid replacements (I161V, G265A, and F330Y) provide resistance against several insecticides. Because temperature disturbs neuronal signaling homeostasis, it was reasoned that the cost of insecticide resistance could be modulated by ambient temperature. Experimental evolution of a natural Drosophila simulans population at hot and cold temperature regimes uncovered a surprisingly strong effect of ambient temperature. In the cold temperature regime, the resistance mutations were strongly counter selected (s = - 0.055), but in a hot environment, the fitness costs of resistance mutations were reduced by almost 50% (s = - 0.031). This unexpected observation is attributed to the advantage of the reduced enzymatic activity of resistance mutations in hot environments. This study shows that fitness costs of insecticide resistance genes are temperature-dependent and suggest that the duration of insecticide-free periods need to be adjusted for different climatic regions to reflect these costs. It is suggested that such environment-dependent fitness effects may be more common than previously assumed and pose a major challenge for modeling climate change.
Li, N., Stanewsky, R., Popay, T., Warman, G. and Cheeseman, J. (2020). The Effect of General Anaesthesia on Circadian Rhythms in Behaviour and Clock Gene Expression of Drosophila melanogaster. Clocks Sleep 2(4): 434-441. PubMed ID: 33113932
General anaesthesia (GA) is implicated as a cause of postoperative sleep disruption and fatigue with part of the disturbance being attributed to a shift of the circadian clock. In this study, Drosophila melanogaster was used as a model to determine how Isoflurane affects the circadian clock at the behavioural and molecular levels. The response of the clock was measured at both of these levels caused by different durations and different concentrations of Isoflurane at circadian time 4 (CT4). Once characterized, the duration and concentration constants (at 2% in air for 6 h) were held and the phase responses were calculated over the entire circadian cycle in both activity and period expression. Phase advances in behaviour were observed during the subjective day, whereas phase delays were associated with subjective night time GA interventions. The corresponding pattern of gene expression preceded the behavioural pattern by approximately four hours. The implications of this effect for clinical and research practice are discussed.
Miyaki, T., Kawasaki, Y., Matsumoto, A., Kakuta, S., Sakai, T. and Ichimura, K. (2020). Nephrocytes are part of the spectrum of filtration epithelial diversity. Cell Tissue Res. PubMed ID: 33191456
The excretory system produces urine by ultrafiltration via a filtration epithelium. Podocytes are widely found as filtration epithelial cells in eucoelomates. In some animal taxa, including insects and crustaceans, nephrocytes serve to separate toxic substances from the body fluid, in addition to podocytes. Drosophila nephrocytes have been recently utilized as a model system to study podocyte function and disease. Pericardial nephrocytes, for example, develop from the cardiogenic mesoderm by the late embryo/early larval stage; they arrange into two rows of 20–25 flanking each side of the heart from the first to the sixth segment. However, functionality and cellular architecture are strikingly different between Drosophila nephrocytes and eucoelomate podocytes, and the phylogenetic relationship between these cells remains enigmatic. Using focused-ion beam-scanning electron microscopy (FIB-SEM) tomography, this study revealed three-dimensional architecture of decapod nephrocytes with unprecedented accuracy-they filled an enormous gap, which can be called "missing link," in the evolutionary diversity of podocytes and nephrocytes. Thus, it is concluded that nephrocytes are part of the spectrum of filtration epithelial diversity in animal phylogeny.
Ito, D., Kawamura, H., Oikawa, A., Ihara, Y., Shibata, T., Nakamura, N., Asano, T., Kawabata, S. I., Suzuki, T. and Masuda, S. (2020). ppGpp functions as an alarmone in metazoa. Commun Biol 3(1): 671. PubMed ID: 33188280
Guanosine 3',5'-bis(pyrophosphate) (ppGpp) functions as a second messenger in bacteria to adjust their physiology in response to environmental changes. In recent years, the ppGpp-specific hydrolase, metazoan SpoT homolog-1 (Mesh1), was shown to have important roles for growth under nutrient deficiency in Drosophila melanogaster. Curiously, however, ppGpp has never been detected in animal cells, and therefore the physiological relevance of this molecule, if any, in metazoans has not been established. This study report the detection of ppGpp in Drosophila and human cells and demonstrates that ppGpp accumulation induces metabolic changes, cell death, and eventually lethality in Drosophila. These results provide the evidence of the existence and function of the ppGpp-dependent stringent response in animals.
Hsu, I. U., Linsley, J. W., Reid, L. E., Hume, R. I., Leflein, A. and Kuwada, J. Y. (2020). Dstac Regulates Excitation-Contraction Coupling in Drosophila Body Wall Muscles. Front Physiol 11: 573723. PubMed ID: 33123029
Stac3 regulates excitation-contraction coupling (EC coupling) in vertebrate skeletal muscles by regulating the L-type voltage-gated calcium channel (Ca(v) channel). Recently a stac-like gene, Dstac, was identified in Drosophila and found to be expressed by both a subset of neurons and muscles. This study shows that Dstac and Dmca1D, the Drosophila L-type Ca(v) channel, are necessary for normal locomotion by larvae. Immunolabeling with specific antibodies against Dstac and Dmca1D found that Dstac and Dmca1D are expressed by larval body-wall muscles. Furthermore, Ca(2+) imaging of muscles of Dstac and Dmca1D deficient larvae found that Dstac and Dmca1D are required for excitation-contraction coupling. Finally, Dstac appears to be required for normal expression levels of Dmca1D in body-wall muscles. These results suggest that Dstac regulates Dmca1D during EC coupling and thus muscle contraction.

Wednesday, December 2nd - Signaling

Kolonko, M., Bystranowska, D., Taube, M., Kozak, M., Bostock, M., Popowicz, G., Ożyhar, A. and Greb-Markiewicz, B. (2020). The intrinsically disordered region of GCE protein adopts a more fixed structure by interacting with the LBD of the nuclear receptor FTZ-F1. Cell Commun Signal 18(1): 180. PubMed ID: 33153474
The Drosophila melanogaster Germ cell-expressed protein (GCE) is a paralog of the juvenile hormone (JH) receptor - Methoprene tolerant protein (MET). Both proteins mediate JH function, preventing precocious differentiation during D. melanogaster development. Despite that GCE and MET are often referred to as equivalent JH receptors, their functions are not fully redundant and show tissue specificity. Both proteins belong to the family of bHLH-PAS transcription factors. The similarity of their primary structure is limited to defined bHLH and PAS domains, while their long C-terminal fragments (GCEC, METC) show significant differences and are expected to determine differences in GCE and MET protein activities. This paper presents the structural characterization of GCEC as a coil-like intrinsically disordered protein (IDP) with highly elongated and asymmetric conformation. In comparison to previously characterized METC, GCEC is less compacted, contains more molecular recognition elements (MoREs) and exhibits a higher propensity for induced folding. The NMR shifts perturbation experiment and pull-down assay clearly demonstrated that the GCEC fragment is sufficient to form an interaction interface with the ligand binding domain (LBD) of the nuclear receptor Fushi Tarazu factor-1 (FTZ-F1). Significantly, these interactions can force GCEC to adopt more fixed structure that can modulate the activity, structure and functions of the full-length receptor. The discussed relation of protein functionality with the structural data of inherently disordered GCEC fragment is a novel look at this protein and contributes to a better understanding of the molecular basis of the functions of the C-terminal fragments of the bHLH-PAS family.
Han, J. H., Kim, Y. and Cho, K. O. (2020). Exosomal arrow (Arr)/lipoprotein receptor protein 6 (LRP6) in Drosophila melanogaster increases the extracellular level of Sol narae (Sona) in a Wnt-independent manner. Cell Death Dis 11(11): 944. PubMed ID: 33139721
Wg/Wnt as a signaling protein binds Frizzled (Fz) and Arrow (Arr), two Wg co-receptors essential for Wg signaling for cell proliferation, differentiation, and cell survival. Arr has a long extracellular region, a single transmembrane domain and an intracellular region. This study reports that a new arrm7 mutant is identified in a genetic screen as a suppressor of lethality induced by overexpression of Sol narae (Sona), a secreted metalloprotease in ADAMTS family involved in Wg signaling. arrm7 allele has a premature stop codon, which encodes Arrm7 protein missing the intracellular region. arrm7 clones show cell death phenotype and overexpression of Arrm7 protein also induces cell death. Levels of extracellular Sona were decreased in both arrm7 and arr2 null clones, demonstrating that Arr increases the level of extracellular Sona. Indeed, Arr but not Arrm7, increased levels of Sona in cytoplasm and exosome fraction by inhibiting the lysosomal degradation pathway. Interestingly, Arr itself was identified in the exosome fraction, demonstrating that Arr is secreted to extracellular space. When Sona-expressing S2 cells were treated with exosomal Arr, the extracellular level of active Sona was increased. These results show that exosomal Arr dictates Sona-expressing cells to increase the level of extracellular Sona. This new function of Arr occurred in the absence of Wg because S2 cells do not express Wg. It is proposed that Arr plays two distinct roles, one as an exosomal protein to increase the level of extracellular Sona in a Wnt-independent manner and the other as a Wg co-receptor in a Wnt-dependent manner.
Kula-Eversole, E., Lee, D. H., Samba, I., Yildirim, E., Levine, D. C., Hong, H. K., Lear, B. C., Bass, J., Rosbash, M. and Allada, R. (2020). Phosphatase of Regenerating Liver-1 Selectively Times Circadian Behavior in Darkness via Function in PDF Neurons and Dephosphorylation of TIMELESS. Curr Biol. PubMed ID: 33157022
The timing of behavior under natural light-dark conditions is a function of circadian clocks and photic input pathways, but a mechanistic understanding of how these pathways collaborate in animals is lacking. This study demonstrates in Drosophila that the Phosphatase of Regenerating Liver-1 (PRL-1) sets period length and behavioral phase gated by photic signals. PRL-1 knockdown in PDF clock neurons dramatically lengthens circadian period. PRL-1 mutants exhibit allele-specific interactions with the light- and clock-regulated gene timeless (tim). Moreover, this study shows that PRL-1 promotes TIM accumulation and dephosphorylation. Interestingly, the PRL-1 mutant period lengthening is suppressed in constant light, and PRL-1 mutants display a delayed phase under short, but not long, photoperiod conditions. Thus, these studies reveal that PRL-1-dependent dephosphorylation of TIM is a core mechanism of the clock that sets period length and phase in darkness, enabling the behavioral adjustment to change day-night cycles.
Girard, V., Goubard, V., Querenet, M., Seugnet, L., Pays, L., Nataf, S., Dufourd, E., Cluet, D., Mollereau, B. and Davoust, N. (2020). Spen modulates lipid droplet content in adult Drosophila glial cells and protects against paraquat toxicity. Sci Rep 10(1): 20023. PubMed ID: 33208773
Glial cells are early sensors of neuronal injury and can store lipids in lipid droplets under oxidative stress conditions. This study investigated the functions of the RNA-binding protein, SPEN/SHARP, in the context of Parkinson's disease (PD). Using a data-mining approach, it was found that SPEN/SHARP is one of many astrocyte-expressed genes that are significantly differentially expressed in the substantia nigra of PD patients compared with control subjects. Interestingly, the differentially expressed genes are enriched in lipid metabolism-associated genes. In a Drosophila model of PD, it was observed that flies carrying a loss-of-function allele of the ortholog split-ends (spen) or with glial cell-specific, but not neuronal-specific, spen knockdown were more sensitive to paraquat intoxication, indicating a protective role for Spen in glial cells. It was also found that Spen is a positive regulator of Notch signaling in adult Drosophila glial cells. Moreover, Spen was required to limit abnormal accumulation of lipid droplets in glial cells in a manner independent of its regulation of Notch signaling. Taken together, these results demonstrate that Spen regulates lipid metabolism and storage in glial cells and contributes to glial cell-mediated neuroprotection.
Ingaramo, M. C., Sanchez, J. A., Perrimon, N. and Dekanty, A. (2020). Fat Body p53 Regulates Systemic Insulin Signaling and Autophagy under Nutrient Stress via Drosophila Upd2 Repression. Cell Rep 33(4): 108321. PubMed ID: 33113367
The tumor suppressor p53 regulates multiple metabolic pathways at the cellular level. However, its role in the context of a whole animal response to metabolic stress is poorly understood. Using Drosophila, this study shows that AMP-activated protein kinase (AMPK)-dependent Dmp53 activation is critical for sensing nutrient stress, maintaining metabolic homeostasis, and extending organismal survival. Under both nutrient deprivation and high-sugar diet, Dmp53 activation in the fat body represses expression of the Drosophila Leptin analog, Unpaired-2 (Upd2), which remotely controls Dilp2 secretion in insulin-producing cells. In starved Dmp53-depleted animals, elevated Upd2 expression in adipose cells and activation of Upd2 receptor Domeless in the brain result in sustained Dilp2 circulating levels and impaired autophagy induction at a systemic level, thereby reducing nutrient stress survival. These findings demonstrate an essential role for the AMPK-Dmp53 axis in nutrient stress responses and expand the concept that adipose tissue acts as a sensing organ that orchestrates systemic adaptation to nutrient status.
Kodra, A., de la Cova, C., Gerhold, A. R. and Johnston, L. A. (2020). Widely used mutants of eiger, encoding the Drosophila Tumor Necrosis factor, carry additional mutations in the NimrodC1 phagocytosis receptor. G3 (Bethesda). PubMed ID: 33127847
The process of apoptosis in epithelia involves activation of caspases, delamination of cells, and degradation of cellular components. Corpses and cellular debris are then rapidly cleared from the tissue by phagocytic blood cells. In studies of the Drosophila TNF, Eiger (Egr) and cell death in wing imaginal discs, the epithelial primordia of fly wings, it was noticed that dying cells appeared to transiently accumulate in egr3 mutant wing discs, raising the possibility that their phagocytic engulfment by hemocytes was impaired. Further investigation revealed that lymph glands and circulating hemocytes from egr3 mutant larvae were completely devoid of NimC1 staining, a marker of phagocytic hemocytes. Genome sequencing uncovered mutations in the NimC1 coding region that are predicted to truncate the NimC1 protein before its transmembrane domain, and provide an explanation for the lack of NimC staining. This work demonstrates the presence of these NimC1 mutations in the widely used egr3 mutant, its sister allele, egr1 , and its parental strain, Regg1(GS9830). As the egr3 and egr1 alleles have been used in numerous studies of immunity and cell death, it may be advisable to re-evaluate their associated phenotypes.

Tuesday, December 1st - RNA and Transposons

Rathore, O. S., Silva, R. D., Ascensao-Ferreira, M., Matos, R., Carvalho, C., Marques, B., Tiago, M. N., Prudencio, P., Andrade, R. P., Roignant, J. Y., Barbosa-Morais, N. L. and Martinho, R. G. (2020). NineTeen Complex-subunit Salsa is required for efficient splicing of a subset of introns and dorsal-ventral patterning. RNA. PubMed ID: 32963109
The NineTeen Complex (NTC), also known as Pre-mRNA-processing factor 19 (Prp19) complex, regulates distinct spliceosome conformational changes necessary for splicing. During Drosophila midblastula transition, splicing is particularly sensitive to mutations in NTC-subunit Fandango, which suggests differential requirements of NTC during development. This study shows that NTC-subunit Salsa, the Drosophila orthologue of human RNA helicase Aquarius (CG31368), is rate-limiting for splicing of a subset of small first introns during oogenesis, including the first intron of gurken. Germ line depletion of Salsa and splice site mutations within gurken first intron both impair adult female fertility and oocyte dorsal-ventral patterning due to an abnormal expression of Gurken. Supporting causality, the fertility and dorsal-ventral patterning defects observed after Salsa depletion could be suppressed by the expression of a gurken construct without its first intron. Altogether these results suggest that one of the key rate-limiting functions of Salsa during oogenesis is to ensure the correct expression and efficient splicing of the first intron of gurken mRNA. Retention of gurken first intron compromises the function of this gene most likely because it undermines the correct structure and function of the transcript 5'UTR.
Cha, I. J., Lee, D., Park, S. S., Chung, C. G., Kim, S. Y., Jo, M. G., Kim, S. Y., Lee, B. H., Lee, Y. S. and Lee, S. B. (2020). Ataxin-2 Dysregulation Triggers a Compensatory Fragile X Mental Retardation Protein Decrease in Drosophila C4da Neurons. Mol Cells 43(10): 870-879. PubMed ID: 33115979
Dendrites require precise and timely delivery of protein substrates to distal areas to ensure the correct morphology and function of neurons. Many of these protein substrates are supplied in the form of ribonucleoprotein (RNP) complex consisting of RNA-binding proteins (RBPs) and mRNAs, which are subsequently translated in distal dendritic areas.This study investigated how Drosophila sensory neurons respond to the dysregulation of a disease-associated RBP, Ataxin-2 (ATX2), which leads to dendritic defects. ATX2 plays a crucial role in spacing dendritic branches for the optimal dendritic receptive fields in Drosophila class IV dendritic arborization (C4da) neurons, where both expression level and subcellular location of ATX2 contribute significantly to this effect. Translational upregulation through the expression of eukaryotic translation initiation factor 4E (eIF4E) further enhanced the ATX2-induced dendritic phenotypes. Additionally, this study found that the expression level of another disease-associated RBP, fragile X mental retardation protein (FMRP), decreased in both cell bodies and dendrites when neurons were faced with aberrant upregulation of ATX2. Finally, this study revealed that the PAM2 motif of ATX2, which mediates its interaction with poly(A)-binding protein (PABP), is potentially necessary for the decrease of FMRP in certain neuronal stress conditions. Collectively, these data suggest that dysregulation of RBPs triggers a compensatory regulation of other functionally-overlapping RBPs to minimize RBP dysregulation-associated aberrations that hinder neuronal homeostasis in dendrites.
Guo, X., Sun, Y., Azad, T., Janse van Rensburg, H. J., Luo, J., Yang, S., Liu, P., Lv, Z., Zhan, M., Lu, L., Zhou, Y., Ma, X., Zhang, X., Yang, X. and Xue, L. (2020). Rox8 promotes microRNA-dependent yki messenger RNA decay. Proc Natl Acad Sci U S A. PubMed ID: 33203680
The Hippo pathway is an evolutionarily conserved regulator of organ growth and tumorigenesis. In Drosophila, oncogenic Ras(V12) cooperates with loss-of-cell polarity to promote Hippo pathway-dependent tumor growth. To identify additional factors that modulate this signaling, a genetic screen was performed utilizing the Drosophila Ras (V12) /lgl (-/-) in vivo tumor model and identified Rox8, a RNA-binding protein (RBP), as a positive regulator of the Hippo pathway. Rox8 overexpression suppresses whereas Rox8 depletion potentiates Hippo-dependent tissue overgrowth, accompanied by altered Yki protein level and target gene expression. Mechanistically, Rox8 directly binds to a target site located in the yki 3' UTR, recruits and stabilizes the targeting of miR-8-loaded RISC, which accelerates the decay of yki messenger RNA (mRNA). Moreover, TIAR, the human ortholog of Rox8, is able to promote the degradation of yki mRNA when introduced into Drosophila and destabilizes YAP mRNA in human cells. Thus,this study provides in vivo evidence that the Hippo pathway is posttranscriptionally regulated by the collaborative action of RBP and microRNA (miRNA), which may provide an approach for modulating Hippo pathway-mediated tumorigenesis.
Treiber, C. D. and Waddell, S. (2020). Transposon expression in the Drosophila brain is driven by neighboring genes and diversifies the neural transcriptome. Genome Res. PubMed ID: 32973040
Somatic transposon expression in neural tissue is commonly considered as a measure of mobilization and has therefore been linked to neuropathology and organismal individuality. This study combined genome sequencing data with single-cell mRNA sequencing of the same, inbred fly strain to map transposon expression in the Drosophila midbrain and found that transposon expression patterns are highly stereotyped. Every detected transposon is resident in at least one cellular gene with a matching expression pattern. Bulk RNA sequencing from fly heads of the same strain revealed that coexpression is a physical link in the form of abundant chimeric transposon-gene mRNAs. 264 genes were identified where transposons introduce cryptic splice sites into the nascent transcript and thereby significantly expand the neural transcript repertoire. Some genes exclusively produce chimeric mRNAs with transposon sequence and on average 11.6% of the mRNAs produced from a given gene are chimeric. Conversely, most transposon-containing transcripts are chimeric, which suggests that somatic expression of these transposons is largely driven by cellular genes. It is proposed that chimeric mRNAs produced by alternative splicing into polymorphic transposons, rather than transposon mobilization, may contribute to functional differences between individual cells and animals.
Wei, L., Lee, S., Majumdar, S., Zhang, B., Sanfilippo, P., Joseph, B., Miura, P., Soller, M. and Lai, E. C. (2020). Overlapping Activities of ELAV/Hu Family RNA Binding Proteins Specify the Extended Neuronal 3' UTR Landscape in Drosophila. Mol Cell 80(1): 140-155. PubMed ID: 33007254
The tissue-specific deployment of highly extended neural 3' UTR isoforms, generated by alternative polyadenylation (APA), is a broad and conserved feature of metazoan genomes. However, the factors and mechanisms that control neural APA isoforms are not well understood. This study shows that three ELAV/Hu RNA binding proteins (Elav, Rbp9, and Fne) have similar capacities to induce a lengthened 3' UTR landscape in an ectopic setting. These factors promote accumulation of chromatin-associated, 3' UTR-extended, nascent transcripts, through inhibition of proximal polyadenylation site (PAS) usage. Notably, Elav represses an unannotated splice isoform of fne, switching the normally cytoplasmic Fne toward the nucleus in elav mutants. This study used genomic profiling to reveal strong and broad loss of neural APA in elav/fne double mutant CNS, the first genetic background to largely abrogate this distinct APA signature. Overall, this study demonstrates how regulatory interplay and functionally overlapping activities of neural ELAV/Hu RBPs drives the neural APA landscape.
Gao, L., Chang, S., Xia, W., Wang, X., Zhang, C., Cheng, L., Liu, X., Chen, L., Shi, Q., Huang, J., Xu, E. Y. and Shan, G. (2020). Circular RNAs from BOULE play conserved roles in protection against stress-induced fertility decline. Sci Adv 6(46). PubMed ID: 33177084
Circular RNAs (circRNAs) are a large family of newly identified transcripts, and their physiological roles and evolutionary significance require further characterization. This study identified circRNAs generated from a conserved reproductive gene, Boule, in species from Drosophila to humans. Flies missing circular Boule (circBoule) RNAs display decreased male fertility, and sperm of circBoule knockout mice exhibit decreased fertilization capacity, when under heat stress conditions. During spermatogenesis, fly circBoule RNAs interact with heat shock proteins (HSPs) Hsc4 and Hsp60C, and mouse circBoule RNAs in sperm interact with HSPA2. circBoule RNAs regulate levels of HSPs by promoting their ubiquitination. The interaction between HSPA2 and circBoule RNAs is conserved in human sperm, and lower levels of the human circBoule RNAs circEx3-6 and circEx2-7 are found in asthenozoospermic sperm. These findings reveal conserved physiological functions of circBoule RNAs in metazoans and suggest that specific circRNAs may be critical modulators of male reproductive function against stresses in animals.
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