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Wednesday, December 8th - Evolution

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Reddiex, A. J. and Chenoweth, S. F. (2021). Integrating genomics and multivariate evolutionary quantitative genetics: a case study of constraints on sexual selection in Drosophila serrata. Proc Biol Sci 288(1960): 20211785. PubMed ID: 34641732
Summary:
In evolutionary quantitative genetics, the genetic variance-covariance matrix, G, and the vector of directional selection gradients, β, are key parameters for predicting multivariate selection responses and genetic constraints. Historically, investigations of G and β have not overlapped with those dissecting the genetic basis of quantitative traits. Thus, it remains unknown whether these parameters reflect pleiotropic effects at individual loci. This study integrates multivariate genome-wide association study (GWAS) with G and β estimation in a well-studied system of multivariate constraint: sexual selection on male cuticular hydrocarbons (CHCs) in Drosophila serrata. In a panel of wild-derived re-sequenced lines, this study augments genome-based restricted maximum likelihood to estimate G alongside multivariate single nucleotide polymorphism (SNP) effects, detecting 532 significant associations from 1 652 276 SNPs. Constraint was evident, with β lying in a direction of G with low evolvability. Interestingly, minor frequency alleles typically increased male CHC-attractiveness suggesting opposing natural selection on β. SNP effects were significantly misaligned with the major eigenvector of G, gmax, but well aligned to the second and third eigenvectors g2 and g3. Potential factors leading to these varied results including multivariate stabilizing selection and mutational bias. This framework may be useful as researchers increasingly access genomic methods to study multivariate selection responses in wild populations (Reddiex, 2021).
Marin, P., Jaquet, A., Picarle, J., Fablet, M., Merel, V., Delignette-Muller, M. L., Ferrarini, M. G., Gibert, P. and Vieira, C. (2021). Phenotypic and Transcriptomic Responses to Stress Differ According to Population Geography in an Invasive Species. Genome Biol Evol 13(9). PubMed ID: 34505904
Summary:
Adaptation to rapid environmental changes must occur within a short-time scale. In this context, studies of invasive species may provide insights into the underlying mechanisms of rapid adaptation as these species have repeatedly encountered and adapted to novel environmental conditions. This study investigated how invasive and noninvasive genotypes of Drosophila suzukii deal with oxidative stress at the phenotypic and molecular levels. Also studied was the impact of transposable element (TE) insertions on the gene expression in response to stress. Results show that flies from invasive areas (France and the United States) live longer in natural conditions than the ones from native Japanese areas. As expected, lifespan for all genotypes was significantly reduced following exposure to paraquat, but this reduction varied among genotypes (genotype-by-environment interaction) with invasive genotypes appearing more affected by exposure than noninvasive ones. A transcriptomic analysis of genotypes upon paraquat treatment detected many genes differentially expressed (DE). Although a small core set of genes were DE in all genotypes following paraquat exposure, much of the response of each genotype was unique. Moreover, it was shown that TEs were not activated after oxidative stress and DE genes were significantly depleted of TEs. In conclusion, it is likely that transcriptomic changes are involved in the rapid adaptation to local environments. This study provides new evidence that in the decade since the invasion from Asia, the sampled genotypes in Europe and the United States of D. suzukii diverged from the ones from the native area regarding their phenotypic and genomic response to oxidative stress (Marin, 2021).
Gomez-Llano, M., Scott, E. and Svensson, E. I. (2021). The importance of pre- and postcopulatory sexual selection promoting adaptation to increasing temperatures. Curr Zool 67(3): 321-327. PubMed ID: 34616924
Summary:
Global temperatures are increasing rapidly affecting species globally. Understanding if and how different species can adapt fast enough to keep up with increasing temperatures is of vital importance. One mechanism that can accelerate adaptation and promote evolutionary rescue is sexual selection. Two different mechanisms by which sexual selection can facilitate adaptation are pre- and postcopulatory sexual selection. However, the relative effects of these different forms of sexual selection in promoting adaptation are unknown. This study presents the results from an experimental study in which fruit flies Drosophila melanogaster were exposed to either no mate choice or 1 of 2 different sexual selection regimes (pre- and postcopulatory sexual selection) for 6 generations, under different thermal regimes. Populations showed evidence of thermal adaptation under precopulatory sexual selection, but this effect was not detected in the postcopulatory sexual selection and the no choice mating regime. This study further demonstrates that sexual dimorphism decreased when flies evolved under increasing temperatures, consistent with recent theory predicting more sexually concordant selection under environmental stress. These results suggest an important role for precopulatory sexual selection in promoting thermal adaptation and evolutionary rescue (Gomez-Llano, 2021).
Harrison, B. R., Hoffman, J. M., Samuelson, A., Raftery, D. and Promislow, D. E. L. (2021). Modular Evolution of the Drosophila Metabolome. Mol Biol Evol. PubMed ID: 34662414
Summary:
Comparative phylogenetic studies offer a powerful approach to study the evolution of complex traits. While much effort has been devoted to the evolution of the genome and to organismal phenotypes, until now relatively little work has been done on the evolution of the metabolome, despite the fact that it is composed of the basic structural and functional building blocks of all organisms. This study explored variation in metabolite levels across 50 million years of evolution in the genus Drosophila, employing a common garden design to measure the metabolome within and among 11 species of Drosophila. This study found that both sex and age have dramatic and evolutionarily conserved effects on the metabolome. This study also found substantial evidence that many metabolite pairs covary after phylogenetic correction, and that such metabolome coevolution is modular. Some of these modules are enriched for specific biochemical pathways and show different evolutionary trajectories, with some showing signs of stabilizing selection. Both observations suggest that functional relationships may ultimately cause such modularity. These coevolutionary patterns also differ between sexes and are affected by age. This study also explored the relevance of modular evolution to fitness by associating modules with lifespan variation measured in the same common garden. Several modules associated with lifespan were found, particularly in the metabolome of older flies. Oxaloacetate levels in older females appear to coevolve with lifespan, and a lifespan-associated module in older females suggests that metabolic associations could underlie 50 million years of lifespan evolution (Harrison, 2021).
Keaney, T. A., Jones, T. M. and Holman, L. (2021). Sexual selection can partly explain low frequencies of Segregation Distorter alleles. Proc Biol Sci 288(1959): 20211190. PubMed ID: 34583584
Summary:
The Segregation Distorter (SD) allele found in Drosophila melanogaster distorts Mendelian inheritance in heterozygous males by causing developmental failure of non-SD spermatids, such that greater than 90% of the surviving sperm carry SD. This within-individual advantage should cause SD to fix, and yet SD is typically rare in wild populations. this study explores whether this paradox can be resolved by sexual selection, by testing if males carrying three different variants of SD suffer reduced pre- or post-copulatory reproductive success. This study finds that males carrying the SD allele are just as successful at securing matings as control males, but that one SD variant (SD-5) reduces sperm competitive ability and increases the likelihood of female remating. The results inform a theoretical model; It was discovered that sexual selection could limit SD to natural frequencies when sperm competitive ability and female remating rate equalled the values observed for SD-5. However, sexual selection was unable to explain natural frequencies of the SD allele when the model was parameterized with the values found for two other SD variants, indicating that sexual selection alone is unlikely to explain the rarity of SD (Keaney, 2021).
Jackson, B. and Charlesworth, B. (2021). Evidence for a force favoring GC over AT at short intronic sites in Drosophila simulans and Drosophila melanogaster. G3 (Bethesda) 11(9). PubMed ID: 34544137
Summary:
Population genetics studies often make use of a class of nucleotide site free from selective pressures, in order to make inferences about population size changes or natural selection at other sites. If such neutral sites can be identified, they offer the opportunity to avoid any confounding effects of selection. This study investigates evolution at putatively neutrally evolving short intronic sites in natural populations of Drosophila melanogaster and Drosophila simulans, in order to understand the properties of spontaneous mutations and the extent of GC-biased gene conversion in these species. Use of data on the genetics of natural populations is advantageous because it integrates information from large numbers of individuals over long timescales. In agreement with direct evidence from observations of spontaneous mutations in Drosophila was found, this study found a bias in the spectrum of mutations toward AT basepairs. In addition, a find that this bias is stronger in the D. melanogaster lineage than in the D. simulans lineage. The evidence for GC-biased gene conversion in Drosophila has been equivocal. This study provides evidence for a weak force favoring GC in both species, which is correlated with the GC content of introns and is stronger in D. simulans than in D. melanogaster (Jackson, 2021).

Tuesday, December 7th - Adult Development

Lam, G., Nam, H. J., Velentzas, P. D., Baehrecke, E. H. and Thummel, C. S. (2021). Drosophila E93 promotes adult development and suppresses larval responses to ecdysone during metamorphosis. Dev Biol. PubMed ID: 34648816.
Summary:
Pulses of the steroid hormone ecdysone act through transcriptional cascades to direct the major developmental transitions during the Drosophila life cycle. These include the prepupal ecdysone pulse, which occurs 10 h after pupariation and triggers the onset of adult morphogenesis and larval tissue destruction. E93 encodes a transcription factor that is specifically induced by the prepupal pulse of ecdysone, supporting a model proposed by earlier work that it specifies the onset of adult development. Although a number of studies have addressed these functions for E93, little is known about its roles in the salivary gland where the E93 locus was originally identified. This study shows that E93 is required for development through late pupal stages, with mutants displaying defects in adult differentiation and no detectable effect on the destruction of salivary glands. RNA-seq analysis demonstrates that E93 regulates genes involved in development and morphogenesis in the salivary glands, but has little effect on cell death gene expression. It was also shown that E93 is required to direct the proper timing of ecdysone-regulated gene expression in salivary glands, and that it suppresses earlier transcriptional programs that occur during larval and prepupal stages. These studies support the model that the stage-specific induction of E93 in late prepupae provides a critical signal that defines the end of larval development and the onset of adult differentiation.
Rand, D. A., Raju, A., Sa¡ez, M., Corson, F. and Siggia, E. D. (2021). Geometry of gene regulatory dynamics. Proc Natl Acad Sci U S A 118(38). PubMed ID: 34518231
Summary:
Embryonic development leads to the reproducible and ordered appearance of complexity from egg to adult. The successive differentiation of different cell types that elaborate this complexity results from the activity of gene networks and was likened by Waddington to a flow through a landscape in which valleys represent alternative fates. Geometric methods allow the formal representation of such landscapes and codify the types of behaviors that result from systems of differential equations. Results from Smale and coworkers imply that systems encompassing gene network models can be represented as potential gradients with a Riemann metric, justifying the Waddington metaphor. This study extends the representation to include parameter dependence and enumerate all three-way cellular decisions realizable by tuning at most two parameters, which can be generalized to include spatial coordinates in a tissue. All diagrams of cell states vs. model parameters are thereby enumerated. The study unifies a number of standard models for spatial pattern formation by expressing them in potential form (i.e., as topographic elevation). Turing systems appear nonpotential, yet in suitable variables the dynamics are low dimensional and potential. A time-independent embedding recovers the original variables. Lateral inhibition is described by a saddle point with many unstable directions. A model for the patterning of the Drosophila eye appears as relaxation in a bistable potential. Geometric reasoning provides intuitive dynamic models for development that are well adapted to fit time-lapse data (Rand, 2021).
Juarez-Carreno, S., Vallejo, D. M., Carranza-Valencia, J., Palomino-Schatzlein, M., Ramon-Canellas, P., Santoro, R., de Hartog, E., Ferres-Marco, D., Romero, A., Peterson, H. P., Ballesta-Illan, E., Pineda-Lucena, A., Dominguez, M. and Morante, J. (2021). Body-fat sensor triggers ribosome maturation in the steroidogenic gland to initiate sexual maturation in Drosophila. Cell Rep 37(2): 109830. PubMed ID: 34644570
Summary:
Fat stores are critical for reproductive success and may govern maturation initiation. This study reports signaling and sensing fat sufficiency for sexual maturation commitment requires the lipid carrier apolipophorin in fat cells and Sema1a in the neuroendocrine prothoracic gland (PG). Larvae lacking apolpp or Sema1a fail to initiate maturation despite accruing sufficient fat stores, and they continue gaining weight until death. Mechanistically, sensing peripheral body-fat levels via the apolipophorin/Sema1a axis regulates endocytosis, endoplasmic reticulum remodeling, and ribosomal maturation for the acquisition of the PG cells' high biosynthetic and secretory capacity. Downstream of apolipophorin/Sema1a, leptin-like upd2 triggers the cessation of feeding and initiates sexual maturation. Human Leptin in the insect PG substitutes for upd2, preventing obesity and triggering maturation downstream of Sema1a. Data shows how peripheral fat levels regulate the control of the maturation decision-making process via remodeling of endomembranes and ribosomal biogenesis in gland cells (Juarez, 2021).
Ostala, C. M., Esteban, N., Lapez-Varea, A. and de Celis, J. F. (2021). Functional requirements of protein kinases and phosphatases in the development of the Drosophila melanogaster wing. G3 (Bethesda). PubMed ID: 34599799
Summary:
Protein kinases and phosphatases constitute a large family of conserved enzymes that control a variety of biological processes by regulating the phosphorylation state of target proteins. They play fundamental regulatory roles during cell cycle progression and signaling, among other key aspects of multicellular development. The complement of protein kinases and phosphatases includes approximately 326 members in Drosophila, and they have been the subject of several functional screens searching for novel components of signaling pathways and regulators of cell division and survival. These approaches have been carried out mostly in cell cultures using RNA interference to evaluate the contribution of each protein in different functional assays, and have contributed significantly to assign specific roles to the corresponding genes. The results are described of an evaluation of the Drosophila complement of kinases and phosphatases using the wing as a system to identify their functional requirements in vivo. This study also describes the results of several modifying screens aiming to identify among the set of protein kinases and phosphatases additional components or regulators of the activities of the Epidermal Growth Factor and Insulin receptors signaling pathways (Ostala, 2021).
Ozsoy, E. D., Yilmaz, M., Patlar, B., Emecen, G., Durmaz, E., Magwire, M. M., Zhou, S., Huang, W., Anholt, R. R. H. and Mackay, T. F. C. (2021). Epistasis for head morphology in Drosophila melanogaster. G3 (Bethesda) 11(10). PubMed ID: 34568933
Summary:
Epistasis-gene-gene interaction-is common for mutations with large phenotypic effects in humans and model organisms. Epistasis impacts quantitative genetic models of speciation, response to natural and artificial selection, genetic mapping, and personalized medicine. However, the existence and magnitude of epistasis between alleles with small quantitative phenotypic effects are controversial and difficult to assess. This study used the Drosophila melanogaster Genetic Reference Panel of sequenced inbred lines to evaluate the magnitude of naturally occurring epistasis modifying the effects of mutations in jing and inv, two transcription factors that have subtle quantitative effects on head morphology as homozygotes. This study found significant epistasis for both mutations and performed single marker genome-wide association analyses to map candidate modifier variants and loci affecting head morphology. A subset of these loci was significantly enriched for a known genetic interaction network, and mutations of the candidate epistatic modifier loci also affect head morphology (Ozsoy, 2021).
Lapez-Varea, A., Ostale, C. M., Vega-Cuesta, P., Ruiz-Gomez, A., Organista, M. F., Martan, M., Hevia, C. F., Molnar, C., de Celis, J., Culi, J., Esteban, N. and de Celis, J. F. (2021). Genome-wide Phenotypic RNAi Screen in the Drosophila Wing: Global Parameters. G3 (Bethesda). PubMed ID: 34599819
Summary:
This study has screened a collection of UAS-RNAi lines targeting 10920 Drosophila protein-coding genes for phenotypes in the adult wing. 3653 genes (33%) were identified whose knock-down causes either larval/pupal lethality or a mutant phenotype affecting the formation of a normal wing. The most frequent phenotypes consist in changes in wing size, vein differentiation and patterning, defects in the wing margin and in the apposition of the dorsal and ventral wing surfaces. This study also defined 16 functional categories encompassing the most relevant aspect of each protein function, and assigned each Drosophila gene to one of these functional groups. This allowed identification of which mutant phenotypes are enriched within each functional group. Finally, this study used previously published gene expression datasets to determine which genes are or are not expressed in the wing disc. Integrating expression, phenotypic and molecular information offers considerable precision to identify the relevant genes affecting wing formation and the biological processes regulated by them (Lapez-Varea, 2021).

Monday, December 6th - RNA and Transposons

Horiuchi, K., Kawamura, T. and Hamakubo, T. (2021). Wilms' Tumor 1-Associating Protein complex regulates alternative splicing and polyadenylation at potential G-quadruplex-forming splice site sequences. J Biol Chem: 101248. PubMed ID: 34582888
Summary:
Wilms' tumor 1-associating protein (WTAP) is a core component of the N6-methyladenosine (m6A)-methyltransferase complex, along with VIRMA, CBLL1, ZC3H13 (KIAA0853), RBM15/15B, and METTL3/14, which generate m6A, a key RNA modification that affects various process of RNA metabolism. WTAP also interacts with splicing factors; however, despite strong evidence suggesting a role of Drosophila WTAP homolog fl(2)d in alternative splicing (AS), its role in splicing regulation in mammalian cells remains elusive. This study demonstrates using RNAi coupled with RNA-seq that WTAP, VIRMA, CBLL1, and ZC3H13 modulate AS, promoting exon skipping and intron retention in AS events that involve short introns/exons with higher GC content and introns with weaker polypyrimidine-tract and branch points. Further analysis of GC-rich sequences involved in AS events regulated by WTAP, together with minigene assay analysis, revealed potential G-quadruplex formation at splice sites where WTAP has an inhibitory effect. this study also found that several AS events occur in the last exon of one isoform of MSL1 and WTAP, leading to competition for polyadenylation. Proteomic analysis also suggested that WTAP/CBLL1 interaction promotes recruitment of the 3'-end processing complex. Taken together, these results indicate that the WTAP complex regulates AS and alternative polyadenylation via inhibitory mechanisms in GC-rich sequences (Horiuchi, 2021).
Prudencio, P., Savisaar, R., Rebelo, K., Goncalo Martinho, R. and Carmo-Fonseca, M. (2021). Transcription and splicing dynamics during early Drosophila development. RNA. PubMed ID: 34667107
Summary:
Widespread co-transcriptional splicing has been demonstrated from yeast to human. However, most studies to date addressing the kinetics of splicing relative to transcription used either Saccharomyces cerevisiae or metazoan cultured cell lines. This study adapted native elongating transcript sequencing technology (NET-seq) to measure co-transcriptional splicing dynamics during the early developmental stages of Drosophila melanogaster embryos. These results reveal the position of RNA polymerase II (Pol II) when both canonical and recursive splicing occur. This study found heterogeneity in splicing dynamics, with some RNAs spliced immediately after intron transcription, whereas for other transcripts no splicing was observed over the first 100 nucleotides of the downstream exon. Introns that show splicing completion before Pol II has reached the end of the downstream exon are necessarily intron-defined. This study included the splicing dynamics of both nascent pre-mRNAs transcribed in the early embryo, which have few and short introns, as well as pre-mRNAs transcribed later in embryonic development, which contain multiple long introns. As expected, this study found a relationship between the proportion of spliced reads and intron size. However, intron definition was observed at all intron sizes. This study further observed that genes transcribed in the early embryo tend to be isolated in the genome whereas genes transcribed later are often overlapped by a neighboring convergent gene. In isolated genes, transcription termination occurred soon after the polyadenylation site, while in overlapped genes Pol II persisted associated with the DNA template after cleavage and polyadenylation of the nascent transcript. Taken together, these data unravels novel dynamic features of Pol II transcription and splicing in the developing Drosophila embryo (Prudencio, 2021).
Nakamura, K., Nakao, T., Mori, T., Ohno, S., Fujita, Y., Masaoka, K., Sakabayashi, K., Mori, K., Tobimatsu, T. and Sera, T. (2021). Necessity of Flanking Repeats R1' and R8' of Human Pumilio1 Protein for RNA Binding. Biochemistry 60(40): 3007-3015. PubMed ID: 34541851
Summary:
Human Pumilio (hPUM see Drosophila Pumilio) is a structurally well-analyzed RNA-binding protein that has been used recently for artificial RNA binding. Structural analysis revealed that amino acids at positions 12, 13, and 16 in the repeats from R1 to R8 each contact one specific RNA base in the eight-nucleotide RNA target. The functions of the N- and C-terminal flanking repeats R1' and R8', however, remain unclear. This study reports how the repeats contribute to overall RNA binding. The first step was to prepare three mutants in which R1' and/or R8' were deleted and then analyzed RNA binding using gel shift assays. The assays showed that all deletion mutants bound to their target less than the original hPUM, but that R1' contributed more than R8', unlike Drosophila PUM. It was further investigated which amino acid residues of R1' or R8' were responsible for RNA binding. With detailed analysis of the protein tertiary structure, this study found a hydrophobic core in each of the repeats. All hydrophobic amino residues in each core to alanine were mutated during this process. The gel shift assays with the resulting mutants revealed that both hydrophobic cores contributed to the RNA binding: especially the hydrophobic core of R1' had a significant influence. In the present study, it was demonstrated that the flanking R1' and R8' repeats are indispensable for RNA binding of hPUM and suggest that hydrophobic R1'-R1 interactions may stabilize the whole hPUM structure (Nakamura, 2021).
Nirala, N. K., Li, Q., Ghule, P. N., Chen, H. J., Li, R., Zhu, L. J., Wang, R., Rice, N. P., Mao, J., Stein, J. L., Stein, G. S., van Wijnen, A. J. and Ip, Y. T. (2021). Hinfp is a guardian of the somatic genome by repressing transposable elements. Proc Natl Acad Sci U S A 118(41). PubMed ID: 34620709
Summary:
Germ cells possess the Piwi-interacting RNA pathway to repress transposable elements and maintain genome stability across generations. Transposable element mobilization in somatic cells does not affect future generations, but nonetheless can lead to pathological outcomes in host tissues. This study shows the loss of function of the conserved zinc-finger transcription factor Hinfp causes dysregulation of many host genes and derepression of most transposable elements. There is also substantial DNA damage in somatic tissues of Drosophila after loss of Hinfp. Interference of transposable element mobilization by reverse-transcriptase inhibitors can suppress some of the DNA damage phenotypes. The key cell-autonomous target of Hinfp in this process is Histone1, which encodes linker histones essential for higher-order chromatin assembly. Transgenic expression of Hinfp or Histone1, but not Histone4 of core nucleosome, is sufficient to rescue the defects in repressing transposable elements and host genes. Loss of Hinfp enhances Ras-induced tissue growth and aging-related phenotypes. Therefore, Hinfp is a physiological regulator of Histone1-dependent silencing of most transposable elements, as well as many host genes, and serves as a venue for studying genome instability, cancer progression, neurodegeneration, and aging (Nirala, 2021).
Roy, M., Viginier, B., Mayeux, C. A., Ratinier, M. and Fablet, M. (2021). Infections by Transovarially Transmitted DMelSV in Drosophila Have No Impact on Ovarian Transposable Element Transcripts but Increase Their Amounts in the Soma. Genome Biol Evol 13(9). PubMed ID: 34498066
Summary:
Transposable elements (TEs) are genomic parasites, which activity is tightly controlled in germline cells. Using Sindbis virus, it was recently demonstrated that viral infections affect TE transcript amounts in somatic tissues. However, the strongest evolutionary impacts are expected in gonads, because that is where the genomes of the next generations lie. This aspect was investigated using the Drosophila melanogaster Sigma virus. It is particularly relevant in the genome/TE interaction given its tropism to ovaries, which is the organ displaying the more sophisticated TE control pathways. Results in Drosophila simulans flies allowed confirmation of the existence of a strong homeostasis of the TE transcriptome in ovaries upon infection, which, however, rely on TE-derived small RNA modulations. In addition, this study performed a meta-analysis of RNA-seq data and propose that the immune pathway that is triggered upon viral infection determines the direction of TE transcript modulation in somatic tissues (Roy, 2021).
Kunar, R. and Roy, J. K. (2021). The mRNA decapping protein 2 (DCP2) is a major regulator of developmental events in Drosophila-insights from expression paradigms. Cell Tissue Res. PubMed ID: 34536141.
Summary:
The Drosophila genome codes for two decapping proteins, DCP1 and DCP2, out of which DCP2 is the active decapping enzyme. The present endeavour explores the endogenous promoter firing, transcript and protein expression of DCP2 in Drosophila wherein, besides a ubiquitous expression across development, an active expression paradigm during dorsal closure and a plausible moonlighting expression in the Corazonin neurons of the larval brain were identified. It was also demonstrated that the ablation of DCP2 leads to embryonic lethality and defects in vital morphogenetic processes whereas a knockdown of DCP2 in the Corazonin neurons reduces the sensitivity to ethanol in adults, thereby ascribing novel regulatory roles to DCP2. These findings unravel novel putative roles for DCP2 and identify it as a candidate for studies on the regulated interplay of essential molecules during early development in Drosophila, nay the living world.

Monday December 5th - Gonads

McDonough-Goldstein, C. E., Whittington, E., McCullough, E. L., Buel, S. M., Erdman, S., Pitnick, S. and Dorus, S. (2021). Pronounced postmating response in the Drosophila female reproductive tract fluid proteome. Mol Cell Proteomics: 100156. PubMed ID: 34597791
Summary:
Fertility depends on the progression of complex and coordinated postmating processes within the extracellular environment of the female reproductive tract (FRT). Molecular interactions between ejaculate and FRT proteins regulate many of these processes, including sperm motility, migration, storage, and modification, along with concurrent changes in the female. Although extensive progress has been made in the proteomic characterization of male-derived components of sperm and seminal fluid, investigations into the FRT have remained more limited. To achieve a comparable level of knowledge regarding female-derived proteins that comprise the reproductive environment, this study utilized semiquantitative mass spectrometry-based proteomics to study the composition of the FRT tissue and, separately, the luminal fluid, before and after mating in Drosophila melanogaster. This approach leveraged whole-fly isotopic labelling to delineate female proteins from those transferred male ejaculate proteins. the results revealed several characteristics that distinguish the FRT fluid proteome from the FRT tissue proteome: the fluid proteome is encoded by genes with higher overall levels of FRT gene expression and tissue specificity, including many genes with enriched expression in the fat body, fluid-biased proteins are enriched for metabolic functions and the fluid exhibits pronounced postmating compositional changes. The dynamic mating-induced proteomic changes in the FRT fluid informs understanding of secretory mechanisms of the FRT, serves as a foundation for establishing female contributions to the ejaculate-female interactions that regulate fertility and highlights the importance of applying proteomic approaches to characterize the composition and dynamics of the FRT environment (McDonough-Goldstein, 2021).
Ohsako, T., Shirakami, M., Oiwa, K., Ibaraki, K., Karr, T. L., Tomaru, M., Sanuki, R. and Takano-Shimizu-Kouno, T. (2021). The Drosophila Neprilysin 4 gene is essential for sperm function following sperm transfer to females. Genes Genet Syst. PubMed ID: 34556622
Summary:
Sperm are modified substantially in passing through both the male and the female reproductive tracts, only thereafter becoming functionally competent to fertilize eggs. Drosophila sperm become motile in the seminal vesicle; after ejaculation, they interact with seminal fluid proteins and undergo biochemical changes on their surface while they are stored in the female sperm storage organs. However, the molecular mechanisms underlying these maturation processes remain largely unknown. This story focused on Drosophila Neprilysin genes, which are the fly orthologs of the mouse Membrane metallo-endopeptidase-like 1 (Mmel1) gene. While Mmel1 knockout male mice have reduced fertility without abnormality in either testis morphology or sperm motility, there are inconsistent results regarding the association of any Neprilysin gene with male fertility in Drosophila. This study examined the association of the Nep1-5 genes with male fertility by RNAi and found that Nep4 gene function is specifically required in germline cells. To investigate this in more detail, this study induced mutations in the Nep4 gene by the CRISPR/Cas9 system and isolated two mutants, both of which were viable and female fertile, but male sterile. The mutant males had normal-looking testes and sperm; during copulation, sperm were transferred to females and stored in the seminal receptacle and paired spermathecae. However, following sperm transfer and storage, three defects were observed for Nep4 mutant sperm. First, sperm were quickly discarded by the females; second, the proportion of eggs fertilized was significantly lower for mutant sperm than for control sperm; and third, most eggs laid did not initiate development after sperm entry. Taking these observations together, this study concluded that the Nep4 gene is essential for sperm function following sperm transfer to females (Ohsako, 2021).
Macartney, E. L., Zeender, V., Meena, A., De Nardo, A. N., Bonduriansky, R. and Lupold, S. (2021). Sperm depletion in relation to developmental nutrition and genotype in Drosophila melanogaster. Evolution. PubMed ID: 34617270
Summary:
Nutrient limitation during development can restrict the ability of adults to invest in costly fitness traits, and genotypes can vary in their sensitivity to developmental nutrition. However, little is known about how genotype and nutrition affect male ability to maintain ejaculate allocation and achieve fertilization across successive matings. Using 17 isogenic lines of Drosophila melanogaster, This study investigated how variation in developmental nutrition affects males' abilities to mate, transfer sperm, and sire offspring when presented with successive virgin females. This study found that, with each successive mating, males required longer to initiate copulation, transferred fewer sperm, and sired fewer offspring. Males reared on a low-nutrient diet transferred fewer sperm than those reared on nutritionally superior diets, but the rate at which males depleted their sperm, as well as their reproductive performance, was largely independent of diet. Genotype and the genotype x diet interaction explained little of the variation in these male reproductive traits. Results show that sperm depletion can occur rapidly and impose substantial fitness costs for D. melanogaster males across multiple genotypes and developmental environments (Macartney, 2021).
Nallasivan, M. P., Haussmann, I. U., Civetta, A. and Soller, M. (2021). Channel nuclear pore protein 54 directs sexual differentiation and neuronal wiring of female reproductive behaviors in Drosophila. BMC Biol 19(1): 226. PubMed ID: 34666772
Summary:
Female reproductive behaviors and physiology change profoundly after mating. In Drosophila, the post-mating response induced by male-derived sex-peptide in females is a well-established model to elucidate how complex innate behaviors are hard-wired into the brain. This study use a genetic approach to further characterize the molecular and cellular architecture of the sex-peptide response in Drosophila females. Screening for mutations that affect the sensitivity to sex-peptide, this study identified the channel nuclear pore protein Nup54 gene as an essential component for mediating the sex-peptide response, with viable mutant alleles leading to the inability of laying eggs and reducing receptivity upon sex-peptide exposure. Nup54 directs correct wiring of eight adult brain neurons that express pickpocket and are required for egg-laying, while additional channel Nups also mediate sexual differentiation. Consistent with links of Nups to speciation, the Nup54 promoter is a hot spot for rapid evolution and promoter variants alter nucleo-cytoplasmic shuttling. These results implicate nuclear pore functionality to neuronal wiring underlying the sex-peptide response and sexual differentiation as a response to sexual conflict arising from male-derived sex-peptide to direct the female post-mating response (Nallasivan, 2021).
Sankaranarayanan, M., Emenecker, R. J., Wilby, E. L., Jahnel, M., Trussina, I., Wayland, M., Alberti, S., Holehouse, A. S. and Weil, T. T. (2021). Adaptable P body physical states differentially regulate bicoid mRNA storage during early Drosophila development. Dev Cell. PubMed ID: 34655524
Summary:
Ribonucleoprotein condensates can exhibit diverse physical states in vitro and in vivo. Despite considerable progress, the relevance of condensate physical states for in vivo biological function remains limited. This study investigated the physical properties of processing bodies (P bodies) and their impact on mRNA storage in mature Drosophila oocytes. This study shows that the conserved DEAD-box RNA helicase Me31B forms viscous P body condensates, which adopt an arrested physical state. This study demonstrates that structurally distinct proteins and protein-protein interactions, together with RNA, regulate the physical properties of P bodies. Using live imaging and in situ hybridization, this study shows that the arrested state and integrity of P bodies support the storage of bicoid (bcd) mRNA and that egg activation modulates P body properties, leading to the release of bcd for translation in the early embryo. Together, this work provides an example of how physical states of condensates regulate cellular function in development (Sankaranarayanan, 2021).
Fajner, V., Giavazzi, F., Sala, S., Oldani, A., Martini, E., Napoletano, F., Parazzoli, D., Cesare, G., Cerbino, R., Maspero, E., Vaccari, T. and Polo, S. (2021). Hecw controls oogenesis and neuronal homeostasis by promoting the liquid state of ribonucleoprotein particles. Nat Commun 12(1): 5488. PubMed ID: 34531401
Summary:
Specialised ribonucleoprotein (RNP) granules are a hallmark of polarized cells, like neurons and germ cells. Among their main functions is the spatial and temporal modulation of the activity of specific mRNA transcripts that allow specification of primary embryonic axes. While RNPs composition and role are well established, their regulation is poorly defined. This study demonstrates that Hecw (CG42797), a newly identified Drosophila ubiquitin ligase, is a key modulator of RNPs in oogenesis and neurons. Hecw depletion leads to the formation of enlarged granules that transition from a liquid to a gel-like state. Loss of Hecw activity results in defective oogenesis, premature aging and climbing defects associated with neuronal loss. At the molecular level, reduced ubiquitination of the Fmrp impairs its translational repressor activity, resulting in altered Orb expression in nurse cells and Profilin in neurons (Fajner, 2021).

Thursday, December 2nd - Chromatin

Shaheen, N., Akhtar, J., Umer, Z., Khan, M. H. F., Bakhtiari, M. H., Saleem, M., Faisal, A. and Tariq, M. (2021). Polycomb Requires Chaperonin Containing TCP-1 Subunit 7 for Maintaining Gene Silencing in Drosophila. Front Cell Dev Biol 9: 727972. PubMed ID: 34660585
Summary:
In metazoans, heritable states of cell type-specific gene expression patterns linked with specialization of various cell types constitute transcriptional cellular memory. Evolutionarily conserved Polycomb group (PcG) and trithorax group (trxG) proteins contribute to the transcriptional cellular memory by maintaining heritable patterns of repressed and active expression states, respectively. Although chromatin structure and modifications appear to play a fundamental role in maintenance of repression by PcG, the precise targeting mechanism and the specificity factors that bind PcG complexes to defined regions in chromosomes remain elusive. This study reports a serendipitous discovery that uncovers an interplay between Polycomb (Pc) and chaperonin containing T-complex protein 1 (TCP-1) subunit 7 (CCT7) of TCP-1 ring complex (TRiC) chaperonin in Drosophila. CCT7 interacts with Pc at chromatin to maintain repressed states of homeotic and non-homeotic targets of PcG, which supports a strong genetic interaction observed between Pc and CCT7 mutants. Depletion of CCT7 results in dissociation of Pc from chromatin and redistribution of an abundant amount of Pc in cytoplasm. It is proposed that CCT7 is an important modulator of Pc, which helps Pc recruitment at chromatin, and compromising CCT7 can directly influence an evolutionary conserved epigenetic network that supervises the appropriate cellular identities during development and homeostasis of an organism.
Laghmach, R., Di Pierro, M. and Potoyan, D. A. (2021). The interplay of chromatin phase separation and lamina interactions in nuclear organization. Biophys J. PubMed ID: 34653387
Summary:
The genetic material of eukaryotes is segregated into transcriptionally active euchromatin and silent heterochromatin compartments. The spatial arrangement of chromatin compartments evolves over the course of cellular life in a process that remains poorly understood. The latest nuclear imaging experiments reveal a number of dynamical signatures of chromatin that are reminiscent of active multi-phase liquids. This includes the observations of viscoelastic response, coherent motions, Ostwald ripening, and coalescence of chromatin compartments. There is also growing evidence that liquid-liquid phase separation of protein and nucleic acid components is the underlying mechanism for the dynamical behavior of chromatin. In order to dissect the organizational and dynamical implications of chromatin's liquid behavior, this study has devised a phenomenological field-theoretic model of the nucleus as a multi-phase condensate of liquid chromatin types. Employing the liquid chromatin model of the Drosophila nucleus, an extensive set of simulations with an objective to shed light on the dynamics and chromatin patterning observed in the latest nuclear imaging experiments. Simulations reveal the emergence of experimentally detected mesoscale chromatin channels and spheroidal droplets which arise from the dynamic interplay of chromatin type to type interactions and intermingling of chromosomal territories. In addition, coherent motions of chromatin domains observed in displacement correlation spectroscopy measurements which are explained within the framework of this model was quantitatively reproduced by phase separation of chromatin types operating within constrained intra-chromosomal and inter-chromosomal boundaries. Finally, this study illuminates the role of heterochromatin-lamina interactions in the nuclear organization by showing that these interactions enhance the mobility of euchromatin and indirectly introduce correlated motions of heterochromatin droplets (Laghmach, 2021).
Shaukat, A., Khan, M. H. F., Ahmad, H., Umer, Z. and Tariq, M. (2021). Interplay Between BALL and CREB Binding Protein Maintains H3K27 Acetylation on Active Genes in Drosophila. Front Cell Dev Biol 9: 740866. PubMed ID: 34650987
Summary:
CREB binding protein (CBP) is a multifunctional transcriptional co-activator that interacts with a variety of transcription factors and acts as a histone acetyltransferase. In Drosophila, CBP mediated acetylation of histone H3 lysine 27 (H3K27ac) is a known hallmark of gene activation regulated by trithorax group proteins (trxG). Recently, it has been shown that a histone kinase Ballchen (BALL) substantially co-localizes with H3K27ac at trxG target loci and is required to maintain gene activation in Drosophila. This study reports a previously unknown interaction between BALL and CBP, which positively regulates H3K27ac. Analysis of genome-wide binding profile of BALL and CBP reveals major overlap and their co-localization at actively transcribed genes. This study shows that BALL biochemically interacts with CBP and depletion of BALL results in drastic reduction in H3K27ac. Together, these results demonstrate a previously unknown synergy between BALL and CBP and reveals a potentially new pathway required to maintain gene activation during development.
Napoletano, F., Ferrari Bravo, G., Voto, I. A. P., Santin, A., Celora, L., Campaner, E., Dezi, C., Bertossi, A., Valentino, E., Santorsola, M., Rustighi, A., Fajner, V., Maspero, E., Ansaloni, F., Cancila, V., Valenti, C. F., Santo, M., Artimagnella, O. B., Finaurini, S., Gioia, U., Polo, S., Sanges, R., Tripodo, C., Mallamaci, A., Gustincich, S., d'Adda di Fagagna, F., Mantovani, F., Specchia, V. and Del Sal, G. (2021). The prolyl-isomerase PIN1 is essential for nuclear Lamin-B structure and function and protects heterochromatin under mechanical stress. Cell Rep 36(11): 109694. PubMed ID: 34525372
Summary:
Chromatin organization plays a crucial role in tissue homeostasis. Heterochromatin relaxation and consequent unscheduled mobilization of transposable elements (TEs) are emerging as key contributors of aging and aging-related pathologies, including Alzheimer's disease (AD) and cancer. However, the mechanisms governing heterochromatin maintenance or its relaxation in pathological conditions remain poorly understood. This study shows that PIN1, the only phosphorylation-specific cis/trans prolyl isomerase, whose loss is associated with premature aging and AD, is essential to preserve heterochromatin. This study demonstrated that the PIN1 function is conserved from Drosophila to humans and prevents TE mobilization-dependent neurodegeneration and cognitive defects. Mechanistically, PIN1 maintains nuclear type-B Lamin structure and anchoring function for heterochromatin protein 1α (HP1α). This mechanism prevents nuclear envelope alterations and heterochromatin relaxation under mechanical stress, which is a key contributor to aging-related pathologies (Napoletano, 2021).
Sharp, K. A., Khoury, M. J., Wirtz-Peitz, F. and Bilder, D. (2021). Evidence for a nuclear role for Drosophila Dlg as a regulator of the NURF complex. Mol Biol Cell 32(21): ar23. PubMed ID: 34495684.
Summary:
Scribble (Scrib), Discs-large (Dlg), and Lethal giant larvae (Lgl) are basolateral regulators of epithelial polarity and tumor suppressors whose molecular mechanisms of action remain unclear. This study used proximity biotinylation to identify proteins localized near Dlg in the Drosophila wing imaginal disc epithelium. In addition to expected membrane- and cytoskeleton-associated protein classes, nuclear proteins were prevalent in the resulting mass spectrometry dataset, including all four members of the nucleosome remodeling factor (NURF) chromatin remodeling complex. Subcellular fractionation demonstrated a nuclear pool of Dlg and proximity ligation confirmed its position near the NURF complex. Genetic analysis showed that NURF activity is also required for the overgrowth of dlg tumors, and this growth suppression correlated with a reduction in Hippo pathway gene expression. Together, these data suggest a nuclear role for Dlg in regulating chromatin and transcription through a more direct mechanism than previously thought.
Ibarra-Morales, D., Rauer, M. Quarato, P. Rabbani, L. Zenk, F. Schulte-Sasse, Cardamon, F. Gomez-Auli, Cecere, G. and Iovino, N. (2021). Toll-Dorsal signaling regulates the spatiotemporal dynamics of yolk granule tubulation during Drosophila cleavage. Nat Commun 12(1):7002. PubMed ID: 34853314 During embryogenesis, the genome shifts from transcriptionally quiescent to extensively active in a process known as Zygotic Genome Activation (ZGA). In Drosophila, the pioneer factor Zelda is known to be essential for the progression of development; still, it regulates the activation of only a small subset of genes at ZGA. However, thousands of genes do not require Zelda, suggesting that other mechanisms exist. By conducting GRO-seq, HiC and ChIP-seq in Drosophila embryos, this study demonstrated that up to 65% of zygotically activated genes are enriched for the histone variant H2A.Z. H2A.Z enrichment precedes ZGA and RNA Polymerase II loading onto chromatin. In vivo knockdown of maternally contributed Domino, a histone chaperone and ATPase, reduces H2A.Z deposition at transcription start sites, causes global downregulation of housekeeping genes at ZGA, and compromises the establishment of the 3D chromatin structure. It is inferred that H2A.Z is essential for the de novo establishment of transcriptional programs during ZGA via chromatin reorganization.

Wednesday, December 1st - Adult Neural Development and Function

Huang, S. K., Whitney, P. H., Dutta, S., Shvartsman, S. Y. and Rushlow, C. A. (2021. Spatial organization of transcribing loci during early genome activation in Drosophila. Curr Biol. PubMed ID: 34614388
Summary:
The early Drosophila embryo provides unique experimental advantages for addressing fundamental questions of gene regulation at multiple levels of organization, from individual gene loci to the entire genome. Using 1.5-h-old Drosophila embryos undergoing the first wave of genome activation, This study detected ~110 discrete "speckles" of RNA polymerase II (RNA Pol II) per nucleus, two of which were larger and localized to the histone locus bodies (HLBs). In the absence of the primary driver of Drosophila genome activation, the pioneer factor Zelda (Zld) 70% fewer speckles were present; however, the HLBs tended to be larger than wild-type (WT) HLBs, indicating that RNA Pol II accumulates at the HLBs in the absence of robust early-gene transcription. This study observed a uniform distribution of distances between active genes in the nuclei of both WT and zld mutant embryos, indicating that early co-regulated genes do not cluster into nuclear sub-domains. However, in instances whereby transcribing genes did come into close 3D proximity (within 400 nm), they were found to have distinct RNA Pol II speckles. In contrast to the emerging model whereby active genes are clustered to facilitate co-regulation and sharing of transcriptional resources, the data support an "individualist" model of gene control at early genome activation in Drosophila. This model is in contrast to a "collectivist" model, where active genes are spatially clustered and share transcriptional resources, motivating rigorous tests of both models in other experimental systems (Huang, 2021).
Flores, C. C., Loschky, S. S., Marshall, W., Spano, G. M., Cenere, M. M., Tononi, G. and Cirelli, C. (2021). Identification of Ultrastructural Signatures of Sleep and Wake in the Fly Brain. Sleep. PubMed ID: 34536282
Summary:
The cellular consequences of sleep loss are poorly characterized. In the pyramidal neurons of mouse frontal cortex this study found that mitochondria and secondary lysosomes occupy a larger proportion of the cytoplasm after chronic sleep restriction compared to sleep, consistent with increased cellular burden due to extended wake. For each morphological parameter the within-animal variance was high, suggesting that the effects of sleep and sleep loss vary greatly among neurons. However, the analysis was based on 4-5 mice/group and a single section/cell. this study applied serial block-face scanning electron microscopy to identify signatures of sleep and sleep loss in the Drosophila brain. Stacks of images were acquired and used to obtain full 3D reconstructions of the cytoplasm and nucleus of 263 Kenyon cells from adult flies collected after a night of sleep (S) or after 11 hours (SD11) or 35 hours (SD35) of sleep deprivation (9 flies/group). Relative to S flies, SD35 flies showed increased density of dark clusters of chromatin and of Golgi apparata and a trend increase in the percent of cell volume occupied by mitochondria, consistent with increased need for energy and protein supply during extended wake. Logistic regression models could assign each neuron to the correct experimental group with good accuracy, but in each cell nuclear and cytoplasmic changes were poorly correlated, and within-fly variance was substantial in all experimental groups. Together, these results support the presence of ultrastructural signatures of sleep and sleep loss but underscore the complexity of their effects at the single-cell level (Flores, 2021).
Lone, S. R., Potdar, S., Venkataraman, A., Sharma, N., Kulkarni, R., Rao, S., Mishra, S., Sheeba, V. and Sharma, V. K. (2021). Mechanosensory Stimulation via Nanchung Expressing Neurons Can Induce Daytime Sleep in Drosophila. J Neurosci. PubMed ID: 34635540
Summary:
The neuronal and genetic bases of sleep, a phenomenon considered crucial for well-being of organisms, has been under investigation using the model organism Drosophila melanogaster. Although sleep is a state where sensory threshold for arousal is greater, it is known that certain kinds of repetitive sensory stimuli, such as rocking, can indeed promote sleep in humans. This study reports that orbital motion-aided mechanosensory stimulation promotes sleep of male and female Drosophila, independent of the circadian clock, but controlled by the homeostatic system. Mechanosensory receptor nanchung (Nan)-expressing neurons in the chordotonal organs mediate this sleep induction: flies in which these neurons are either silenced or ablated display significantly reduced sleep induction on mechanosensory stimulation. Transient activation of the Nan-expressing neurons also enhances sleep levels, confirming the role of these neurons in sleep induction. This study also reveals that certain regions of the antennal mechanosensory and motor center in the brain are involved in conveying information from the mechanosensory structures to the sleep centers. Thus, for the first time, this study shows that a circadian clock-independent pathway originating from peripherally distributed mechanosensors can promote daytime sleep of flies Drosophila melanogaster (Lone, 2021).
Krzeptowski, W., Walkowicz, L., Krzeptowska, E., Motta, E., Witek, K., Szramel, J., Al Abaquita, T., Baster, Z., Rajfur, Z., Rosato, E., Stratoulias, V., Heino, T. I. and Pyza, E. M. (2021). Mesencephalic Astrocyte-Derived Neurotrophic Factor Regulates Morphology of Pigment-Dispersing Factor-Positive Clock Neurons and Circadian Neuronal Plasticity in Drosophila melanogaster. Front Physiol 12: 705183. PubMed ID: 34646147
Summary:
Mesencephalic Astrocyte-derived Neurotrophic Factor (MANF) is one of a few neurotrophic factors described in Drosophila melanogaster (DmMANF) but its function is still poorly characterized. The present study found that DmMANF is expressed in different clusters of clock neurons. In particular, the PDF-positive large (l-LNv) and small (s-LNv) ventral lateral neurons, the CRYPTOCHROME-positive dorsal lateral neurons (LNd), the group 1 dorsal neurons posterior (DN1p) and different tim-positive cells in the fly's visual system. Importantly, DmMANF expression in the ventral lateral neurons is not controlled by the clock nor it affects its molecular mechanism. However, silencing DmMANF expression in clock neurons affects the rhythm of locomotor activity in light:dark and constant darkness conditions. Such phenotypes correlate with abnormal morphology of the dorsal projections of the s-LNv and with reduced arborizations of the l-LNv in the medulla of the optic lobe. Additionally, it was shown that DmMANF is important for normal morphology of the L2 interneurons in the visual system and for the circadian rhythm in the topology of their dendritic tree. These results indicate that DmMANF is important not only for the development of neurites but also for maintaining circadian plasticity of neurons.
Lin, H. W., Chen, C. C., de Belle, J. S., Tully, T. and Chiang, A. S. (2021). CREBA and CREBB in two identified neurons gate long-term memory formation in Drosophila. Proc Natl Acad Sci U S A 118(37). PubMed ID: 34507985
Summary:
Episodic events are frequently consolidated into labile memory but are not necessarily transferred to persistent long-term memory (LTM). Regulatory mechanisms leading to LTM formation are poorly understood, however, especially at the resolution of identified neurons. This study demonstrates enhanced LTM following aversive olfactory conditioning in Drosophila when the transcription factor cyclic AMP response element binding protein A (CREBA) is induced in just two dorsal-anterior-lateral (DAL) neurons. These experiments show that this process is regulated by protein-gene interactions in DAL neurons: (1) crebA transcription is induced by training and repressed by crebB overexpression, (2) CREBA bidirectionally modulates LTM formation, (3) crebA overexpression enhances training-induced gene transcription, and (4) increasing membrane excitability enhances LTM formation and gene expression. These findings suggest that activity-dependent gene expression in DAL neurons during LTM formation is regulated by CREB proteins.
Almeida, M. P., Lago Solis, B., Stickley, L., Feidler, A. and Nagoshi, E. (2021). Neurofibromin 1 in mushroom body neurons mediates circadian wake drive through activating cAMP-PKA signaling. Nat Commun 12(1): 5758. PubMed ID: 34599173
Summary:
Various behavioral and cognitive states exhibit circadian variations in animals across phyla including Drosophila melanogaster, in which only ~0.1% of the brain's neurons contain circadian clocks. Clock neurons transmit the timing information to a plethora of non-clock neurons via poorly understood mechanisms. This study addresses the molecular underpinning of this phenomenon by profiling circadian gene expression in non-clock neurons that constitute the mushroom body, the center of associative learning and sleep regulation. This study shows that circadian clocks drive rhythmic expression of hundreds of genes in mushroom body neurons, including the Neurofibromin 1 (Nf1) tumor suppressor gene and Pka-C1. Circadian clocks also drive calcium rhythms in mushroom body neurons via NF1-cAMP/PKA-C1 signaling, eliciting higher mushroom body activity during the day than at night, thereby promoting daytime wakefulness. These findings reveal the pervasive, non-cell-autonomous circadian regulation of gene expression in the brain and its role in sleep (Almeida, 2021).

Tuesday, November 3th - Gonads

Huang, W., Liu, Z. and Rong, Y. S. (2021). Dynamic localization of DNA topoisomerase I and its functional relevance during Drosophila development. G3 (Bethesda) 11(9). PubMed ID: 34544118
Summary:
DNA topoisomerase I (Top1) maintains chromatin conformation during transcription. While Top1 is not essential in simple eukaryotic organisms such as yeast, it is required for the development of multicellular organisms. In fact, tissue and cell-type-specific functions of Top1 have been suggested in the fruit fly Drosophila. A better understanding of Top1's function in the context of development is important as Top1 inhibitors are among the most widely used anticancer drugs. As a step toward such a better understanding. Consistent with prior results, Top1 is highly enriched at the nucleolus in transcriptionally active polyploid cells, and this enrichment responds to perturbation of transcription. In diploid cells, this study uncovered evidence for Top1 foci formation at genomic regions not limited to the active rDNA locus, suggestive of novel regulation of Top1 recruitment. In the male germline, Top1 is highly enriched at the paired rDNA loci on sex chromosomes suggesting that it might participate in regulating their segregation during meiosis. Results from RNAi-mediated Top1 knockdown lend support to this hypothesis. This study has provided one of the most comprehensive descriptions of Top1 localization during animal development (Huang, 2021).
Huang, Y. C., Chen, K. H., Chen, Y. Y., Tsao, L. H., Yeh, T. H., Chen, Y. C., Wu, P. Y., Wang, T. W. and Yu, J. Y. (2021). βPS-Integrin acts downstream of Innexin 2 in modulating stretched cell morphogenesis in the Drosophila ovary. G3 (Bethesda) 11(9). PubMed ID: 34544125
Summary:
During oogenesis, a group of specialized follicle cells, known as stretched cells (StCs), flatten drastically from cuboidal to squamous shape. While morphogenesis of epithelia is critical for organogenesis, genes and signaling pathways involved in this process remain to be revealed. In addition to formation of gap junctions for intercellular exchange of small molecules, gap junction proteins form channels or act as adaptor proteins to regulate various cellular behaviors. In invertebrates, gap junction proteins are Innexins. Knockdown of Innexin 2 but not other Innexins expressed in follicle cells attenuates StC morphogenesis. Interestingly, blocking of gap junctions with an inhibitor carbenoxolone does not affect StC morphogenesis, suggesting that Innexin 2 might control StCs flattening in a gap-junction-independent manner. An excessive level of βPS-Integrin encoded by myospheroid is detected in Innexin 2 mutant cells specifically during StC morphogenesis. Simultaneous knockdown of Innexin 2 and myospheroid partially rescues the morphogenetic defect resulted from Innexin 2 knockdown. Furthermore, reduction of βPS-Integrin is sufficient to induce early StCs flattening. Taken together, this data suggests that βPS-Integrin acts downstream of Innexin 2 in modulating StCs morphogenesis (Huang, 2021).
Jang, S., Lee, J., Mathews, J., Ruess, H., Williford, A. O., Rangan, P., Betran, E. and Buszczak, M. (2021). The Drosophila ribosome protein S5 paralog RpS5b promotes germ cell and follicle cell differentiation during oogenesis. Development 148(19). PubMed ID: 34495316
Summary:
Emerging evidence suggests that ribosome heterogeneity may have important functional consequences in the translation of specific mRNAs within different cell types and under various conditions. Ribosome heterogeneity comes in many forms, including post-translational modification of ribosome proteins (RPs), absence of specific RPs and inclusion of different RP paralogs. The Drosophila genome encodes two RpS5 paralogs: RpS5a and RpS5b. While RpS5a is ubiquitously expressed, RpS5b exhibits enriched expression in the reproductive system. Deletion of RpS5b results in female sterility marked by developmental arrest of egg chambers at stages 7-8, disruption of vitellogenesis and posterior follicle cell (PFC) hyperplasia. While transgenic rescue experiments suggest functional redundancy between RpS5a and RpS5b, molecular, biochemical and ribo-seq experiments indicate that RpS5b mutants display increased rRNA transcription and RP production, accompanied by increased protein synthesis. Loss of RpS5b results in microtubule-based defects and in mislocalization of Delta and Mindbomb1, leading to failure of Notch pathway activation in PFCs. The results indicate that germ cell-specific expression of RpS5b promotes proper egg chamber development by ensuring the homeostasis of functional ribosomes (Jang, 2021).
Kenny, A., Morgan, M. B., Mohr, S. and Macdonald, P. M. (2021). Knock down analysis reveals critical phases for specific oskar noncoding RNA functions during Drosophila oogenesis. G3 (Bethesda). PubMed ID: 34586387
Summary:
The oskar transcript, acting as a noncoding RNA, contributes to a diverse set of pathways in the Drosophila ovary, including karyosome formation, positioning of the microtubule organizing center, integrity of certain ribonucleoprotein particles, control of nurse cell divisions, restriction of several proteins to the germline, and progression through oogenesis. How oskar mRNA acts to perform these functions remains unclear. This study use a knock down approach to identify the critical phases when oskar is required for three of these functions. The existing transgenic shRNA for removal of oskar mRNA in the germline targets a sequence overlapping a regulatory site bound by Bruno1 protein to confer translational repression, and was ineffective during oogenesis. Novel transgenic shRNAs targeting other sites were effective at strongly reducing oskar mRNA levels and reproducing phenotypes associated with the absence of the mRNA. Using GAL4 drivers active at different developmental stages of oogenesis, this study found that early loss of oskar mRNA reproduced defects in karyosome formation and positioning of the microtubule organizing center, but not arrest of oogenesis. Loss of oskar mRNA at later stages was required to prevent progression through oogenesis. The noncoding function of oskar mRNA is thus required for more than a single event (Kenny, 2021).
Reilein, A., Kogan, H. V., Misner, R., Park, K. S. and Kalderon, D. (2021). Adult stem cells and niche cells segregate gradually from common precursors that build the adult Drosophila ovary during pupal development. Proc Natl Acad Sci U S A 118(39). PubMed ID: Elife 10. PubMed ID: 34590579
Summary:
Production of proliferative follicle cells (FCs) and quiescent escort cells (ECs) by follicle stem cells (FSCs) in adult Drosophila ovaries is regulated by niche signals from anterior (cap cells, ECs) and posterior (polar FCs) sources. This study shows that ECs, FSCs, and FCs develop from common pupal precursors, with different fates acquired by progressive separation of cells along the AP axis and a graded decline in anterior cell proliferation. ECs, FSCs, and most FCs derive from intermingled cell (IC) precursors interspersed with germline cells. Precursors also accumulate posterior to ICs before engulfing a naked germline cyst projected out of the germarium to form the first egg chamber and posterior polar FC signaling center. Thus, stem and niche cells develop in appropriate numbers and spatial organization through regulated proliferative expansion together with progressive establishment of spatial signaling cues that guide adult cell behavior, rather than through rigid early specification events.
Luo, W., Liu, S., Zhang, W., Yang, L., Huang, J., Zhou, S., Feng, Q., Palli, S. R., Wang, J., Roth, S. and Li, S. (2021). Juvenile hormone signaling promotes ovulation and maintains egg shape by inducing expression of extracellular matrix genes. Proc Natl Acad Sci U S A 118(39). PubMed ID: 34544864
Summary:
It is well documented that the juvenile hormone (JH) can function as a gonadotropic hormone that stimulates vitellogenesis by activating the production and uptake of vitellogenin in insects. This study describes a phenotype associated with mutations in the Drosophila JH receptor genes, Met and Gce: the accumulation of mature eggs with reduced egg length in the ovary. JH signaling is mainly activated in ovarian muscle cells and induces laminin gene expression in these cells. Meanwhile, JH signaling induces collagen IV gene expression in the adult fat body, from which collagen IV is secreted and deposited onto the ovarian muscles. Laminin locally and collagen IV remotely contribute to the assembly of ovarian muscle extracellular matrix (ECM); moreover, the ECM components are indispensable for ovarian muscle contraction. Furthermore, ovarian muscle contraction externally generates a mechanical force to promote ovulation and maintain egg shape. This work reveals an important mechanism for JH-regulated insect reproduction (Luo, 2021).

Monday, November 29th - Adult Neural Development and Function

Shrestha, B. R., Burgos, A. and Grueber, W. B. (2021). The Immunoglobulin Superfamily Member Basigin Is Required for Complex Dendrite Formation in Drosophila. Front Cell Neurosci 15: 739741. PubMed ID: 34803611

Coordination of dendrite growth with changes in the surrounding substrate occurs widely in the nervous system and is vital for establishing and maintaining neural circuits. However, the molecular basis of this important developmental process remains poorly understood. To identify potential mediators of neuron-substrate interactions important for dendrite morphogenesis, this study undertook an expression pattern-based screen in Drosophila larvae, which revealed many proteins with expression in dendritic arborization (da) sensory neurons and in neurons and their epidermal substrate. Reporters for Basigin, a cell surface molecule of the immunoglobulin (Ig) superfamily previously implicated in cell-cell and cell-substrate interactions, are expressed in da sensory neurons and epidermis. Loss of Basigin in da neurons led to defects in morphogenesis of the complex dendrites of class IV da neurons. Classes of sensory neurons with simpler branching patterns were unaffected by loss of Basigin. Structure-function analyses showed that a juxtamembrane KRR motif is critical for this function. Furthermore, knock down of Basigin in the epidermis led to defects in dendrite elaboration of class IV neurons, suggesting a non-autonomous role. Together, these findings support a role for Basigin in complex dendrite morphogenesis and interactions between dendrites and the adjacent epidermis.

Fulgham, C. V., Dreyer, A. P., Nasseri, A., Miller, A. N., Love, J., Martin, M. M., Jabr, D. A., Saurabh, S. and Cavanaugh, D. J. (2021). Central and Peripheral Clock Control of Circadian Feeding Rhythms. J Biol Rhythms: 7487304211045835. PubMed ID: 34547954
Summary:
Many behaviors exhibit ~24-h oscillations under control of an endogenous circadian timing system.Most circadian research in Drosophila has focused on the generation of locomotor activity rhythms, but a fundamental question is how the circadian clock orchestrates multiple distinct behavioral outputs. This study has investigated the cells and circuits mediating circadian control of feeding behavior. This study shows that the presence of feeding rhythms requires molecular clock function in the ventrolateral clock neurons of the central brain. This study further demonstrate that the speed of molecular clock oscillations in these neurons dictates the free-running period length of feeding rhythms. In contrast to the effects observed with central clock cell manipulations, This study shows that genetic abrogation of the molecular clock in the fat body, a peripheral metabolic tissue, is without effect on feeding behavior. Under these conditions, the period of feeding rhythms tracks with molecular oscillations in central brain clock cells, consistent with a primary role of the brain clock in dictating the timing of feeding behavior. Finally, despite a lack of effect of fat body selective manipulations, this study found that flies with simultaneous disruption of molecular clocks in multiple peripheral tissues (but with intact central clocks) exhibit decreased feeding rhythm strength and reduced overall food intake. This study concluded that both central and peripheral clocks contribute to the regulation of feeding rhythms, with a particularly dominant, pacemaker role for specific populations of central brain clock cells (Fulgham, 2021).
Guo, L., Wu, Y., Chang, H., Zhang, Z., Tang, H., Yu, Y., Xin, L., Liu, Y. and He, Y. (2021). Structure of cell-cell adhesion mediated by the Down syndrome cell adhesion molecule. Proc Natl Acad Sci U S A 118(39). PubMed ID: 34531300
Summary:
The Down syndrome cell adhesion molecule (DSCAM) belongs to the immunoglobulin superfamily (IgSF) and plays important roles in neural development. It has a large ectodomain, including 10 Ig-like domains and 6 fibronectin III (FnIII) domains. Previous data have shown that DSCAM can mediate cell adhesion by forming homophilic dimers between cells and contributes to self-avoidance of neurites or neuronal tiling, which is important for neural network formation. However, the organization and assembly of DSCAM at cell adhesion interfaces has not been fully understood. This study combined electron microscopy and other biophysical methods to characterize the structure of the DSCAM-mediated cell adhesion and generate three-dimensional views of the adhesion interfaces of DSCAM by electron tomography. The results show that mouse DSCAM forms a regular pattern at the adhesion interfaces. The Ig-like domains contribute to both trans homophilic interactions and cis assembly of the pattern, and the FnIII domains are crucial for the cis pattern formation as well as the interaction with the cell membrane. By contrast, no obvious assembly pattern is observed at the adhesion interfaces mediated by mouse DSCAML1 or Drosophila DSCAMs, suggesting the different structural roles and mechanisms of DSCAMs in mediating cell adhesion and neural network formation (Guo, 2021).
Kaldun, J. C., Lone, S. R., Humbert Camps, A. M., Fritsch, C., Widmer, Y. F., Stein, J. V., Tomchik, S. M. and Sprecher, S. G. (2021). Dopamine, sleep, and neuronal excitability modulate amyloid-beta-mediated forgetting in Drosophila. PLoS Biol 19(10): e3001412. PubMed ID: 34613972
Summary:
Alzheimer disease (AD) is one of the main causes of age-related dementia and neurodegeneration. However, the onset of the disease and the mechanisms causing cognitive defects are not well understood. Aggregation of amyloidogenic peptides is a pathological hallmark of AD and is assumed to be a central component of the molecular disease pathways. Pan-neuronal expression of Aβ42 Arctic peptides in Drosophila melanogaster results in learning and memory defects. Surprisingly, targeted expression to the mushroom bodies, a center for olfactory memories in the fly brain, does not interfere with learning but accelerates forgetting. This study shows that reducing neuronal excitability either by feeding Levetiracetam or silencing of neurons in the involved circuitry ameliorates the phenotype. Furthermore, inhibition of the Rac-regulated forgetting pathway could rescue the Aβ42Arctic-mediated accelerated forgetting phenotype. Similar effects are achieved by increasing sleep, a critical regulator of neuronal homeostasis. Results provide a functional framework connecting forgetting signaling and sleep, which are critical for regulating neuronal excitability and homeostasis and are therefore a promising mechanism to modulate forgetting caused by toxic Aβ peptides (Kaldun, 2021).
Kohn, J. R., Portes, J. P., Christenson, M. P., Abbott, L. F. and Behnia, R. (2021). Flexible filtering by neural inputs supports motion computation across states and stimuli. Curr Biol. PubMed ID: 34670114
Summary:
Sensory systems flexibly adapt their processing properties across a wide range of environmental and behavioral conditions. Such variable processing complicates attempts to extract a mechanistic understanding of sensory computations. This is evident in the highly constrained, canonical Drosophila motion detection circuit, where the core computation underlying direction selectivity is still debated despite extensive studies. This study measured the filtering properties of neural inputs to the OFF motion-detecting T5 cell in Drosophila. Stimulus-dependent changes in the shape of these signals, which become more biphasic under specific conditions. Summing these inputs within the framework of a connectomic-constrained model of the circuit demonstrates that these shapes are sufficient to explain T5 responses to various motion stimuli. Thus, these stimulus- and state-dependent measurements reconcile motion computation with the anatomy of the circuit. These findings provide a clear example of how a basic circuit supports flexible sensory computation (Kohn, 2021).
Jia, J., He, L., Yang, J., Shuai, Y., Yang, J., Wu, Y., Liu, X., Chen, T., Wang, G., Wang, X., Song, X., Ding, Z., Zhu, Y., Zhang, L., Chen, P. and Qin, H. (2021). A pair of dopamine neurons mediate chronic stress signals to induce learning deficit in Drosophila melanogaster. Proc Natl Acad Sci U S A 118(42). PubMed ID: 34654742
Summary:
Chronic stress could induce severe cognitive impairments. Despite extensive investigations in mammalian models, the underlying mechanisms remain obscure. This study shows that chronic stress could induce dramatic learning and memory deficits in Drosophila melanogaster The chronic stress-induced learning deficit (CSLD) is long lasting and associated with other depression-like behaviors. This study demonstrates that excessive dopaminergic activity provokes susceptibility to CSLD. Remarkably, a pair of PPL1-γ1pedc dopaminergic neurons that project to the mushroom body (MB) γ1pedc compartment play a key role in regulating susceptibility to CSLD so that stress-induced PPL1-γ1pedc hyperactivity facilitates the development of CSLD. Consistently, the mushroom body output neurons (MBON) of the γ1pedc compartment, MBON-γ1pedc>&alpha/β neurons, are important for modulating susceptibility to CSLD. Imaging studies showed that dopaminergic activity is necessary to provoke the development of chronic stress-induced maladaptations in the MB network. The data supports PPL1-γ1pedc mediates chronic stress signals to drive allostatic maladaptations in the MB network that lead to CSLD.

Friday, November 26th - Signaling

Hughes, M. P., Goldschmidt, L. and Eisenberg, D. S. (2021). Prevalence and species distribution of the low-complexity, amyloid-like, reversible, kinked segment (LARKS) structural motif in amyloid-like fibrils. J Biol Chem: 101194. PubMed ID: 34537246
Summary:
Membraneless Organelles (MLOs) are vital and dynamic reaction centers in cells that compartmentalize the cytoplasm in the absence of a membrane. Multivalent interactions between protein Low-Complexity Domains (LCDs) contribute to MLO organization. Previously, This study used computational methods to identify structural motifs termed Low-complexity Amyloid-like Reversible Kinked Segments (LARKS) that promote phase-transition to form hydrogels and that are common in human proteins that participate in MLOs. This study searched for LARKS in the proteomes of six model organisms: Homo sapiens, Drosophila melanogaster, Plasmodium falciparum, Saccharomyces cerevisiae, Mycobacterium tuberculosis, and Escherichia coli to gain an understanding of the distribution of LARKS in the proteomes of various species. This study found that LARKS are abundant in M. tuberculosis, D. melanogaster, and H. sapiens, but not in S. cerevisiae or P. falciparum. LARKS have high glycine content, which enables kinks to form as exemplified by the known LARKS-rich amyloidogenic structures of TDP43, FUS, and hnRNPA2, three proteins that are known to participate in MLOs. These results support the idea of LARKS as an evolved structural motif. Based on these results, this study also established the LARKSdb webserver, which permits users to search for LARKS in their protein sequences of interest (Hughes, 2021).
Karim, M. S., Madamanchi, A., Dutko, J. A., Mullins, M. C. and Umulis, D. M. (2021). Heterodimer-heterotetramer formation mediates enhanced sensor activity in a biophysical model for BMP signaling. PLoS Comput Biol 17(9): e1009422. PubMed ID: 34591841
Summary:
Numerous stages of organismal development rely on the cellular interpretation of gradients of secreted morphogens including members of the Bone Morphogenetic Protein (BMP) family through transmembrane receptors. Early gradients of BMPs drive dorsal/ventral patterning throughout the animal kingdom in both vertebrates and invertebrates. Growing evidence in Drosophila, zebrafish, murine and other systems suggests that BMP ligand heterodimers are the primary BMP signaling ligand, even in systems in which mixtures of BMP homodimers and heterodimers are present. Signaling by heterodimers occurs through a hetero-tetrameric receptor complex comprising of two distinct type one BMP receptors and two type II receptors. This study developed a kinetic model for BMP tetramer formation based on current measurements for binding rates and affinities. This study finds that contrary to a common hypothesis, heterodimer-heterotetramer formation is not kinetically favored over the formation of homodimer-tetramer complexes under physiological conditions of receptor and ligand concentrations and therefore other mechanisms, potentially including differential kinase activities of the formed heterotetramer complexes, must be the cause of heterodimer-heterotetramer signaling primacy. Further, although BMP complex assembly favors homodimer and homomeric complex formation over a wide range of parameters, ignoring these signals and instead relying on the heterodimer improves the range of morphogen interpretation in a broad set of conditions, suggesting a performance advantage for heterodimer signaling in patterning multiple cell types in a gradient (Karim, 2021).
Kawasaki, H., Okano, H., Nedachi, T., Nakagawa-Yagi, Y., Hara, A. and Ishida, N. (2021). Effects of an electric field on sleep quality and life span mediated by ultraviolet (UV)-A/blue light photoreceptor CRYPTOCHROME in Drosophila. Sci Rep 11(1): 20543. PubMed ID: 34654874
Summary:
Although electric fields (EF) exert beneficial effects on animal wound healing, differentiation, cancers and rheumatoid arthritis, the molecular mechanisms of these effects have remained unclear about a half century. This study aimed to elucidate the molecular mechanisms underlying EF effects in Drosophila melanogaster as a genetic animal model. This study shows that the sleep quality of wild type (WT) flies was improved by exposure to a 50-Hz (35 kV/m) constant electric field during the day time, but not during the night time. The effect was undetectable in cryptochrome mutant (cryb) flies. Exposure to a 50-Hz electric field under low nutrient conditions elongated the lifespan of male and female WT flies by ~18%, but not of several cry mutants and cry RNAi strains. Metabolome analysis indicated that the adenosine triphosphate (ATP) content was higher in intact WT than cry gene mutant strains exposed to an electric field. A putative magnetoreceptor protein and UV-A/blue light photoreceptor, CRYPTOCHROME (CRY) is involved in electric field (EF) receptors in animals. The present findings constitute hitherto unknown genetic evidence of a CRY-based system that is electric field sensitive in animals (Kawasaki, 2021).
Jevitt, A., Huang, Y. C., Zhang, S. M., Chatterjee, D., Wang, X. F., Xie, G. Q. and Deng, W. M. (2021). Modeling Notch-Induced Tumor Cell Survival in the Drosophila Ovary Identifies Cellular and Transcriptional Response to Nuclear NICD Accumulation. Cells 10(9). PubMed ID: 34571871
Summary:
Notch is a conserved developmental signaling pathway that is dysregulated in many cancer types, most often through constitutive activation. Tumor cells with nuclear accumulation of the active Notch receptor, NICD, generally exhibit enhanced survival while patients experience poorer outcomes. To understand the impact of NICD accumulation during tumorigenesis, this study developed a tumor model using the Drosophila ovarian follicular epithelium. Using this system the study demonstrated that NICD accumulation contributed to larger tumor growth, reduced apoptosis, increased nuclear size, and fewer incidents of DNA damage without altering ploidy. Using bulk RNA sequencing key genes were identified involved in both a pre- and post- tumor response to NICD accumulation. Among these are genes involved in regulating double-strand break repair, chromosome organization, metabolism, like raptor, this study experimentally validated contributes to early Notch-induced tumor growth. Finally, using single-cell RNA sequencing identified follicle cell-specific targets in NICD-overexpressing cells which contribute to DNA repair and negative regulation of apoptosis. This valuable tumor model for nuclear NICD accumulation in adult Drosophila follicle cells has allowed a better understand the specific contribution of nuclear NICD accumulation to cell survival in tumorigenesis and tumor progression (Jevitt, 2021).
Han, Y. and Jiang, J. (2022). Cell-Based Assays for Smoothened Ubiquitination and Sumoylation. Methods Mol Biol 2374: 139-147. PubMed ID: 34562249
Summary:
The Hedgehog (Hh) family of secreted proteins governs embryonic development and adult tissue homeostasis by regulating the abundance, localization, and activity of the GPCR family protein Smoothened (Smo). Smo trafficking and subcellular accumulation are controlled by multiple posttranslational modifications (PTMs) including phosphorylation, ubiquitination, and sumoylation, which appears to be conserved from Drosophila to mammals. Smo ubiquitination is dynamically regulated by E3 ubiquitin ligases and deubiquitinases (dubs) and is opposed by Hh signaling. By contrast, Smo sumoylation is stimulated by Hh, which counteracts Smo ubiquitination by recruiting the dub USP8. This study describes cell-base assays for Smo ubiquitination and its regulation by Hh and the E3 ligases in Drosophila. Also described are assays for Smo sumoylation in both Drosophila and mammalian cultured cells (Han, 2022).
Kim, J., Chuang, H. C., Wolf, N. K., Nicolai, C. J., Raulet, D. H., Saijo, K. and Bilder, D. (2021). Tumor-induced disruption of the blood-brain barrier promotes host death. Dev Cell 56(19): 2712-2721.e2714. PubMed ID: 34496290
Summary:
Cancer patients often die from symptoms that manifest at a distance from any tumor. Mechanisms underlying these systemic physiological perturbations, called paraneoplastic syndromes, may benefit from investigation in non-mammalian systems. Using a non-metastatic Drosophila adult model, this study found that malignant-tumor-produced cytokines drive widespread host activation of JAK-STAT signaling and cause premature lethality. STAT activity is particularly high in cells of the blood-brain barrier (BBB), where it induces aberrant BBB permeability. Remarkably, inhibiting STAT in the BBB not only rescues barrier function but also extends the lifespan of tumor-bearing hosts. This study identified BBB damage in other pathological conditions that cause elevated inflammatory signaling, including obesity and infection, where BBB permeability also regulates host survival. IL-6-dependent BBB dysfunction is further seen in a mouse tumor model, and it again promotes host morbidity. Therefore, BBB alterations constitute a conserved lethal tumor-host interaction that also underlies other physiological morbidities (Kim, 2021).

Wednesday, November 25th - Embryonic Development

Graham, P. L., Fischer, M. D., Giri, A. and Pick, L. (2021). The fushi tarazu zebra element is not required for Drosophila viability or fertility. G3 (Bethesda) 11(11). PubMed ID: 34518886
Summary:
Expression of genes in precisely controlled spatiotemporal patterns is essential for embryonic development. Much understanding of mechanisms regulating gene expression comes from the study of cis-regulatory elements (CREs) that direct expression of reporter genes in transgenic organisms. This reporter-transgene approach identifies genomic regions sufficient to drive expression but fails to provide information about quantitative and qualitative contributions to endogenous expression, although such conclusions are often inferred. This study evaluated the endogenous function of a classic Drosophila CRE, the fushi tarazu (ftz) zebra element. ftz is a pair-rule segmentation gene expressed in seven stripes during embryogenesis, necessary for formation of alternate body segments. Reporter transgenes identified the promoter-proximal zebra element as a major driver of the seven ftz stripes. This study generated a precise genomic deletion of the zebra element (ftzδZ) to assess its role in the context of native chromatin and neighboring CREs, expecting large decreases in ftz seven-stripe expression. However, significant reduction in expression was found for only one stripe, ftz stripe 4, expressed at ∼25% of wild type levels in ftz&deltaZ homozygotes. Defects in corresponding regions of ftz&deltaZ mutants suggest this level of expression borders the threshold required to promote morphological segmentation. Further, This study established true-breeding lines of homozygous ftz&deltaZ flies, demonstrating that the body segments missing in the mutants are not required for viability or fertility. These results highlight the different types of conclusions drawn from different experimental designs and emphasize the importance of examining transcriptional regulatory mechanisms in the context of the native genomic environment (Graham, 2021).
York-Andersen, A. H., Wood, B. W., Wilby, E. L., Berry, A. S. and Weil, T. T. (2021). Osmolarity-regulated swelling initiates egg activation in Drosophila. Open Biol 11(8): 210067. PubMed ID: 34343463.
Summary:
Egg activation is a series of highly coordinated processes that prepare the mature oocyte for embryogenesis. Typically associated with fertilization, egg activation results in many downstream outcomes, including the resumption of the meiotic cell cycle, translation of maternal mRNAs and cross-linking of the vitelline membrane. While some aspects of egg activation, such as initiation factors in mammals and environmental cues in sea animals, have been well-documented, the mechanics of egg activation in insects are less well-understood. For many insects, egg activation can be triggered independently of fertilization. In Drosophila melanogaster, egg activation occurs in the oviduct resulting in a single calcium wave propagating from the posterior pole of the oocyte. This study used physical manipulations, genetics and live imaging to demonstrate the requirement of a volume increase for calcium entry at egg activation in ex vivo mature Drosophila oocytes. The addition of water, modified with sucrose to a specific osmolarity, is sufficient to trigger the calcium wave in the mature oocyte and the downstream events associated with egg activation. T he swelling process is regulated by the conserved osmoregulatory channels, aquaporins and DEGenerin/Epithelial Na(+) channels. Furthermore, through pharmacological and genetic disruption, this study reveals a concentration-dependent requirement of transient receptor potential M channels to transport calcium, most probably from the perivitelline space, across the plasma membrane into the mature oocyte. These data establish osmotic pressure as a mechanism that initiates egg activation in Drosophila and are consistent with previous work from evolutionarily distant insects, including dragonflies and mosquitos, and show remarkable similarities to the mechanism of egg activation in some plants.
Diaz-Cuadros, M., Pourquie, O. and El-Sherif, E. (2021). Patterning with clocks and genetic cascades: Segmentation and regionalization of vertebrate versus insect body plans. PLoS Genet 17(10): e1009812. PubMed ID: 34648490
Summary:
Oscillatory and sequential processes have been implicated in the spatial patterning of many embryonic tissues. For example, molecular clocks delimit segmental boundaries in vertebrates and insects and mediate lateral root formation in plants, whereas sequential gene activities are involved in the specification of regional identities of insect neuroblasts, vertebrate neural tube, vertebrate limb, and insect and vertebrate body axes. These processes take place in various tissues and organisms, and, hence, raise the question of what common themes and strategies they share. Two processes were reviewed that rely on the spatial regulation of periodic and sequential gene activities: segmentation and regionalization of the anterior-posterior (AP) axis of animal body plans. To study these processes in species that belong to 2 different phyla: vertebrates and insects. By contrasting 2 different processes (segmentation and regionalization) in species that belong to 2 distantly related phyla (arthropods and vertebrates), This study elucidates the deep logic of patterning by oscillatory and sequential gene activities. Furthermore, in some of these organisms (e.g., the fruit fly Drosophila), a mode of AP patterning has evolved that seems not to overtly rely on oscillations or sequential gene activities, providing an opportunity to study the evolution of pattern formation mechanisms (Diaz-Cuadros, 2021).
Velagala, V. and Zartman, J. (2021). Pinching and pushing: fold formation in the Drosophila dorsal epidermis. Biophys J. PubMed ID: 34461105
Summary:
Epithelial folding is a fundamental morphogenetic process that shapes planar epithelial sheets into complex three-dimensional structures. Multiple mechanisms can generate epithelial folds, including apical constriction, which acts locally at the cellular level, differential growth on the tissue scale, or buckling due to compression from neighboring tissues. This study investigated the formation of dorsally-located epithelial folds at segment boundaries during the late stages of Drosophila embryogenesis. The fold formation at the segment boundaries was found to occur through the juxtaposition of two key morphogenetic processes: local apical constriction and tissue-level compressive forces from posterior segments. Further, it was found that epidermal spreading and fold formation are accompanied by spatiotemporal pulses of Hedgehog signaling. A computational model that incorporates the local forces generated from the differential tensions of the apical, basal, and lateral sides of the cell and active forces generated within the whole tissue recapitulates the overall fold formation process in wildtype and Hedgehog overexpression conditions. In sum, this work demonstrates how epithelial folding depends on multiple, separable physical mechanisms to generate the final morphology of the dorsal epidermis. This work illustrates the modularity of morphogenetic unit operations that occur during epithelial morphogenesis.
Wyatt, B. H., Amin, N. M., Bagley, K., Wcisel, D. J., Dush, M. K., Yoder, J. A. and Nascone-Yoder, N. M. (2021). Single-minded 2 is required for left-right asymmetric stomach morphogenesis. Development 148(17). PubMed ID: 34486651.
Summary:
The morphogenesis of left-right (LR) asymmetry is a crucial phase of organogenesis. In the digestive tract, the development of anatomical asymmetry is first evident in the leftward curvature of the stomach. To elucidate the molecular events that shape this archetypal laterality, transcriptome analyses was performed of the left versus right sides of the developing stomach in frog embryos. Besides the known LR gene pitx2, the only gene found to be expressed asymmetrically throughout all stages of curvature was single-minded 2 (sim2), a Down Syndrome-related transcription factor and homolog of a Drosophila gene (sim) required for LR asymmetric looping of the fly gut. sim2 was shown to function downstream of LR patterning cues to regulate key cellular properties and behaviors in the left stomach epithelium that drive asymmetric curvature. These results reveal unexpected convergent cooption of single-minded genes during the evolution of LR asymmetric morphogenesis, and have implications for dose-dependent roles of laterality factors in non-laterality-related birth defects.
Wat, L. W., Chowdhury, Z. S., Millington, J. W., Biswas, P. and Rideout, E. J. (2021). Sex determination gene transformer regulates the male-female difference in Drosophila fat storage via the adipokinetic hormone pathway. Elife 10. PubMed ID: 34672260.
Summary:
Sex differences in whole-body fat storage exist in many species. For example, Drosophila females store more fat than males. Yet, the mechanisms underlying this sex difference in fat storage remain incompletely understood. This study identified transformer (tra) in regulating the male-female difference in fat storage. Normally, a functional Tra protein is present only in females, where it promotes female sexual development. This study shows that loss of Tra in females reduced whole-body fat storage, whereas gain of Tra in males augmented fat storage. Tra's role in promoting fat storage was largely due to its function in neurons, specifically the Adipokinetic hormone (Akh)-producing cells (APCs). This analysis of Akh pathway regulation revealed a male bias in APC activity and Akh pathway function, where this sex-biased regulation influenced the sex difference in fat storage by limiting triglyceride accumulation in males. Importantly, Tra loss in females increased Akh pathway activity, and genetically manipulating the Akh pathway rescued Tra-dependent effects on fat storage. This identifies sex-specific regulation of Akh as one mechanism underlying the male-female difference in whole-body triglyceride levels, and provides important insight into the conserved mechanisms underlying sexual dimorphism in whole-body fat storage.

Tuesday, November 23rd - Adult Physiology

Gu, X., Chen, W., Perry, T., Batterham, P. and Hoffmann, A. A. (2021). Genomic knockout of hsp23 both decreases and increases fitness under opposing thermal extremes in Drosophila melanogaster. Insect Biochem Mol Biol 139: 103652. PubMed ID: 34562590
Summary:
Under exposure to harmful environmental stresses, organisms exhibit a general stress response involving upregulation of the expression of heat shock proteins (HSPs) which is thought to be adaptive. Small heat shock proteins (sHSPs) are key components of this response, although shsp genes may have other essential roles in development. However, the upregulation of expression of a suite of genes under stress may not necessarily be evidence of an adaptive response to stress that involves those genes. To explore this issue, the CRISPR/Cas9 system was used to investigate pleiotropic effects of the hsp23 gene in Drosophila melanogaster. Transgenic flies carrying a pCFD5 plasmid containing sgRNAs were created to generate a complete knockout of the hsp23 gene. The transgenic line lacking hsp23 showed an increased hatch rate and no major fitness costs under an intermediate temperature used for culturing the flies. In addition, hsp23 knockout affected tolerance to hot and cold temperature extremes but in opposing directions; knockout flies had reduced tolerance to cold, but increased tolerance to heat. Despite this, hsp23 expression (in wild type flies) was increased under both hot and cold conditions. The hsp23 gene was required for heat hardening at the pupal stage, but not at the 1st-instar larval stage, even though the gene was upregulated in wild type controls at that life stage. The phenotypic effects of hsp23 were not compensated for by expression changes in other shsps. This study shows that the fitness consequences of an hsp gene knockout depends on environmental conditions, with potential fitness benefits of gene loss even under conditions when the gene is normally upregulated (Gu, 2021).
Francis, D., Ghazanfar, S., Havula, E., Krycer, J. R., Strbenac, D., Senior, A., Minard, A. Y., Geddes, T., Nelson, M. E., Weiss, F., Stockli, J., Yang, J. Y. H. and James, D. E. (2021). Genome-wide analysis in Drosophila reveals diet-by-gene interactions and uncovers diet-responsive genes. G3 (Bethesda) 11(10). PubMed ID: 34568906
Summary:
GeThis study sought to understand how such gene-diet interactions influenced nutrient storage and utilization, a major determinant of metabolic disease. In this study 178 inbred strains were subjected from the Drosophila genetic reference panel (DGRP) to diets varying in sugar, fat, and protein. This study assessed starvation resistance, a holistic phenotype of nutrient storage and utilization that can be robustly measured. Diet influenced the starvation resistance of most strains, but the effect varied markedly between strains such that some displayed better survival on a high carbohydrate diet (HCD) compared to a high-fat diet while others had opposing responses, illustrating a considerable gene x diet interaction. This demonstrates that genetics plays a major role in diet responses. Furthermore, heritability analysis revealed that the greatest genetic variability arose from diets either high in sugar or high in protein. To uncover the genetic variants that contribute to the heterogeneity in starvation resistance, this study mapped 566 diet-responsive SNPs in 293 genes, 174 of which have human orthologs. Using whole-body knockdown, this study identified two genes that were required for glucose tolerance, storage, and utilization. Strikingly, flies in which the expression of one of these genes, CG4607 a putative homolog of a mammalian glucose transporter, was reduced at the whole-body level, displayed lethality on a HCD. This study provides evidence that there is a strong interplay between diet and genetics in governing survival in response to starvation, a surrogate measure of nutrient storage efficiency and obesity. It is likely that a similar principle applies to higher organisms thus supporting the case for nutrigenomics as an important health strategy (Ghazanfar, 2021).
Sujkowski, A. and Wessells, R. (2021). Exercise and Sestrin Mediate Speed and Lysosomal Activity in Drosophila by Partially Overlapping Mechanisms. Cells 10(9). PubMed ID: 34572128
Summary:
Chronic exercise is widely recognized as an important contributor to healthspan in humans and in diverse animal models. Recently, it has been demonstrated that Sestrins, a family of evolutionarily conserved exercise-inducible proteins, are critical mediators of exercise benefits in flies and mice. Knockout of Sestrins prevents exercise adaptations to endurance and flight in Drosophila, and similarly prevents benefits to endurance and metabolism in exercising mice. In contrast, overexpression of dSestrin in muscle mimics several of the molecular and physiological adaptations characteristic of endurance exercise. This study extended those observations to examine the impact of dSestrin on preserving speed and increasing lysosomal activity. dSestrin was found to be a critical factor driving exercise adaptations to climbing speed, but is not absolutely required for exercise to increase lysosomal activity in Drosophila. The role of Sestrin in increasing speed during chronic exercise requires both the TORC2/AKT axis and the PGC1α homolog Spargel, while dSestrin requires interactions with TORC1 to cell-autonomously increase lysosomal activity. These results highlight the conserved role of Sestrins as key factors that drive diverse physiological adaptations conferred by chronic exercise.
Davies, L. R., Loeschcke, V., Schou, M. F., Schramm, A. and Kristensen, T. N. (2021). The importance of environmental microbes for Drosophila melanogaster during seasonal macronutrient variability. Sci Rep 11(1): 18850. PubMed ID: 34552121
Summary:
There is little information on how macronutrient composition and bacterial communities in natural food sources vary across seasons in nature and on how these factors affect the fitness components of insects. In this study, diet samples from an orchard compost heap, which is a natural habitat for many Drosophila species and other arthropods, were collected over 9 months covering all seasons in a temperate climate. It developed D. melanogaster on diet samples and investigated stress resistance and life-history traits as well as the microbial community of flies and compost. Nutrient and microbial community analysis of the diet samples showed marked differences in macronutrient composition and microbial community across seasons. However, except for the duration of development on these diet samples and Critical Thermal maximum, fly stress resistance and life-history traits were unaffected. The resulting differences in the fly microbial community were also more stable and less diverse than the microbial community of the diet samples. This study suggests that when D. melanogaster are exposed to a vastly varying nutritional environment with a rich, diverse microbial community, the detrimental consequences of an unfavourable macronutrient composition are offset by the complex interactions between microbes and nutrients (Davis, 2021).
de Brito Sanchez, G., Exposito Munoz, A., Chen, L., Huang, W., Su, S. and Giurfa, M. (2021). Adipokinetic hormone (AKH), energy budget and their effect on feeding and gustatory processes of foraging honey bees. Sci Rep 11(1): 18311. PubMed ID: 34526585
Summary:
The adipokinetic hormone (AKH) of insects is considered an equivalent of the mammalian hormone glucagon as it induces fast mobilization of carbohydrates and lipids from the fat body upon starvation. Yet, in foraging honey bees, which lack fat body storage for carbohydrates, it was suggested that AKH may have lost its original function. This study the energy budget of bee foragers was manipulated to determine the effect of AKH on appetitive responses. As AKH participates in a cascade leading to acceptance of unpalatable substances in starved Drosophila, This study also assessed its effect on foragers presented with sucrose solution spiked with salicin. Starved and partially-fed bees were topically exposed with different doses of AKH to determine if this hormone modifies food ingestion and sucrose responsiveness. Studies found a significant effect of the energy budget (i.e. starved vs. partially-fed) on the decision to ingest or respond to both pure sucrose solution and sucrose solution spiked with salicin, but no effect of AKH per se. These results are consistent with a loss of function of AKH in honey bee foragers, in accordance with a social life that implies storing energy resources in the hive, in amounts that exceed individual needs (Sanchez, 2021).
Shell, B. C., Luo, Y., Pletcher, S. and Grotewiel, M. (2021). Expansion and application of dye tracers for measuring solid food intake and food preference in Drosophila. Sci Rep 11(1): 20044. PubMed ID: 34625601
Summary:
The Drosophila model is used to investigate the effects of diet on physiology as well as the effects of genetic pathways, neural systems and environment on feeding behavior. Previous work showed that Blue 1 works well as a dye tracer to track consumption of agar-based media in Drosophila in a method called Consumption-Excretion (Con-Ex. This study describes Orange 4 as a novel dye for use in Con-Ex studies that expands the utility of this method. Con-Ex experiments using Orange 4 detect the predicted effects of starvation, mating status, strain, and sex on feeding behavior in flies. Orange 4 is consumed and excreted into vials linearly with time in Con-Ex experiments, the number of replicates required to detect differences between groups when using Orange 4 is comparable to that for Blue 1, and excretion of the dye reflects the volume of consumed dye. In food preference studies using Orange 4 and Blue 1 as a dye pair, flies decreased their intake of food laced with the aversive tastants caffeine and NaCl as determined using Con-Ex or a more recently described modification called EX-Q. These results indicate that Orange 4 is suitable for Con-Ex experiments, has comparable utility to Blue 1 in Con-Ex studies, and can be paired with Blue 1 to assess food preference via both Con-Ex and EX-Q.

Monday, November 22nd - Signaling

Uckun, E., Wolfstetter, G., Anthonydhason, V., Kumar Sukumar, S., Umapathy, G., Molander, L., Fuchs, J. and Palmer, R. H. (2021). In vivo profiling of the Alk proximitome in the developing Drosophila brain. J Mol Biol: 167282. PubMed ID: 34624297
Summary:
Anaplastic lymphoma kinase (Alk) is an evolutionary conserved receptor tyrosine kinase belonging to the insulin receptor superfamily. In addition to its well-studied role in cancer, numerous studies have revealed that Alk-signaling is associated with a variety of complex traits such as: regulation of growth and metabolism, hibernation, regulation of neurotransmitters, synaptic coupling, axon targeting, decision making, memory formation and learning, alcohol use disorder, as well as steroid hormone metabolism. This study used BioID-based in vivo proximity labeling to identify molecules that interact with Alk in the Drosophila CNS. To do this, CRISPR/Cas9 induced homology-directed repair (HDR) was used to modify the endogenous Alk locus to produce first and next generation Alk::BioID chimeras. This approach allowed identification of Alk proximitomes under physiological conditions and without overexpression. The results show that the next generation of BioID proteins (TurboID and miniTurbo) outperform the first generation BirA* fusion in terms of labeling speed and efficiency. LC-MS3-based BioID screening of Alk(TurboID) and Alk(miniTurbo) larval brains revealed an extensive neuronal Alk proximitome identifying numerous potential components of Alk signaling complexes. Validation of Alk proximitome candidates further revealed co-expression of Stardust (Sdt), Discs large 1 (Dlg1), Syntaxin (Syx) and Rugose (Rg) with Alk in the CNS and identified the protein-tyrosine-phosphatase Corkscrew (Csw) as a modulator of Alk signaling.
Yang, S., Constantin, O. M., Sachidanandan, D., Hofmann, H., Kunz, T. C., Kozjak-Pavlovic, V., Oertner, T. G., Nagel, G., Kittel, R. J., Gee, C. E. and Gao, S. (2021). PACmn for improved optogenetic control of intracellular cAMP. BMC Biol 19(1): 227. PubMed ID: 34663304
Summary:
Cyclic adenosine monophosphate (cAMP) is a ubiquitous second messenger that transduces extracellular signals in virtually all eukaryotic cells. The soluble Beggiatoa photoactivatable adenylyl cyclase (bPAC) rapidly raises cAMP in blue light and has been used to study cAMP signaling pathways cell-autonomously. The aim of this study was to engineer a plasma membrane-anchored PAC with no dark activity (i.e., no cAMP accumulation in the dark) that rapidly increases cAMP when illuminated. Using expression in Xenopus oocytes, natural PACs and confirmed bPAC were compared as the best starting point for protein engineering efforts. Several modifications were identified that reduce bPAC dark activity. Mutating a phenylalanine to tyrosine at residue 198 substantially decreased dark cyclase activity, which increased 7000-fold when illuminated. Whereas Drosophila larvae expressing bPAC in mechanosensory neurons show nocifensive-like behavior even in the dark, larvae expressing improved soluble (e.g., bPAC(R278A)) and membrane-anchored PACs exhibited nocifensive responses only when illuminated. The plasma membrane-anchored PAC (PACmn) had an undetectable dark activity which increased >4000-fold in the light. PACmn does not raise resting cAMP nor, when expressed in hippocampal neurons, affect cAMP-dependent kinase (PKA) activity in the dark, but rapidly and reversibly increases cAMP and PKA activity in the soma and dendrites upon illumination. The peak responses to brief (2 s) light flashes exceeded the responses to forskolin-induced activation of endogenous cyclases and returns to baseline within seconds (cAMP) or ~10 min (PKA). PACmn is a valuable optogenetic tool for precise cell-autonomous and transient stimulation of cAMP signaling pathways in diverse cell types.
Gao, J., Zhao, B. R., Zhang, H., You, Y. L., Li, F. and Wang, X. W. (2021). Interferon functional analog activates antiviral Jak/Stat signaling through integrin in an arthropod. Cell Rep 36(13): 109761. PubMed ID: 34592151
Summary:
Drosophila Vago is a small antiviral peptide. Its ortholog in Culex mosquito was found to be an interferon-like cytokine that limits virus replication through activating Jak/Stat signaling. However, this activation is independent of Domeless, the sole homolog of vertebrate type I cytokine receptor. How Vago activates the Jak/Stat pathway remains unknown. Herein, this process is dependent on integrin in kuruma shrimp (Marsupenaeus japonicus). Shrimp Vago-like (MjVago-L) plays an antiviral role by activating the Jak/Stat pathway and inducing Stat-regulated Ficolin. Blocking integrin abrogates the role of MjVago-L. The interaction between MjVago-L and integrin β3 is confirmed. An Asp residue in MjVago-L is found critical for the interaction and MjVago-L's antiviral role. Moreover, Fak, a key adaptor of integrin signaling, mediates MjVago-L-induced Jak/Stat activation. Therefore, this study reveals that integrin, as the receptor of MjVago-L, mediates Jak/Stat activation. The establishment of the MjVago-L/integrin/Fak/Jak/Stat/Ficolin axis provides insights into antiviral cytokine signaling in invertebrates. (Gao, 2021)
De Munck, S., Provost, M., Kurikawa, M., Omori, I., Mukohyama, J., Felix, J., Bloch, Y., Abdel-Wahab, O., Bazan, J. F., Yoshimi, A. and Savvides, S. N. (2021). Structural basis of cytokine-mediated activation of ALK family receptors. Nature. PubMed ID: 34646012
Summary:
Anaplastic lymphoma kinase (ALK) and the related leukocyte tyrosine kinase (LTK) are recently deorphanized receptor tyrosine kinases. Together with their activating cytokines, ALKAL1 and ALKAL2, they are involved in neural development, cancer band autoimmune diseases. Furthermore, mammalian ALK recently emerged as a key regulator of energy expenditure and weight gain, consistent with a metabolic role for Drosophila ALK. This study shows that the cytokine-binding segments of human ALK and LTK comprise a novel architectural chimera of a permuted TNF-like module that braces a glycine-rich subdomain featuring a hexagonal lattice of long polyglycine type II helices. The cognate cytokines ALKAL1 and ALKAL2 are monomeric three-helix bundles, yet their binding to ALK and LTK elicits similar dimeric assemblies with two-fold symmetry, that sent a single cytokine molecule proximal to the cell membrane. This study showed that the membrane-proximal EGF-like domain dictates the apparent cytokine preference of ALK. A structural and mechanistic blueprint is proposedfor complexes of ALK family receptors, and thereby the repertoire of ligand-mediated dimerization mechanisms adopted by receptor tyrosine kinases is extended (De Munck, 2021).
Wang, J., Liu, Q., Gong, Y. and Jin, L. H. (2021). Anchor maintains gut homeostasis by restricting the JNK and Notch pathways in Drosophila. J Insect Physiol 134: 104309. PubMed ID: 34496279
Summary:
The adult Drosophila intestinal epithelium must be tightly regulated to maintain regeneration and homeostasis. The dysregulation of the regenerative capacity is frequently associated with intestinal diseases such as inflammation and tumorigenesis. This study shows that the G protein-coupled receptor Anchor maintains Drosophila adult midgut homeostasis by restricting Jun-N-terminal kinase (JNK) and Notch pathway activity. anchor inactivation resulted in aberrant JNK pathway activation, which led to excessive enteroblast (EB) production and premature enterocyte (EC) differentiation. In addition, increased Notch levels promoted premature EC differentiation following the loss of anchor. This defect induced by the loss of anchor ultimately caused sensitivity to stress or environmental challenge in adult flies. Taken together, these results demonstrate that the activity of anchor is essential to coordinate stem cell differentiation and proliferation to maintain intestinal homeostasis.
Toddie-Moore, D. J., Montanari, M. P., Tran, N. V., Brik, E. M., Antson, H., Salazar-Ciudad, I. and Shimmi, O. (2021).. Mechano-chemical feedback mediated competition for BMP signalling leads to pattern formation. Dev Biol 481: 43-51. PubMed ID: 34555363
Summary:
Developmental patterning is thought to be regulated by conserved signalling pathways. Initial patterns are often broad before refining to only those cells that commit to a particular fate. However, the mechanisms by which pattern refinement takes place remain to be addressed. Using the posterior crossvein (PCV) of the Drosophila pupal wing as a model, into which bone morphogenetic protein (BMP) ligand is extracellularly transported to instruct vein patterning, this study investigate how pattern refinement is regulated. It was found that BMP signalling induces apical enrichment of Myosin II in developing crossvein cells to regulate apical constriction. Live imaging of cellular behaviour indicates that changes in cell shape are dynamic and transient, only being maintained in those cells that retain vein fate competence after refinement. Disrupting cell shape changes throughout the PCV inhibits pattern refinement. In contrast, disrupting cell shape in only a subset of vein cells can result in a loss of BMP signalling. It is proposed that mechano-chemical feedback leads to competition for the developmental signal which plays a critical role in pattern refinement.

Friday, November 19th - Early Neural Development

Carreira-Rosario, A., York, R. A., Choi, M., Doe, C. Q. and Clandinin, T. R. (2021). Mechanosensory input during circuit formation shapes Drosophila motor behavior through patterned spontaneous network activity. Curr Biol. PubMed ID: 34478644
Summary:
Neural activity sculpts circuit wiring in many animals. In vertebrates, patterned spontaneous network activity (PaSNA) generates sensory maps and establishes local circuits. However, it remains unclear how PaSNA might shape neuronal circuits and behavior in invertebrates. Previous work in the developing Drosophila embryo discovered intrinsic muscle activity that did not require synaptic transmission, and hence was myogenic, preceding PaSNA. These studies, however, monitored muscle movement, not neural activity, and were therefore unable to observe how myogenic activity might relate to subsequent neural network engagement. This study use calcium imaging to directly record neural activity and characterize the emergence of PaSNA. The spatiotemporal properties of PaSNA are highly stereotyped across embryos, arguing for genetic programming. Neural activity begins well before it becomes patterned, emerging during the myogenic stage. Remarkably, inhibition of mechanosensory input, as well as inhibition of muscle contractions, results in premature and excessive PaSNA, demonstrating that muscle movement serves as a brake on this process. Finally, transient mechanosensory inhibition during PaSNA, followed by quantitative modeling of larval behavior, shows that mechanosensory modulation during development is required for proper larval foraging. This work provides a foundation for using the Drosophila embryo to study the role of PaSNA in circuit formation, provides mechanistic insight into how PaSNA is entrained by motor activity, and demonstrates that spontaneous network activity is essential for locomotor behavior. These studies argue that sensory feedback during the earliest stages of circuit formation can sculpt locomotor behaviors through innate motor learning.
Howard, L. J., Reichert, M. C. and Evans, T. A. (2021). The Slit-binding Ig1 domain is required for multiple axon guidance activities of Drosophila Robo2. Genesis: e23443. PubMed ID: 34411419
Summary:
Drosophila Robo2 is a member of the evolutionarily conserved Roundabout (Robo) family of axon guidance receptors. Robo receptors signal midline repulsion in response to Slit ligands, which bind to the N-terminal Ig1 domain in most family members. In the Drosophila embryonic ventral nerve cord, Robo1 and Robo2 signal Slit-dependent midline repulsion, while Robo2 also regulates the medial-lateral position of longitudinal axon pathways and acts non-autonomously to promote midline crossing of commissural axons. While Robo2 signals midline repulsion in response to Slit, it is less clear whether Robo2's other activities are also Slit-dependent. To determine which of Robo2's axon guidance roles depend on its Slit-binding Ig1 domain, this study used a clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based strategy to replace the endogenous robo2 gene with a robo2 variant lacking the Ig1 domain (robo2ΔIg1). The expression and localization of Robo2ΔIg1 protein with full-length Robo2 in embryonic neurons in vivo and examine its ability to substitute for Robo2 to mediate midline repulsion and lateral axon pathway formation. This study found that the removal of the Ig1 domain from Robo2ΔIg1 disrupts both of these axon guidance activities. In addition, it was found that the Ig1 domain of Robo2 is required for its proper subcellular localization in embryonic neurons, a role that is not shared by the Ig1 domain of Robo1. Finally, it is reported that although FasII-positive lateral axons are misguided in embryos expressing Robo2$Delta;'Ig1, the axons that normally express Robo2 are correctly guided to the lateral zone, suggesting that Robo2 may guide lateral longitudinal axons through a cell non-autonomous mechanism.
Chaudhari, K., Gorla, M., Chang, C., Kania, A. and Bashaw, G. J. (2021) . Robo recruitment of the Wave regulatory complex plays an essential and conserved role in midline repulsion. Elife 10. PubMed ID: 33843588
Summary:
The Roundabout (Robo) guidance receptor family induces axon repulsion in response to its ligand Slit by inducing local cytoskeletal changes; however, the link to the cytoskeleton and the nature of these cytoskeletal changes are poorly understood. This study shows that the heteropentameric Scar/Wave Regulatory Complex (WRC), which drives Arp2/3-induced branched actin polymerization, is a direct effector of Robo signaling. Biochemical evidence shows that Slit triggers WRC recruitment to the Robo receptor's WRC-interacting receptor sequence (WIRS) motif. In Drosophila embryos, mutants of the WRC enhance Robo1-dependent midline crossing defects. Additionally, mutating Robo1's WIRS motif significantly reduces receptor activity in rescue assays in vivo, and CRISPR-Cas9 mutagenesis shows that the WIRS motif is essential for endogenous Robo1 function. Finally, axon guidance assays in mouse dorsal spinal commissural axons and gain-of-function experiments in chick embryos demonstrate that the WIRS motif is also required for Robo1 repulsion in mammals. Together, these data support an essential conserved role for the WIRS-WRC interaction in Robo1-mediated axon repulsion.
Gallicchio, L., Griffiths-Jones, S. and Ronshaugen, M. (2021). Single-cell visualization of mir-9a and Senseless co-expression during Drosophila melanogaster embryonic and larval peripheral nervous system development. G3 (Bethesda) 11(1). PubMed ID: 33561238
Summary:
The Drosophila melanogaster peripheral nervous system (PNS) comprises the sensory organs that allow the fly to detect environmental factors such as temperature and pressure. PNS development is a highly specified process where each sensilla originates from a single sensory organ precursor (SOP) cell. One of the major genetic orchestrators of PNS development is Senseless, which encodes a zinc finger transcription factor (Sens). Sens is both necessary and sufficient for SOP differentiation. Senseless expression and SOP number are regulated by the microRNA miR-9a. However, the reciprocal dynamics of Senseless and miR-9a are still obscure. By coupling single-molecule FISH with immunofluorescence, it was possible to visualize transcription of the mir-9a locus and expression of Sens simultaneously. During embryogenesis, it was shown that the expression of mir-9a in SOP cells is rapidly lost as Senseless expression increases. However, this mutually exclusive expression pattern is not observed in the third instar imaginal wing disk, where some Senseless-expressing cells show active sites of mir-9a transcription. These data challenge and extend previous models of Senseless regulation and show complex co-expression dynamics between mir-9a and Senseless. The differences in this dynamic relationship between embryonic and larval PNS development suggest a possible switch in miR-9a function. This work brings single-cell resolution to the understanding of dynamic regulation of PNS development by Senseless and miR-9a (Gallicchiom 2021).
Valdes-Aleman, J., Fetter, R. D., Sales, E. C., Heckman, E. L., Venkatasubramanian, L., Doe, C. Q., Landgraf, M., Cardona, A. and Zlatic, M. (2020). Comparative Connectomics Reveals How Partner Identity, Location, and Activity Specify Synaptic Connectivity in Drosophila. Neuron. PubMed ID: 33120017
Summary:
The mechanisms by which synaptic partners recognize each other and establish appropriate numbers of connections during embryonic development to form functional neural circuits are poorly understood. This study combined electron microscopy reconstruction, functional imaging of neural activity, and behavioral experiments to elucidate the roles of (1) partner identity, (2) location, and (3) activity in circuit assembly in the embryonic nerve cord of Drosophila. Postsynaptic partners were found to be able to find and connect to their presynaptic partners even when these have been shifted to ectopic locations or silenced. However, orderly positioning of axon terminals by positional cues and synaptic activity is required for appropriate numbers of connections between specific partners, for appropriate balance between excitatory and inhibitory connections, and for appropriate functional connectivity and behavior. This study reveals with unprecedented resolution the fine connectivity effects of multiple factors that work together to control the assembly of neural circuits (Valdes-Aleman, 2021).
Houssin, E., Pinot, M., Bellec, K. and Le Borgne, R. (2021). Par3 cooperates with Sanpodo for the assembly of Notch clusters following asymmetric division of Drosophila sensory organ precursor cells . Elife 10. PubMed ID: 34596529
Summary:
In multiple cell lineages, Delta-Notch signalling regulates cell fate decisions owing to unidirectional signalling between daughter cells. In Drosophila pupal sensory organ lineage, Notch regulates the intra-lineage pIIa/pIIb fate decision at cytokinesis. Notch and Delta that localise apically and basally at the pIIa-pIIb interface are expressed at low levels and their residence time at the plasma membrane is in the order of minutes. How Delta can effectively interact with Notch to trigger signalling from a large plasma membrane area remains poorly understood. this study reports the signalling interface possesses a unique apico-basal polarity with Par3/Bazooka localising in the form of nano-clusters at the apical and basal level. Notch is preferentially targeted to the pIIa-pIIb interface, where it co-clusters with Bazooka and its cofactor Sanpodo. Clusters whose assembly relies on Bazooka and Sanpodo activities are also positive for Neuralized, the E3 ligase required for Delta activity. This study proposes that the nano-clusters act as snap buttons at the new pIIa-pIIb interface to allow efficient intra-lineage signalling (Houssin, 2021).

Thursday, November 19th - Adult Neural Development and function

Tanaka, N. K., Hirao, T., Chida, H. and Ejima, A. (2021). A sexually dimorphic olfactory neuron mediates fixed action transition during courtship ritual in Drosophila melanogaster. J Neurosci. PubMed ID: 34649953
Summary:
Animals perform a series of actions in a fixed order during ritualistic innate behaviors. Although command neurons and sensory pathways responding to external stimuli that trigger these behaviors have been identified, how each action is induced in a fixed order in response to multimodal sensory stimuli remains unclear. In this study, the sexually dimorphic lateral antennal lobe tract projection neuron 4 (lPN4) in male Drosophila melanogaster mediates the expression of a fixed behavioral action pattern at the beginning of the courtship ritual, in which a male taps a female body and then extends a wing unilaterally to produce a courtship song. Blocking the synaptic output of lPN4 caused an increase in the ratio of male flies that extended a wing unilaterally without tapping the female body, whereas excitation of lPN4 suppressed the transition from the tapping phase to the unilateral wing extension phase. Real-time calcium imaging showed that lPN4 is activated by a volatile pheromone, palmitoleic acid, whose responses were inhibited by simultaneous gustatory stimulation with female cuticular hydrocarbons, showing the existence of an "AND-gate" for multimodal sensory inputs during male courtship behaviors. These results suggest that the function of lPN4 is to suppress unilateral wing extension while responding to a female smell, which is released by appropriate contact chemosensory inputs received when tapping a female. As the female smell also promotes male courtship behaviors, the olfactory system is ready to simultaneously promote and suppress the progress of courtship actions while responding to a female smell (Tanaka, 2021).
Driscoll, M., Buchert, S. N., Coleman, V., McLaughlin, M., Nguyen, A. and Sitaraman, D. (2021). Compartment specific regulation of sleep by mushroom body requires GABA and dopaminergic signaling. Sci Rep 11(1): 20067. PubMed ID: 34625611
Summary:
Sleep is a fundamental behavioral state important for survival and is universal in animals with sufficiently complex nervous systems. Biogenic amines like dopamine, serotonin and norepinephrine have been shown to be critical for sleep regulation across species but the precise circuit mechanisms underlying how amines control persistence of sleep, arousal and wakefulness remain unclear. The fruit fly, Drosophila melanogaster, provides a powerful model system for the study of sleep and circuit mechanisms underlying state transitions and persistence of states to meet the organisms motivational and cognitive needs. In Drosophila, two neuropils in the central brain, the mushroom body (MB) and the central complex (CX) have been shown to influence sleep homeostasis and receive aminergic neuromodulator input critical to sleep-wake switch. Dopamine neurons (DANs) are prevalent neuromodulator inputs to the MB but the mechanisms by which they interact with and regulate sleep- and wake-promoting neurons within MB are unknown. This study investigated the role of subsets of PAM-DANs that signal wakefulness and project to wake-promoting compartments of the MB. This study found that PAM-DANs are GABA responsive and require GABA(A)-Rdl receptor in regulating sleep. In mapping the pathways downstream of PAM neurons innervating γ5 and β'2 MB compartments it was found that wakefulness is regulated by both DopR1 and DopR2 receptors in downstream Kenyon cells (KCs) and mushroom body output neurons (MBONs). Taken together, a dopamine modulated sleep microcircuit has been identified within the mushroom body that has previously been shown to convey information about positive and negative valence critical for memory formation. These studies will pave the way for understanding how flies balance sleep, wakefulness and arousal (Buchert, 2021).
Cruz, T. L., Perez, S. M. and Chiappe, M. E. (2021). Fast tuning of posture control by visual feedback underlies gaze stabilization in walking Drosophila. Curr Biol. PubMed ID: 34499851
Summary:
Locomotion requires a balance between mechanical stability and movement flexibility to achieve behavioral goals despite noisy neuromuscular systems, but rarely is it considered how this balance is orchestrated. This study combined virtual reality tools with quantitative analysis of behavior to examine how Drosophila uses self-generated visual information (reafferent visual feedback) to control gaze during exploratory walking. It was found that flies execute distinct motor programs coordinated across the body to maximize gaze stability. However, the presence of inherent variability in leg placement relative to the body jeopardizes fine control of gaze due to posture-stabilizing adjustments that lead to unintended changes in course direction. Surprisingly, whereas visual feedback is dispensable for head-body coordination, studies found that self-generated visual signals tune postural reflexes to rapidly prevent turns rather than to promote compensatory rotations, a long-standing idea for visually guided course control. Together, these findings support a model in which visual feedback orchestrates the interplay between posture and gaze stability in a manner that is both goal dependent and motor-context specific (Cruz, 2021).
Guangming, G., Mei, C., Chenchen, Z., Wei, X. and Junhua, G. (2021). Improved analysis method of neuromuscular junction in Drosophila larvae by transmission electron microscopy. Anat Sci Int. PubMed ID: 34661863
Summary:
The Drosophila neuromuscular junction is an excellent model for neuroscience research. However, the distribution of neuromuscular junctions is very diffuse, and it is not easy to accurately locate during ultrathin sectioning, which seriously interferes with the ultrastructural analysis under electron microscopy that only has a small field of view. This study reports an efficient method for acquiring the ultrastructural picture of neuromuscular junctions in Drosophila larva under electron microscopy. The procedure was as follows: first, the larval sample of body wall muscle was placed between the metal mesh and was dehydrated with alcohol and infiltrated with epoxy resin to prevent the sample from curling or bending, after it was dissected and fixed into thin slices. Second, the sample was embedded in resin into a flat sheet to facilitate the positioning of the muscles. Third, carefully and gradually remove the excess resin and the cuticle of the larvae, cut off both ends of the special body segment, and trim the excess specific muscles according to the recommended ratio of trimming muscles, which would reduce the workload exponentially. At last, the trimmed sample were prepared into serial about 1000 ultrathin sections that was about total 80 microns thickness, and 30-40 sections were gathered into a grid to stain with lead citrate and uranyl acetate. This method could also be applied to the other small and thin samples such as the Drosophila embryo, ventral nerve cord and brain (Guangming, 2021).
Chen, C., Agrawal, S., Mark, B., Mamiya, A., Sustar, A., Phelps, J. S., Lee, W. A., Dickson, B. J., Card, G. M. and Tuthill, J. C. (2021). Functional architecture of neural circuits for leg proprioception in Drosophila. Curr Biol. PubMed ID: 34637749
Summary:
To effectively control their bodies, animals rely on feedback from proprioceptive mechanosensory neurons. In the Drosophila leg, different proprioceptor subtypes monitor joint position, movement direction, and vibration. How these diverse sensory signals are integrated by central proprioceptive circuits was investigated in this study. Signals for leg joint position and directional movement were found to converge in second-order neurons, revealing pathways for local feedback control of leg posture. Distinct populations of second-order neurons integrate tibia vibration signals across pairs of legs, suggesting a role in detecting external substrate vibration. In each pathway, the flow of sensory information is dynamically gated and sculpted by inhibition. Overall, the results reveal parallel pathways for processing of internal and external mechanosensory signals, which are proposed to mediate feedback control of leg movement and vibration sensing, respectively. The existence of a functional connectivity map also provides a resource for interpreting connectomic reconstruction of neural circuits for leg proprioception (Chen, 2021).
Fenk, L. M., Kim, A. J. and Maimon, G. (2021). Suppression of motion vision during course-changing, but not course-stabilizing, navigational turns. Curr Biol. PubMed ID: 34644548
Summary:
From mammals to insects, locomotion has been shown to strongly modulate visual-system physiology. Does the manner in which a locomotor act is initiated change the modulation observed? Patch-clamp recordings were performed from motion-sensitive visual neurons in tethered, flying Drosophila. This study observed motor-related signals in flies performing flight turns in rapid response to looming discs and also during spontaneous turns, but motor-related signals were weak or non-existent in the context of turns made in response to brief pulses of unidirectional visual motion (i.e., optomotor responses). Thus, the act of a locomotor turn is variably associated with modulation of visual processing. These results can be understood via the following principle: suppress visual responses during course-changing, but not course-stabilizing, navigational turns. This principle is likely to apply broadly-even to mammals-whenever visual cells whose activity helps to stabilize a locomotor trajectory or the visual gaze angle are targeted for motor modulation (Fenk, 2021).

Wednesday, November 17th - Transcriptional Regulation

Dibaeinia, P. and Sinha, S. (2021). Deciphering enhancer sequence using thermodynamics-based models and convolutional neural networks. Nucleic Acids Res 49(18): 10309-10327. PubMed ID: 34508359
Summary:
Deciphering the sequence-function relationship encoded in enhancers holds the key to interpreting non-coding variants and understanding mechanisms of transcriptomic variation. Several quantitative models exist for predicting enhancer function and underlying mechanisms; however, there has been no systematic comparison of these models characterizing their relative strengths and shortcomings. In this study a rich data set was interrogated of neuroectodermal enhancers in Drosophila, representing cis- and trans- sources of expression variation, with a suite of biophysical and machine learning models. In this study was performed rigorous comparisons of thermodynamics-based models implementing different mechanisms of activation, repression and cooperativity. Moreover, this study developed a convolutional neural network (CNN) model, called CoNSEPT, that learns enhancer 'grammar' in an unbiased manner. CoNSEPT is the first general-purpose CNN tool for predicting enhancer function in varying conditions, such as different cell types and experimental conditions, and it was shown that such complex models can suggest interpretable mechanisms. Model-based evidence was found for mechanisms previously established for the studied system, including cooperative activation and short-range repression. The data also favored one hypothesized activation mechanism over another and suggested an intriguing role for a direct, distance-independent repression mechanism. This modeling shows that while fundamentally different models can yield similar fits to data, they vary in their utility for mechanistic inference (Dibaeinia, 2021).
Eremina, M. A., Menshanov, P. N., Shishkina, O. D. and Gruntenko, N. E. (2021). The transcription factor dfoxo controls the expression of insulin pathway genes and lipids content under heat stress in Drosophila melanogaster. Vavilovskii Zhurnal Genet Selektsii 25(5): 465-471. PubMed ID: 34595369
Summary:
The insulin/insulin-like growth factor signaling (IIS) pathway is one of the key elements in an organism's response to unfavourable conditions. To identify the properties of interaction of two key IIS pathway components under heat stress in D. melanogaster (the forkhead box O transcription factor (dfoxo) and insulin-like peptide 6 (dilp6), which intermediates the dfoxo signal sent from the fat body to the insulin-producing cells of the brain where DILPs1-5 are synthesized), the expression of the genes dilp6, dfoxo and insulin-like receptor gene (dInR) was analyzed in females of strains carrying the hypomorphic mutation dilp6 and hypofunctional mutation foxo (BG01018). This study found that neither mutation influenced dfoxo expression and its uprise under short-term heat stress, but both of them disrupted the stress response of the dilp6 and dInR genes. To reveal the role of identified disruptions in metabolism control and feeding behaviour, this study analysed the effect of the dilp6 and foxo (BG01018) mutations on total lipids content and capillary feeding intensity in the imago under normal conditions and under short-term heat stress. Both mutations caused an increase in these parameters under normal conditions and prevented decrease in total lipids content following heat stress observed in the control strain. In mutants, feeding intensity was increased under normal conditions; and decreased following short-term heat stress in all studied strains for the first 24 h of observation, and in dilp6 41 strain, for 48 h. Thus, it can conclude that dfoxo takes part in regulating the IIS pathway response to heat stress as well as the changes in lipids content caused by heat stress, and this regulation is mediated by dilp6. At the same time, the feeding behaviour of imago might be controlled by dfoxo and dilp6 under normal conditions, but not under heat stress (Eremina, 2021).
Banzai, K. and Izumi, S. (2021). Cis-regulatory elements of the cholinergic gene locus in the silkworm Bombyx mori. Insect Mol Biol. PubMed ID: 34549831
Summary:
Genes of choline acetyltransferase (ChAT) and vesicular acetylcholine transporter are encoded in the same gene locus, called the cholinergic gene locus. They are essential in cholinergic neurons to maintain their functional phenotype. The genomic structure of the cholinergic gene locus is conserved among invertebrates to mammals. However, the cholinergic gene expression in a specific subset of neurons is unknown in insects except for Drosophila melanogaster. This study analysed the upstream sequence of cholinergic gene locus in the silkworm Bombyx mori to identify specific cis-regulatory regions. Multiple enhancer regions were found that are localized within 1 kb upstream of the cholinergic gene locus. The combination of promoter assays using small deletions and bioinformatic analysis among insect species illuminates two conserved sequences in the cis-regulatory region: TGACGTA and CCAAT, which are known as the cAMP response element and CAAT box, respectively. Dibutyryl-cAMP, an analogue of cAMP, influences the expression of ChAT in B. mori. Tissue-specific expression analysis of transcriptional factors identified potential candidates that control the cholinergic gene locus expression. This investigation provides new insight into the regulation mechanism of cholinergic neuron-specific gene machinery in this lepidopteran insect.
Verma, S., Pathak, R. U. and Mishra, R. K. (2021). Genomic organization of the autonomous regulatory domain of eyeless locus in Drosophila melanogaster. G3 (Bethesda). PubMed ID: 34570231
Summary:
In Drosophila, expression of eyeless (ey) gene is restricted to the developing eyes and central nervous system. However, the flanking genes, myoglianin (myo), and bent (bt) have different temporal and spatial expression patterns as compared to the ey. How distinct regulation of ey is maintained is mostly unknown. Earlier work identified a boundary element intervening myo and ey genes (ME boundary) that prevents the crosstalk between the cis-regulatory elements of myo and ey genes. The present study further searched for the cis-elements that define the domain of ey and maintain its expression pattern. Another boundary element was identified between ey and bt, the EB boundary. The EB boundary separates the regulatory landscapes of ey and bt genes. The two boundaries, ME and EB, show a long-range interaction as well as interact with the nuclear architecture. This suggests functional autonomy of the ey locus and its insulation from differentially regulated flanking regions. This study also identified a new Polycomb Response Element, the ey-PRE, within the ey domain. The expression state of the ey gene, once established during early development is likely to be maintained with the help of ey-PRE. This study proposes a general regulatory mechanism by which a gene can be maintained in a functionally independent chromatin domain in gene-rich euchromatin.
Waymack, R., Gad, M. and Wunderlich, Z. (2021). Molecular competition can shape enhancer activity in the Drosophila embryo. iScience 24(9): 103034. PubMed ID: 34568782
Summary:
Transgenic reporters allow the measurement of regulatory DNA activity in vivo and consequently have long been useful tools for studying enhancers. Despite their utility, few studies have investigated the effects these reporters may have on the expression of other genes. Understanding these effects is required to accurately interpret reporter data and characterize gene regulatory mechanisms. By measuring the expression of Kruppel (Kr) enhancer reporters in live Drosophila embryos, it was found that reporters inhibit one another's expression and that of a nearby endogenous gene. Using synthetic transcription factor (TF) binding site arrays, evidence is presented that competition for TFs is partially responsible for the observed transcriptional inhibition. A simple thermodynamic model was developed that predicts competition of the measured magnitude specifically when TF binding is restricted to distinct nuclear subregions. These findings underline an unexpected role of the non-homogenous nature of the nucleus in regulating gene expression.
Vuong-Brender, T. T., Flynn, S., Vallis, Y. and de Bono, M. (2021). Neuronal calmodulin levels are controlled by CAMTA transcription factors. Elife 10. PubMed ID: 34499028
Summary:
The ubiquitous Ca(2+) sensor calmodulin (CaM) binds and regulates many proteins, including ion channels, CaM kinases, and calcineurin, according to Ca(2+)-CaM levels. What regulates neuronal CaM levels, is, however, unclear. CaM-binding transcription activators (CAMTAs) are ancient proteins expressed broadly in nervous systems and whose loss confers pleiotropic behavioral defects in flies, mice, and humans. Using Caenorhabditis elegans and Drosophila, this study showed that CAMTAs control neuronal CaM levels. The behavioral and neuronal Ca(2+) signaling defects in mutants lacking camt-1, the sole C. elegans CAMTA, can be rescued by supplementing neuronal CaM. CAMT-1 binds multiple sites in the CaM promoter and deleting these sites phenocopies camt-1. These data suggest CAMTAs mediate a conserved and general mechanism that controls neuronal CaM levels, thereby regulating Ca(2+) signaling, physiology, and behavior.

Tuesday, November 16th - Disease Models

Shilpa, O., Anupama, K. P., Antony, A. and Gurushankara, H. P. (2021). Lead (Pb)-induced oxidative stress mediates sex-specific autistic-like behaviour in Drosophila melanogaster. Mol Neurobiol. PubMed ID: 34528217
Summary:
Autism spectrum disorder (ASD) is a highly prevalent neurodevelopmental disorder characterised by three main behavioural symptoms: abnormal social interaction, verbal and non-verbal communication impairments, and repetitive and restricted activities or interests. Even though the exact aetiology of ASD remains unknown, studies have shown a link between genetics and environmental pollutants. Heavy metal lead (Pb), the environmental pollutant, is associated with ASD. Pb may also exhibit sex-specific ASD behaviour, as has been demonstrated in the global human populations. Drosophila melanogaster as a model has been used in the present study to understand the involvement of Pb-induced oxidative stress in developing ASD behaviour. The larval feeding technique has been employed to administer different Pb concentrations (0.2-0.8 mM) to Oregon-R (ORR), superoxide dismutase (Sod), or catalase (Cat) antioxidants overexpressed or knockdown flies. Adult Drosophila (5-day old) were used for Pb content, biochemical, and behavioural analysis. Pb accumulated in the Drosophila brain induces oxidative stress and exhibited a human autistic-like behaviour such as reduced climbing, increased grooming, increased social spacing, and decreased learning and memory in a sex-specific manner. Pb-induced autistic-like behaviour was intensified in Sod or Cat-knockdown flies, whereas Sod or Cat-overexpressed flies overcome that behavioural alterations. These results unequivocally proved that Pb-induced oxidative stress causes ASD behaviour of humans in Drosophila. Thus, Drosophila is used as a model organism to analyse ASD-like human behaviour and underlines the importance of using antioxidant therapy in alleviating ASD symptoms in children.
Cragnaz, L., Spinelli, G., De Conti, L., Bureau, E. A., Brownlees, J., Feiguin, F., Romano, V., Skoko, N., Klima, R., Kettleborough, C. A., Baralle, F. E. and Baralle, M. (2021). Thioridazine reverts the phenotype in cellular and Drosophila models of amyotrophic lateral sclerosis by enhancing TDP-43 aggregate clearance. Neurobiol Dis 160: 105515. PubMed ID: 34571136
Summary:
Brain inclusions mainly composed of misfolded and aggregated TAR DNA binding protein 43 (TDP-43), are characteristic hallmarks of amyotrophic lateral sclerosis (ALS). Irrespective of the role played by the inclusions, their reduction represents an important therapeutic pathway that is worth exploring. Their removal can either lead to the recovery of TDP-43 function by removing the self-templating conformers that sequester the protein in the inclusions, and/or eliminate any potential intrinsic toxicity of the aggregates. The search for curative therapies has been hampered by the lack of ALS models for use in high-throughput screening. The study adapted, optimised, and extensively characterised a previous ALS cellular model for such use. The model demonstrated efficient aggregation of endogenous TDP-43, and concomitant loss of its splicing regulation function. The study provided a proof-of-principle for its eventual use in high-throughput screening using compounds of the tricyclic family and showed that recovery of TDP-43 function can be achieved by the enhanced removal of TDP-43 aggregates by these compounds. It was then observed that the degradation of the aggregates occurs independent of the autophagy pathway beyond autophagosome-lysosome fusion, but requires a functional proteasome pathway. The in vivo translational effect of the cellular model was tested with two of these compounds in a Drosophila model expressing a construct analogous to the cellular model, where thioridazine significantly improved the locomotive defect. These findings have important implications as thioridazine cleared TDP-43 aggregates and recovered TDP-43 functionality. This study also highlights the importance of a two-stage, in vitro and in vivo model system to cross-check the search for small molecules that can clear TDP-43 aggregates in TDP-43 proteinopathies (Cragnaz, 2021).
Santabarbara-Ruiz, P. and Leopold, P. (2021). An Oatp transporter-mediated steroid sink promotes tumor-induced cachexia in Drosophila. Dev Cell 56(19): 2741-2751.e2747. PubMed ID: 34610327
Summary:
Cancer cachexia is associated with many types of tumors and is characterized by a combination of anorexia, loss of body weight, catabolic alterations, and systemic inflammation. A tumor model was developed in Drosophila larvae that causes cachexia-like syndrome, and it was found that cachectic larvae show reduced levels of the circulating steroid ecdysone (Ec). Artificially importing Ec in the tumor through the use of the EcI/Oatp74D importer aggravated cachexia, whereas feeding animals with Ec rescued cachectic defects. This suggests that a steroid sink induced by the tumor promotes catabolic alterations in healthy tissues. This study found that Oatp33Eb, a member of the Oatp transporter family, is specifically induced in tumors promoting cachexia. The overexpression of Oatp33Eb in noncachectic tumors induced cachexia, whereas its inhibition in cachectic tumors restored circulating Ec and reversed cachectic alterations. Oatp transporters are induced in several types of hormone-dependent tumors, and this result suggests that a similar sink effect could modify hormonal balance in cachectic cancer patients.
Choi, H. J., Lee, J. Y., Cha, S. J., Han, Y. J., Yoon, J. H., Kim, H. J. and Kim, K. (2021). FUS-induced neurotoxicity is prevented by inhibiting GSK-3model in a Drosophila model of amyotrophic lateral sclerosis. Hum Mol Genet. PubMed ID: 34605896
Summary:
Amyotrophic lateral sclerosis (ALS)-linked mutations in fused in sarcoma (FUS) lead to the formation of cytoplasmic aggregates in neurons. They are believed play a critical role in the pathogenesis of FUS-associated ALS. Therefore, the clearance and degradation of cytoplasmic FUS aggregates in neurons may be considered a therapeutic strategy for ALS. This study reports GSK-3β as a potential modulator of FUS-induced toxicity. RNAi-mediated knockdown of Drosophila ortholog Shaggy in FUS-expressing flies suppresses defective phenotypes, including retinal degeneration, motor defects, motor neuron degeneration, and mitochondrial dysfunction. Furthermore, it was found that cytoplasmic FUS aggregates were significantly reduced by Shaggy knockdown. In addition, studies found that the levels of FUS proteins were significantly reduced by co-overexpression of Slimb, a F-box protein, in FUS-expressing flies, indicating that Slimb is critical for the suppressive effect of Shaggy/GSK-3β inhibition on FUS-induced toxicity in Drosophila. These findings revealed a novel mechanism of neuronal protective effect through SCFSlimb-mediated FUS degradation via GSK-3β inhibition, and provided in vivo evidence of the potential for modulating FUS-induced ALS progression using GSK-3β inhibitors (Choi, 2021).
Dabbara, H., Schultz, A. and Im, S. H. (2021). Drosophila insulin receptor regulates diabetes-induced mechanical nociceptive hypersensitivity. MicroPubl Biol 2021. PubMed ID: 34549177
Summary:
Painful diabetic neuropathy (PDN) is one of the predominant complications of diabetes that causes numbness, tingling, and extreme pain sensitivity. Understanding the mechanisms of PDN pathogenesis is important for patient treatments. This study reports Drosophila models of diabetes-induced mechanical nociceptive hypersensitivity. Type 2 diabetes-like conditions and loss of insulin receptor function in multidendritic sensory neurons lead to mechanical nociceptive hypersensitivity. Furthermore, it was found that restoring insulin signaling in multidendritic sensory neurons can block diabetes-induced mechanical nociceptive hypersensitivity. This work highlights the critical role of insulin signaling in nociceptive sensory neurons in the regulation of diabetes-induced nociceptive hypersensitivities (Dabbara, 2021).
Elovsson, G., Bergkvist, L. and Brorsson, A. C. (2021). Exploring Aβ Proteotoxicity and Therapeutic Candidates Using Drosophila melanogaster. Int J Mol Sci 22(19). PubMed ID: 34638786
Summary:
Alzheimer's disease is a widespread and devastating neurological disorder associated with proteotoxic events caused by the misfolding and aggregation of the amyloid-β peptide. To find therapeutic strategies to combat this disease, Drosophila melanogaster has proved to be an excellent model organism that is able to uncover anti-proteotoxic candidates due to its outstanding genetic toolbox and resemblance to human disease genes. In this review, it highlights the use of Drosophila melanogaster to both study the proteotoxicity of the amyloid-β peptide and to screen for drug candidates. Expanding the knowledge of how the etiology of Alzheimer's disease is related to proteotoxicity and how drugs can be used to block disease progression will hopefully shed further light on the field in the search for disease-modifying treatments.

Monday, November 15th - Synapse and Vesicles

Chan, E. H. Y., Zhou, Y., Aerne, B. L., Holder, M. V., Weston, A., Barry, D. J., Collinson, L. and Tapon, N. (2021). RASSF8-mediated transport of Echinoid via the exocyst promotes Drosophila wing elongation and epithelial ordering. Development 148(20). PubMed ID: 34532737
Summary:
Cell-cell junctions are dynamic structures that maintain cell cohesion and shape in epithelial tissues. During development, junctions undergo extensive rearrangements to drive the epithelial remodelling required for morphogenesis. This is particularly evident during axis elongation, where neighbour exchanges, cell-cell rearrangements and oriented cell divisions lead to large-scale alterations in tissue shape. Polarised vesicle trafficking of junctional components by the exocyst complex has been proposed to promote junctional rearrangements during epithelial remodelling, but the receptors that allow exocyst docking to the target membranes remain poorly understood. Here, this study shows that the adherens junction component Ras Association domain family 8 (RASSF8) is required for the epithelial re-ordering that occurs during Drosophila pupal wing proximo-distal elongation. The exocyst component Sec15 was identfied as a RASSF8 interactor. Loss of RASSF8 elicits cytoplasmic accumulation of Sec15 and Rab11-containing vesicles. These vesicles also contain the nectin-like homophilic adhesion molecule Echinoid, the depletion of which phenocopies the wing elongation and epithelial packing defects observed in RASSF8 mutants. Thus, these results suggest that RASSF8 promotes exocyst-dependent docking of Echinoid-containing vesicles during morphogenesis.
Dulac, A., Issa, A. R., Sun, J., Matassi, G., Jonas, C., Cherif-Zahar, B., Cattaert, D. and Birman, S. (2021). A Novel Neuron-Specific Regulator of the V-ATPase in Drosophila. eNeuro 8(5). PubMed ID: 34620624
Summary:
The V-ATPase is a highly conserved enzymatic complex that ensures appropriate levels of organelle acidification in virtually all eukaryotic cells. While the general mechanisms of this proton pump have been well studied, little is known about the specific regulations of neuronal V-ATPase. This study characterized CG31030, a previously uncharacterized Drosophila protein predicted from its sequence homology to be part of the V-ATPase family. In contrast to its ortholog ATP6AP1/VhaAC45 which is ubiquitous, it was observed that CG31030 expression is apparently restricted to all neurons, and using CRISPR/Cas9-mediated gene tagging, that it is mainly addressed to synaptic terminals. CG31030 is essential for fly survival and this protein co-immunoprecipitates with identified V-ATPase subunits, and in particular ATP6AP2. Using a genetically-encoded pH probe (VMAT-pHluorin) and electrophysiological recordings at the larval neuromuscular junction, it showed that CG31030 knock-down induces a major defect in synaptic vesicle acidification and a decrease in quantal size, which is the amplitude of the postsynaptic response to the release of a single synaptic vesicle. These defects were associated with severe locomotor impairments. Overall, these data indicate that CG31030, which was renamed VhaAC45-related protein (VhaAC45RP), is a specific regulator of neuronal V-ATPase in Drosophila that is required for proper synaptic vesicle acidification and neurotransmitter release (Dulac, 2021).
Neuman, S. D., Lee, A. R., Selegue, J. E., Cavanagh, A. T. and Bashirullah, A. (2021). A novel function for Rab1 and Rab11 during secretory granule maturation. J Cell Sci 134(15). PubMed ID: 34342349
Summary:
Regulated exocytosis is an essential process whereby specific cargo proteins are secreted in a stimulus-dependent manner. Cargo-containing secretory granules are synthesized in the trans-Golgi network (TGN); after budding from the TGN, granules undergo modifications, including an increase in size. These changes occur during a poorly understood process called secretory granule maturation. This study leveraged the Drosophila larval salivary glands as a model to characterize a novel role for Rab GTPases during granule maturation. Secretory granules were found to increase in size ∼300-fold between biogenesis and release, and loss of Rab1 or Rab11 reduces granule size. Surprisingly, it wax found that Rab1 and Rab11 localize to secretory granule membranes. Rab11 associates with granule membranes throughout maturation, and Rab11 recruits Rab1. In turn, Rab1 associates specifically with immature granules and drives granule growth. In addition to roles in granule growth, both Rab1 and Rab11 appear to have additional functions during exocytosis; Rab11 function is necessary for exocytosis, while the presence of Rab1 on immature granules may prevent precocious exocytosis. Overall, these results highlight a new role for Rab GTPases in secretory granule maturation.
Sechi, S., Karimpour-Ghahnavieh, A., Frappaolo, A., Di Francesco, L., Piergentili, R., Schinina, E., D'Avino, P. P. and Giansanti, M. G. (2021).. Identification of GOLPH3 Partners in Drosophila Unveils Potential Novel Roles in Tumorigenesis and Neural Disorders. Cells 10(9). PubMed ID: 34571985
Summary:
Golgi phosphoprotein 3 (GOLPH3) is a highly conserved peripheral membrane protein localized to the Golgi apparatus and the cytosol. GOLPH3 binding to Golgi membranes depends on phosphatidylinositol 4-phosphate [PI(4)P] and regulates Golgi architecture and vesicle trafficking. GOLPH3 overexpression has been correlated with poor prognosis in several cancers, but the molecular mechanisms that link GOLPH3 to malignant transformation are poorly understood. It was recently shown that PI(4)P-GOLPH3 couples membrane trafficking with contractile ring assembly during cytokinesis in dividing Drosophila spermatocytes. This study used affinity purification coupled with mass spectrometry (AP-MS) to identify the protein-protein interaction network (interactome) of Drosophila GOLPH3 in testes. Analysis of the GOLPH3 interactome revealed enrichment for proteins involved in vesicle-mediated trafficking, cell proliferation and cytoskeleton dynamics. In particular, it was found that dGOLPH3 interacts with the Drosophila orthologs of Fragile X mental retardation protein and Ataxin-2, suggesting a potential role in the pathophysiology of disorders of the nervous system. These findings suggest novel molecular targets associated with GOLPH3 that might be relevant for therapeutic intervention in cancers and other human diseases.
Neuman, S. D., Jorgensen, J. R., Cavanagh, A. T., Smyth, J. T., Selegue, J. E., Emr, S. D. and Bashirullah, A. (2022). The Hob proteins are novel and conserved lipid-binding proteins at ER-PM contact sites. J Cell Sci 135(5). PubMed ID: 34415038
Summary:
Membrane contact sites are critical junctures for organelle signaling and communication. Endoplasmic reticulum-plasma membrane (ER-PM) contact sites were the first membrane contact sites to be described; however, the protein composition and molecular function of these sites is still emerging. This study leverage yeast and Drosophila model systems to uncover a novel role for the Hobbit (Hob) proteins at ER-PM contact sites. Hobbit was found to localize to ER-PM contact sites in both yeast cells and the Drosophila larval salivary glands, and this localization is mediated by an N-terminal ER membrane anchor and conserved C-terminal sequences. The C-terminus of Hobbit binds to plasma membrane phosphatidylinositols, and the distribution of these lipids is altered in hobbit mutant cells. Notably, the Hobbit protein is essential for viability in Drosophila, providing one of the first examples of a membrane contact site-localized lipid binding protein that is required for development.
Yang, K., Liu, M., Feng, Z., Rojas, M., Zhou, L., Ke, H. and Pastor-Pareja, J. C. (2021). ER exit sites in Drosophila display abundant ER-Golgi vesicles and pearled tubes but no megacarriers. Cell Rep 36(11): 109707. PubMed ID: 34525362
Summary:
Secretory cargos are collected at endoplasmic reticulum (ER) exit sites (ERES) before transport to the Golgi apparatus. Decades of research have provided many details of the molecular events underlying ER-Golgi exchanges. Essential questions, however, remain about the organization of the ER-Golgi interface in cells and the type of membrane structures mediating traffic from ERES. To investigate these, transgenic tagging in Drosophila flies, 3D-structured illumination microscopy (SIM) and focused ion beam scanning electron microscopy (FIB-SEM) were used to characterize ERES-Golgi units in collagen-producing fat body, imaginal discs, and imaginal discs overexpressing ERES determinant Tango1. Facing ERES was fiybd a pre-cis-Golgi region, equivalent to the vertebrate ER-Golgi intermediate compartment (ERGIC), involved in both anterograde and retrograde transport. This pre-cis-Golgi is continuous with the rest of the Golgi, not a separate compartment or collection of large carriers, for which no evidence is found. This study observed, however, many vesicles, as well as pearled tubules connecting ERES and Golgi.

Friday, November 12th - Evolution

Blondel, L., Besse, S., Rivard, E. L., Ylla, G. and Extavour, C. G. (2021). Evolution of a cytoplasmic determinant: evidence for the biochemical basis of functional evolution of the novel germ line regulator oskar. Mol Biol Evol. PubMed ID: 34550378
Summary:
Germ line specification is essential in sexually reproducing organisms. Despite their critical role, the evolutionary history of the genes that specify animal germ cells is heterogeneous and dynamic. In many insects, the gene oskar is required for the specification of the germ line. However, the germ line role of oskar is thought to be a derived role resulting from co-option from an ancestral somatic role. To address how evolutionary changes in protein sequence could have led to changes in the function of Oskar protein that enabled it to regulate germ line specification, oskar orthologs were sought in 1565 publicly available insect genomic and transcriptomic datasets. The earliest-diverging lineage in which an oskar ortholog was identified was the order Zygentoma (silverfish and firebrats), suggesting that oskar originated before the origin of winged insects. Some order-specific trends in oskar sequence evolution were noted, including whole gene duplications, clade-specific losses, and rapid divergence. An alignment of all known 379 Oskar sequences revealed new highly conserved residues as candidates that promote dimerization of the LOTUS domain. Moreover, regions were identified of the OSK domain with conserved predicted RNA binding potential. Furthermore, it was shown that despite a low overall amino acid conservation, the LOTUS domain shows higher conservation of predicted secondary structure than the OSK domain. Finally, new key amino acids in the LOTUS domain are suggested that may be involved in the previously reported Oskar-Vasa physical interaction that is required for its germ line role.
Xia, S., Ventura, I. M., Blaha, A., Sgromo, A., Han, S., Izaurralde, E. and Long, M. (2021). Rapid Gene evolution in an ancient post-transcriptional and translational regulatory system compensates for meiotic X chromosomal inactivation. Mol Biol Evol. PubMed ID: 34626117
Summary:
It is conventionally assumed that conserved pathways evolve slowly with little participation of gene evolution. Nevertheless, it has been recently observed that young genes can take over fundamental functions in essential biological processes, for example, development and reproduction. It is unclear how newly duplicated genes are integrated into ancestral networks and reshape the conserved pathways of important functions. This study investigated the origination and function of two autosomal genes that evolved recently in Drosophila: Poseidon (CG2053) and Zeus, which were created by RNA-based duplications from the X-linked CAF40, a subunit of the conserved CCR4-NOT deadenylase complex involved in post-transcriptional and translational regulation. Knockdown and knockout assays show that the two genes quickly evolved critically important functions in viability and male fertility. Moreover, transcriptome analysis demonstrates that the three genes have a broad and distinct effect in the expression of hundreds of genes, with almost half of the differentially expressed genes being perturbed exclusively by one paralog, but not the others. Co-immunoprecipitation and tethering assays show that the CAF40 paralog Poseidon maintains the ability to interact with the CCR4-NOT deadenylase complex and might act in post-transcriptional mRNA regulation. The rapid gene evolution in the ancient post-transcriptional and translational regulatory system may be driven by evolution of sex chromosomes to compensate for the meiotic X chromosomal inactivation (MXCI) in Drosophila.
Shaw, K. H., Dent, C. I., Johnson, T. K., Anderson, A., de Bruyne, M. and Warr, C. G. (2021). Natural variation at the Drosophila melanogaster Or22 odorant receptor locus is associated with changes in olfactory behaviour. Open Biol 11(9): 210158. PubMed ID: 34582710
Summary:
In insects, many critical olfactory behaviours are mediated by the large odorant receptor (Or) gene family, which determines the response properties of different classes of olfactory receptor neurons (ORNs). While ORN responses are generally conserved within and between Drosophila species, variant alleles of the D. melanogaster Or22 locus have previously been shown to alter the response profile of an ORN class called ab3A. These alleles show potential clinal variation, suggesting that selection is acting at this locus. This study investigated if the changes seen in ab3A responses lead to changes in olfactory-related behaviours. Variation at the Or22 locus and in the ab3A neurons were not fully compensated for by other ORNs and lead to overall changes in antennal odorant detection. It was further shown that this correlates with differences in odorant preference behaviour and with differences in oviposition site preference, with flies that have the chimaeric short allele strongly preferring to oviposit on banana. These findings indicate that variation at the Or22 locus leads to changes in olfactory-driven behaviours, and add support to the idea that the ab3A neurons are of especial importance to the ecology of Drosophila flies.
Scott, A. M., Dworkin, I. and Dukas, R. (2021). Evolution of sociability by artificial selection. Evolution. PubMed ID: 34605553
Summary:
There has been extensive research on the ecology and evolution of social life in animals that live in groups. Less attention, however, has been devoted to apparently solitary species, even though recent research indicates that they also possess complex social behaviors. To address this knowledge gap, this study artificially selected on sociability, defined as the tendency to engage in nonaggressive activities with others, in fruit flies. The goal was to quantify the factors that determine the level of sociability and the traits correlated with this feature. After 25 generations of selection, the high-sociability lineages showed sociability scores about 50% higher than did the low-sociability lineages. Experiments using the evolved lineages indicated that there were no differences in mating success between flies from the low and high lineages. Both males and females from the low lineages, however, were more aggressive than males and females from the high lineages. Finally, the evolved lineages maintained their sociability scores after 10 generations of relaxed selection, suggesting no costs to maintaining low and high sociability, at least under the settings used in this study. Sociability is a complex trait, which is currently being assessed through genomic work on the evolved lineages.
Schiller, E. A. and Bergstralh, D. T. (2021). Interaction between Discs large and Pins/LGN/GPSM2: A comparison across species. Biol Open. PubMed ID: 34596678
Summary:
The orientation of the mitotic spindle determines the direction of cell division, and therefore contributes to tissue shape and cell fate. Interaction between the multifunctional scaffolding protein Discs large (Dlg) and the canonical spindle orienting factor GPSM2 (called Pins in Drosophila and LGN in vertebrates) has been established in bilaterian models, but its function remains unclear. A phylogenetic approach was used to test whether the interaction is obligate in animals, and in particular whether Pins/LGN/GPSM2 evolved in multicellular organisms as a Dlg-binding protein. This study shows that Dlg diverged in C. elegans and the syncytial sponge O. minuta and proposes that this divergence may correspond to differences in spindle orientation requirements between these organisms and the canonical pathways described in bilaterians. It was also demonstrated that Pins/LGN/GPSM2 is present in basal animals, but the established Dlg-interaction site cannot be found in either Placozoa or Porifera. These results suggest that the interaction between Pins/LGN/GPSM2 and Dlg appeared in Cnidaria, and it is therefore speculated that it may have evolved to promote accurate division orientation in the nervous system. This work reveals the evolutionary history of the Pins/LGN/GPSM2-Dlg interaction and suggests new possibilities for its importance in spindle orientation during epithelial and neural tissue development.
Santos, M. A., Carromeu-Santos, A., Quina, A. S., Santos, M., Matos, M. and Simoes, P. (2021).. No evidence for short-term evolutionary response to a warming environment in Drosophila. Evolution. PubMed ID: 34617283
Summary:
Adaptive evolution is key in mediating responses to global warming and may sometimes be the only solution for species to survive. Such evolution will expectedly lead to changes in the populations' thermal reaction norm and improve their ability to cope with stressful conditions. Conversely, evolutionary constraints might limit the adaptive response. This study tests these expectations by performing a real-time evolution experiment in historically differentiated Drosophila subobscura populations. The phenotypic change was addressed after nine generations of evolution in a daily fluctuating environment with average constant temperature, or in a warming environment with increasing average and amplitude temperature across generations. The results showed that (1) evolution under a global warming scenario does not lead to a noticeable change in the thermal response; (2) historical background appears to be affecting responses under the warming environment, particularly at higher temperatures; and (3) thermal reaction norms are trait dependent: although lifelong exposure to low temperature decreases fecundity and productivity but not viability, high temperature causes negative transgenerational effects on productivity and viability, even with high fecundity. These findings in such an emblematic organism for thermal adaptation studies raise concerns about the short-term efficiency of adaptive responses to the current rising temperatures.

Thursday, November 11th - RNA and Transposons

Macosek, J., Simon, B., Linse, J. B., Jagtap, P. K. A., Winter, S. L., Foot, J., Lapouge, K., Perez, K., Rettel, M., Ivanovic, M. T., Masiewicz, P., Murciano, B., Savitski, M. M., Loedige, I., Hub, J. S., Gabel, F. and Hennig, J. (2021). Structure and dynamics of the quaternary hunchback mRNA translation repression complex. Nucleic Acids Res. PubMed ID: 34329466
Summary:
A key regulatory process during Drosophila development is the localized suppression of the hunchback mRNA translation at the posterior, which gives rise to a hunchback gradient governing the formation of the anterior-posterior body axis. This suppression is achieved by a concerted action of Brain Tumour (Brat), Pumilio (Pum) and Nanos. Each protein is necessary for proper Drosophila development. The RNA contacts have been elucidated for the proteins individually in several atomic-resolution structures. However, the interplay of all three proteins during RNA suppression remains a long-standing open question. This study characterize the quaternary complex of the RNA-binding domains of Brat, Pum and Nanos with hunchback mRNA by combining NMR spectroscopy, SANS/SAXS, XL/MS with MD simulations and ITC assays. The quaternary hunchback mRNA suppression complex comprising the RNA binding domains is flexible with unoccupied nucleotides functioning as a flexible linker between the Brat and Pum-Nanos moieties of the complex. Moreover, the presence of the Pum-HD/Nanos-ZnF complex has no effect on the equilibrium RNA binding affinity of the Brat RNA binding domain. This is in accordance with previous studies, which showed that Brat can suppress mRNA independently and is distributed uniformly throughout the embryo.
Chin, A. and Lecuyer, E. (2021). Puromycin Labeling Coupled with Proximity Ligation Assays to Define Sites of mRNA Translation in Drosophila Embryos and Human Cells. Methods Mol Biol 2381: 267-284. PubMed ID: 34590282
Summary:
Genetic mutations, whether they occur within protein-coding or noncoding regions of the genome, can affect various aspects of gene expression by influencing the complex network of intra- and intermolecular interactions that occur between cellular nucleic acids and proteins. One aspect of gene expression control that can be impacted is the intracellular trafficking and translation of mRNA molecules. To study the occurrence and dynamics of translational regulation, researchers have developed approaches such as genome-wide ribosome profiling and artificial reporters that enable single molecule imaging. In this study, A complementary and optimized approach that combines puromycin labeling with a proximity ligation assay (Puro-PLA) to define sites of translation of specific mRNAs in tissues or cells. This method can be used to study the mechanisms driving the translation of select mRNAs and to access the impact of genetic mutations on local protein synthesis. This approach involves the treatment of cell or tissue specimens with puromycin to label nascently translated peptides, rapid fixation, followed by immunolabeling with appropriate primary and secondary antibodies coupled to PLA oligonucleotide probes, ligation, amplification, and signal detection via fluorescence microscopy. Puro-PLA can be performed at small scale in individual tubes or in chambered slides, or in a high-throughput setup with 96-well plate, for both in situ and in vitro experimentation (Chin, 2021).
Ullastres, A., Merenciano, M. and Gonzalez, J. (2021). Regulatory regions in natural transposable element insertions drive interindividual differences in response to immune challenges in Drosophila. Genome Biol 22(1): 265. PubMed ID: 34521452
Summary:
Variation in gene expression underlies interindividual variability in relevant traits including immune response. However, the genetic variation responsible for these gene expression changes remains largely unknown. Among the non-coding variants that could be relevant, transposable element insertions are promising candidates as they have been shown to be a rich and diverse source of cis-regulatory elements. This work used a population genetics approach to identify transposable element insertions likely to increase the tolerance of Drosophila melanogaster to bacterial infection by affecting the expression of immune-related genes. This study identified 12 insertions associated with allele-specific expression changes in immune-related genes. three of these insertions were experimentally validate including one likely to be acting as a silencer, one as an enhancer, and one with a dual role as enhancer and promoter. The direction in the change of gene expression associated with the presence of several of these insertions is consistent with an increased survival to infection. Indeed, for one of the insertions, it was shown that this is the case by analyzing both natural populations and CRISPR/Cas9 mutants in which the insertion is deleted from its native genomic context. It was shown that transposable elements contribute to gene expression variation in response to infection in D. melanogaster and that this variation is likely to affect their survival capacity. Because the role of transposable elements as regulatory elements is not restricted to Drosophila, transposable elements are likely to play a role in immune response in other organisms as well.
Li, D., Ge, Y., Zhao, Z., Zhu, R., Wang, X. and Bi, X. (2021). Distinct and Coordinated Regulation of Small Non-coding RNAs by E2f1 and p53 During Drosophila Development and in Response to DNA Damage. Front Cell Dev Biol 9: 695311. PubMed ID: 34368144
Summary:
Small non-coding RNAs (ncRNAs), including microRNAs (miRNAs) and PIWI-interacting RNAs (piRNAs), play a pivotal role in biological processes. A comprehensive quantitative reference of small ncRNAs expression during development and in DNA damage response (DDR) would significantly advance understanding of their roles. This study systemically analyzed the expression profile of miRNAs and piRNAs in wild-type flies, e2f1 mutant, p53 mutant and e2f1 p53 double mutant during development and after X-ray irradiation. By using small RNA sequencing and bioinformatic analysis, it was found that both miRNAs and piRNAs were expressed in a dynamic mode and formed 4 distinct clusters during development. Notably, the expression pattern of miRNAs and piRNAs was changed in e2f1 mutant at multiple developmental stages, while retained in p53 mutant, indicating a critical role of E2f1 played in mediating small ncRNAs expression. Moreover, differentially expressed (DE) small ncRNAs were identified in e2f1 mutant and p53 mutant after X-ray irradiation. Furthermore, the binding motif of E2f1 and p53 was mapped around the small ncRNAs. The data suggested that E2f1 and p53 work differently yet coordinately to regulate small ncRNAs expression, and E2f1 may play a major role to regulate miRNAs during development and after X-ray irradiation. Collectively, these results provide comprehensive characterization of small ncRNAs, as well as the regulatory roles of E2f1 and p53 in small ncRNAs expression, during development and in DNA damage response, which reveal new insights into the small ncRNAs biology.
Sankaranarayanan, M., Emenecker, R. J., Wilby, E. L., Jahnel, M., Trussina, I., Wayland, M., Alberti, S., Holehouse, A. S. and Weil, T. T. (2021). Adaptable P body physical states differentially regulate bicoid mRNA storage during early Drosophila development. Dev Cell. PubMed ID: 34655524
Summary:
Ribonucleoprotein condensates can exhibit diverse physical states in vitro and in vivo. Despite considerable progress, the relevance of condensate physical states for in vivo biological function remains limited. This study investigated the physical properties of processing bodies (P bodies) and their impact on mRNA storage in mature Drosophila oocytes. The conserved DEAD-box RNA helicase Me31B forms viscous P body condensates, which adopt an arrested physical state. Structurally distinct proteins and protein-protein interactions, together with RNA, regulate the physical properties of P bodies. Using live imaging and in situ hybridization, this study shows that the arrested state and integrity of P bodies support the storage of bicoid (bcd) mRNA and that egg activation modulates P body properties, leading to the release of bcd for translation in the early embryo. Together, this work provides an example of how physical states of condensates regulate cellular function in development.
Zhang, G., Yu, T., Parhad, S. S., Ho, S., Weng, Z. and Theurkauf, W. E. (2021). piRNA-independent transposon silencing by the Drosophila THO complex. Dev Cell 56(18): 2623-2635.e2625. PubMed ID: 34547226
Summary:
piRNAs guide Piwi/Panoramix-dependent H3K9me3 chromatin modification and transposon silencing during Drosophila germline development. The THO RNA export complex is composed of Hpr1, Tho2, and Thoc5-7. Null thoc7 mutations, which displace Thoc5 and Thoc6 from a Tho2-Hpr1 subcomplex, reduce expression of a subset of germline piRNAs and increase transposon expression, suggesting that THO silences transposons by promoting piRNA biogenesis. This study shows that the thoc7-null mutant combination increases transposon transcription but does not reduce anti-sense piRNAs targeting half of the transcriptionally activated transposon families. These mutations also fail to reduce piRNA-guided H3K9me3 chromatin modification or block Panoramix-dependent silencing of a reporter transgene, and unspliced transposon transcripts co-precipitate with THO through a Piwi- and Panoramix-independent mechanism. Mutations in piwi also dominantly enhance germline defects associated with thoc7-null alleles. THO thus functions in a piRNA-independent transposon-silencing pathway, which acts cooperatively with Piwi to support germline development.

Wednesday, November 10th - Immune Response

Tang, R., Huang, W., Guan, J., Liu, Q., Beerntsen, B. T. and Ling, E. (2021). Drosophila H2Av negatively regulates the activity of the IMD pathway via facilitating Relish SUMOylation. PLoS Genet 17(8): e1009718. PubMed ID: 34370736
Summary:
Insects depend on the innate immune response for defense against a wide array of pathogens. Central to Drosophila immunity are antimicrobial peptides (AMPs), released into circulation when pathogens trigger either of the two widely studied signal pathways, Toll or IMD. The Toll pathway responds to infection by Gram-positive bacteria and fungi while the IMD pathway is activated by Gram-negative bacteria. During activation of the IMD pathway, the NF-κB-like transcription factor Relish is phosphorylated and then cleaved, which is crucial for IMD-dependent AMP gene induction. This study shows that loss-of-function mutants of the unconventional histone variant H2Av upregulate IMD-dependent AMP gene induction in germ-free Drosophila larvae and adults. After careful dissection of the IMD pathway, it was found that Relish has an epistatic relationship with H2Av. In the H2Av mutant larvae, SUMOylation is down-regulated, suggesting a possible role of SUMOylation in the immune phenotype. Eventually it was demonstrated that Relish is mostly SUMOylated on amino acid K823. Loss of the potential SUMOylation site leads to significant auto-activation of Relish in vivo. Further work indicated that H2Av regulates Relish SUMOylation after physically interacting with Su(var)2-10, the E3 component of the SUMOylation pathway. Biochemical analysis suggested that SUMOylation of Relish prevents its cleavage and activation. These findings suggest a new mechanism by which H2Av can negatively regulate, and thus prevent spontaneous activation of IMD-dependent AMP production, through facilitating SUMOylation of the NF-κB like transcription factor Relish.
Zhou, H., Li, S., Wu, S., Jin, P. and Ma, F. (2021). LncRNA-CR11538 Decoys Dif/Dorsal to Reduce Antimicrobial Peptide Products for Restoring Drosophila Toll Immunity Homeostasis. Int J Mol Sci 22(18). PubMed ID: 34576280
Summary:
Avoiding excessive or insufficient immune responses and maintaining homeostasis are critical for animal survival. Although many positive or negative modulators involved in immune responses have been identified, little has been reported to date concerning whether the long non-coding RNA (lncRNA) can regulate Drosophila immunity response. Firstly, this study discovered that the overexpression of lncRNA-CR11538 can inhibit the expressions of antimicrobial peptides Drosomycin (Drs) and Metchnikowin (Mtk) in vivo, thereby suppressing the Toll signaling pathway. Secondly, the results demonstrate that lncRNA-CR11538 can interact with transcription factors Dif/Dorsal in the nucleus based on both subcellular localization and RIP analyses. Thirdly, the findings reveal that lncRNA-CR11538 can decoy Dif/Dorsal away from the promoters of Drs and Mtk to repress their transcriptions by ChIP-qPCR and dual luciferase report experiments. Fourthly, the dynamic expression changes of Drs, Dif, Dorsal and lncRNA-CR11538 in wild-type flies (w(1118)) at different time points after M. luteus stimulation disclose that lncRNA-CR11538 can help Drosophila restore immune homeostasis in the later period of immune response. Overall, this study reveals a novel mechanism by which lncRNA-CR11538 serves as a Dif/Dorsal decoy to downregulate antimicrobial peptide expressions for restoring Drosophila Toll immunity homeostasis, and provides a new insight into further studying the complex regulatory mechanism of animal innate immunity.
Wang, L., Lin, J., Yu, J., Yang, K., Sun, L., Tang, H. and Pan, L. (2021). Downregulation of Perilipin1 by the Immune Deficiency Pathway Leads to Lipid Droplet Reconfiguration and Adaptation to Bacterial Infection in Drosophila. J Immunol 207(9): 2347-2358. PubMed ID: 34588219l
Summary:
Lipid droplets (LDs), the highly dynamic intracellular organelles, are critical for lipid metabolism. Dynamic alterations in the configurations and functions of LDs during innate immune responses to bacterial infections and the underlying mechanisms, however, remain largely unknown. This study traced the time-course morphology of LDs in fat bodies of Drosophila after transient bacterial infection. Detailed analysis shows that perilipin1 (plin1), a core gene involved in the regulation of LDs, is suppressed by the immune deficiency signaling, one major innate immune pathway in Drosophila. During immune activation, downregulated plin1 promotes the enlargement of LDs, which in turn alleviates immune reaction-associated reactive oxygen species stress. Thus, the growth of LDs is likely an active adaptation to maintain redox homeostasis in response to immune deficiency activation. Therefore, this study provides evidence that plin1 serves as a modulator on LDs' reconfiguration in regulating infection-induced pathogenesis, and plin1 might be a potential therapeutic target for coordinating inflammation resolution and lipid metabolism.
Prakash, P., Roychowdhury-Sinha, A. and Goto, A. (2021). Verloren negatively regulates the expression of IMD pathway dependent antimicrobial peptides in Drosophila. Sci Rep 11(1): 15549. PubMed ID: 34330981
Summary:
Drosophila immune deficiency (IMD) pathway is similar to the human tumor necrosis factor receptor (TNFR) signaling pathway and is preferentially activated by Gram-negative bacterial infection. Recent studies highlighted the importance of IMD pathway regulation as it is tightly controlled by numbers of negative regulators at multiple levels. This study reports a new negative regulator of the IMD pathway, Verloren (Velo). Silencing of Velo led to constitutive expression of the IMD pathway dependent antimicrobial peptides (AMPs), and Escherichia coli stimulation further enhanced the AMP expression. Epistatic analysis indicated that Velo knock-down mediated AMP upregulation is dependent on the canonical members of the IMD pathway. The immune fluorescent study using overexpression constructs revealed that Velo resides both in the nucleus and cytoplasm, but the majority (~ 75%) is localized in the nucleus. It was also observed from in vivo analysis that Velo knock-down flies exhibit significant upregulation of the AMP expression and reduced bacterial load. Survival experiments showed that Velo knock-down flies have a short lifespan and are susceptible to the infection of pathogenic Gram-negative bacteria, P. aeruginosa. Taken together, these data suggest that Velo is an additional new negative regulator of the IMD pathway, possibly acting in both the nucleus and cytoplasm.
Vincent, C. M. and Dionne, M. S. (2021). Disparate regulation of IMD signaling drives sex differences in infection pathology in Drosophila melanogaster. Proc Natl Acad Sci U S A 118(32). PubMed ID: 34341118
Summary:
Male and female animals exhibit differences in infection outcomes. One possible source of sexually dimorphic immunity is the sex-specific costs of immune activity or pathology, but little is known about the independent effects of immune- versus microbe-induced pathology and whether these may differ for the sexes. By measuring metabolic and physiological outputs in Drosophila melanogaster with wild-type and mutant immune responses, this study tested whether the sexes are differentially impacted by these various sources of pathology and identified a critical regulator of this difference. The sexes exhibit differential immune activity but similar bacteria-derived metabolic pathology. Female-specific immune-inducible expression of PGRP-LB, a negative regulator of the immune deficiency (IMD) pathway, enables females to reduce immune activity in response to reductions in bacterial numbers. In the absence of PGRP-LB, females are more resistant to infection, confirming the functional importance of this regulation and suggesting that female-biased immune restriction comes at a cost.
Shahrestani, P., King, E., Ramezan, R., Phillips, M., Riddle, M., Thornburg, M., Greenspan, Z., Estrella, Y., Garcia, K., Chowdhury, P., Malarat, G., Zhu, M., Rottshaefer, S. M., Wraight, S., Griggs, M., Vandenberg, J., Long, A. D., Clark, A. G. and Lazzaro, B. P. (2021). The molecular architecture of Drosophila melanogaster defense against Beauveria bassiana explored through evolve and resequence and quantitative trait locus mapping. G3 (Bethesda). PubMed ID: 34534291
Summary:
Little is known about the genetic architecture of antifungal immunity in natural populations. Using two population genetic approaches, Quantitative Trait Locus (QTL) Mapping and Evolve and Resequence (E&R), this study explored D. melanogaster immune defense against infection with the fungus Beauveria bassiana. Immune defense was highly variable both in the recombinant inbred lines from the Drosophila Synthetic Population Resource used for QTL mapping and in the synthetic outbred populations used in the E&R study. Survivorship of infection improved dramatically over just 10 generations in the E&R study, and continued to increase for an additional 9 generations, revealing a trade-off with uninfected longevity. Populations selected for increased defense against B. bassiana evolved cross resistance to a second, distinct B. bassiana strain but not to bacterial pathogens. The QTL mapping study revealed that sexual dimorphism in defense depends on host genotype, and the E&R study indicated that sexual dimorphism also depends on the specific pathogen to which the host is exposed. Both the QTL mapping and E&R experiments generated lists of potentially causal candidate genes, although these lists were non-overlapping.

Tuesday, November 9th - Adult Physiology

Chrostek, E., Martins, N., Marialva, M. S. and Teixeira, L. (2021). Wolbachia-Conferred Antiviral Protection Is Determined by Developmental Temperature. mBio: e0292320. PubMed ID: 34488458
Summary:
Wolbachia is a maternally transmitted bacterium that is widespread in arthropods and filarial nematodes and confers strong antiviral protection in Drosophila melanogaster and other arthropods. Wolbachia-transinfected Aedes aegypti mosquitoes are currently being deployed to fight transmission of dengue and Zika viruses. However, the mechanism of antiviral protection and the factors influencing are still not fully understood. Studies show that temperature modulates Wolbachia-conferred protection in Drosophila melanogaster. Temperature after infection directly impacts Drosophila C virus (DCV) replication and modulates Wolbachia protection. At higher temperatures, viruses proliferate more and are more lethal, while Wolbachia confers lower protection. Strikingly, host developmental temperature is a determinant of Wolbachia-conferred antiviral protection. While there is strong protection when flies develop from egg to adult at 25°C, the protection is highly reduced or abolished when flies develop at 18°C. However, Wolbachia-induced changes during development are not sufficient to limit virus-induced mortality, as Wolbachia is still required to be present in adults at the time of infection. This developmental effect is general, since it was present in different host genotypes, Wolbachia variants, and upon infection with different viruses. Overall, this study showed that Wolbachia-conferred antiviral protection is temperature dependent, being present or absent depending on the environmental conditions. This interaction likely impacts Wolbachia-host interactions in nature and, as a result, frequencies of host and symbionts in different climates. Dependence of Wolbachia-mediated pathogen blocking on developmental temperature could be used to dissect the mechanistic bases of protection and influence the deployment of Wolbachia to prevent transmission of arboviruses.
Szlachcic, E. and Czarnoleski, M. (2021). Thermal and Oxygen Flight Sensitivity in Ageing Drosophila melanogaster Flies: Links to Rapamycin-Induced Cell Size Changes. Biology (Basel) 10(9). PubMed ID: 34571738
Summary:
Ectotherms can become physiologically challenged when performing oxygen-demanding activities (e.g., flight) across differing environmental conditions, specifically temperature and oxygen levels. Achieving a balance between oxygen supply and demand can also depend on the cellular composition of organs, which either evolves or changes plastically in nature; however, this hypothesis has rarely been examined, especially in tracheated flying insects. The relatively large cell membrane area of small cells should increase the rates of oxygen and nutrient fluxes in cells; however, it does also increase the costs of cell membrane maintenance. To address the effects of cell size on flying insects, the wing-beat frequency was measured in two cell-size phenotypes of Drosophila melanogaster when flies were exposed to two temperatures (warm/hot) combined with two oxygen conditions (normoxia/hypoxia). The cell-size phenotypes were induced by rearing 15 isolines on either standard food (large cells) or rapamycin-enriched food (small cells). Rapamycin supplementation (downregulation of TOR activity) produced smaller flies with smaller wing epidermal cells. Flies generally flapped their wings at a slower rate in cooler (warm treatment) and less-oxygenated (hypoxia) conditions, but the small-cell-phenotype flies were less prone to oxygen limitation than the large-cell-phenotype flies and did not respond to the different oxygen conditions under the warm treatment. It is suggested that ectotherms with small-cell life strategies can maintain physiologically demanding activities (e.g., flight) when challenged by oxygen-poor conditions, but this advantage may depend on the correspondence among body temperatures, acclimation temperatures and physiological thermal limits.
Sujkowski, A. and Wessells, R. (2021). Exercise and Sestrin Mediate Speed and Lysosomal Activity in Drosophila by Partially Overlapping Mechanisms. Cells 10(9). PubMed ID: 34572128
Summary:
Chronic exercise is widely recognized as an important contributor to healthspan in humans and in diverse animal models. Sestrins, a family of evolutionarily conserved exercise-inducible proteins, are critical mediators of exercise benefits in flies and mice. Knockout of Sestrins prevents exercise adaptations to endurance and flight in Drosophila, and similarly prevents benefits to endurance and metabolism in exercising mice. In contrast, overexpression of dSestrin in muscle mimics several of the molecular and physiological adaptations characteristic of endurance exercise. This study extends those observations to examine the impact of dSestrin on preserving speed and increasing lysosomal activity. dSestrin was found to be a critical factor driving exercise adaptations to climbing speed, but is not absolutely required for exercise to increase lysosomal activity in Drosophila. The role of Sestrin in increasing speed during chronic exercise requires both the TORC2/AKT axis and the PGC1α homolog spargel, while dSestrin requires interactions with TORC1 to cell-autonomously increase lysosomal activity. These results highlight the conserved role of Sestrins as key factors that drive diverse physiological adaptations conferred by chronic exercise.
Chen, P., De Schutter, K., Serna, S., Chen, S., Yang, Q., Reichardt, N. C., Van Damme, E. J. M. and Smagghe, G. (2021). Glycosylation reduces the glycan-independent immunomodulatory effect of recombinant Orysata lectin in Drosophila S2 cells. Sci Rep 11(1): 17958. PubMed ID: 34504130
Summary:
Several plant lectins, or carbohydrate-binding proteins, interact with glycan moieties on the surface of immune cells, thereby influencing the immune response of these cells. Orysata, a mannose-binding lectin from rice, has been reported to exert immunomodulatory activities on insect cells. While the natural lectin is non-glycosylated, recombinant Orysata produced in the yeast Pichia pastoris (YOry) is modified with a hyper-mannosylated N-glycan. Since it is unclear whether this glycosylation can affect the YOry activity, non-glycosylated rOrysata was produced in Escherichia coli (BOry). In a comparative analysis, both recombinant Orysata proteins were tested for their carbohydrate specificity on a glycan array, followed by the investigation of the carbohydrate-dependent agglutination of red blood cells (RBCs) and the carbohydrate-independent immune responses in Drosophila melanogaster S2 cells. Although YOry and BOry showed a similar carbohydrate-binding profiles, lower concentration of BOry were sufficient for the agglutination of RBCs and BOry induced stronger immune responses in S2 cells. The data are discussed in relation to different hypotheses explaining the weaker responses of glycosylated YOry. In conclusion, these observations contribute to the understanding how post-translational modification can affect protein function, and provide guidance in the selection of the proper expression system for the recombinant production of lectins (Chen, 2021).
Coni, S., Falconio, F. A., Marzullo, M., Munafò, M., Zuliani, B., Mosti, F., Fatica, A., Ianniello, Z., Bordone, R., Macone, A., Agostinelli, E., Perna, A., Matkovic, T., Sigrist, S., Silvestri, G., Canettieri, G. and Ciapponi, L. (2021). Translational control of polyamine metabolism by CNBP is required for Drosophila locomotor function. Elife 10. PubMed ID: 34517941
Summary:
Microsatellite expansions of CCTG repeats in the cellular nucleic acid-binding protein (CNBP) gene leads to accumulation of toxic RNA and have been associated with myotonic dystrophy type 2 (DM2). However, it is still unclear whether the dystrophic phenotype is also linked to CNBP decrease, a conserved CCHC-type zinc finger RNA-binding protein that regulates translation and is required for mammalian development. This study showed that depletion of Drosophila CNBP in muscles causes ageing-dependent locomotor defects that are correlated with impaired polyamine metabolism. studies demonstrate that the levels of ornithine decarboxylase (ODC) and polyamines are significantly reduced upon dCNBP depletion. Of note, studies show a reduction of the CNBP-polyamine axis in muscles from DM2 patients. Mechanistically, studies provide evidence that dCNBP controls polyamine metabolism through binding dOdc mRNA and regulating its translation. Remarkably, the locomotor defect of dCNBP-deficient flies is rescued by either polyamine supplementation or dOdc1 overexpression. Studies suggest that this dCNBP function is evolutionarily conserved in vertebrates with relevant implications for CNBP-related pathophysiological conditions.
Coquerel, Q. R. R., Ddmares, F., Geldenhuys, W. J., Le Ray, A. M., Brdard, D., Richomme, P., Legros, C., Norris, E. and Bloomquist, J. R. (2021).. Toxicity and mode of action of the aporphine plant alkaloid liriodenine on the insect GABA receptor. Toxicon 201: 141-147. PubMed ID: 34474068
Summary:
Liriodenine is a biologically active plant alkaloid with multiple effects on mammals, fungi, and bacteria, but has never been evaluated for insecticidal activity. Accordingly, liriodenine was applied topically in ethanolic solutions to adult female Anopheles gambiae, and found to be mildly toxic. Its lethality was synergized in mixtures with dimethyl sulfoxide and piperonyl butoxide. Recordings from the ventral nerve cord of larval Drosophila melanogaster showed that liriodenine was neuroexcitatory and reversed the inhibitory effect of 1 mM GABA at effective concentrations of 20-30 μM. GABA antagonism on the larval nervous system was equally expressed on both susceptible and cyclodiene-resistant rdl preparations. Acutely isolated neurons from Periplaneta americana were studied under patch clamp and inhibition of GABA-induced currents with an IC(50) value of about 1 μM were observed. In contrast, bicuculline did not reverse the effects of GABA on cockroach neurons, as expected. In silico molecular models suggested reasonable structural concordance of liriodenine and bicuculline and isosteric hydrogen bond acceptor sites. This study is the first assessing of the toxicology of liriodenine on insects and implicates the GABA receptor as one likely neuronal target, where liriodenine might be considered an active chemical analog of bicuculline.

Monday, November 8 - Chromatin and Chromosomes

Yannuzzi, I., Butler, M. A., Fernandez, J. and LaRocque, J. R. (2021). The Role of Drosophila CtIP in Homology-Directed Repair of DNA Double-Strand Breaks. Genes (Basel) 12(9). PubMed ID: 34573412
Summary:
DNA double-strand breaks (DSBs) are a particularly genotoxic type of DNA damage that can result in chromosomal aberrations. Thus, proper repair of DSBs is essential to maintaining genome integrity. DSBs can be repaired by non-homologous end joining (NHEJ), where ends are processed before joining through ligation. Alternatively, DSBs can be repaired through homology-directed repair, either by homologous recombination (HR) or single-strand annealing (SSA). Both types of homology-directed repair are initiated by DNA end resection. In cultured human cells, the protein CtIP has been shown to play a role in DNA end resection through its interactions with CDK, BRCA1, DNA2, and the MRN complex. To elucidate the role of CtIP in a multicellular context, CRISPR/Cas9 genome editing was used to create a DmCtIPΔ allele in Drosophila melanogaster. Using the DSB repair reporter assay direct repeat of white (DR-white), a two-fold decrease in HR in DmCtIPΔ/Δ mutants was observed when compared to heterozygous controls. However, analysis of HR gene conversion tracts (GCTs) suggests DmCtIP plays a minimal role in determining GCT length. To assess the function of DmCtIP on both short (~550 bp) and long (~3.6 kb) end resection, modified homology-directed SSA repair assays were implemented, resulting in a two-fold decrease in SSA repair in both short and extensive end resection requirements in the DmCtIPΔ/Δ mutants compared to heterozygote controls. Through these analyses, the importance was affirmed of end resection on DSB repair pathway choice in multicellular systems, the function of DmCtIP in short and extensive DNA end resection was described, and the impact of end resection on GCT length during HR was determined.
Vernizzi, L. and Lehner, C. F. (2021). Bivalent individualization during chromosome territory formation in Drosophila spermatocytes by controlled condensin II protein activity and additional force generators. PLoS Genet 17(10): e1009870. PubMed ID: 34669718
Summary:
Chromosome pairing during meiosis must avoid interlocking of non-homologous chromosomes. In spermatocytes of Drosophila, extensive non-homologous associations arise from the coalescence of the large blocks of pericentromeric heterochromatin into a chromocenter and from centromere clustering. Nevertheless, during territory formation, bivalents are moved apart into spatially separate subnuclear regions. The condensin II subunits, Cap-D3 and Cap-H2, have been implicated, but the remarkable separation of bivalents during interphase might require more than just condensin II. For further characterization of this process, time-lapse imaging was applied using fluorescent markers of centromeres, telomeres and DNA satellites in pericentromeric heterochromatin. The dynamics of the disruption of centromere clusters and the chromocenter are described in normal spermatocytes. Mutations in Cap-D3 and Cap-H2 abolish chromocenter disruption, resulting in excessive chromosome missegregation during meiosis I. Chromocenter persistence in the mutants is not mediated by the special system, which conjoins homologs in compensation for the absence of crossovers in Drosophila spermatocytes. However, overexpression of Cap-H2 precluded conjunction between autosomal homologs, resulting in random segregation of univalents. Interestingly, Cap-D3 and Cap-H2 mutant spermatocytes displayed conspicuous stretching of the chromocenter, as well as occasional chromocenter disruption, suggesting that territory formation might involve forces unrelated to condensin II. While the molecular basis of these forces remains to be clarified, they are not destroyed by inhibitors of F actin and microtubules. These results indicate that condensin II activity promotes chromosome territory formation in co-operation with additional force generators and that careful co-ordination with alternative homolog conjunction is crucial.
Yoneda, M., Yasui, K., Nakagawa, T., Hattori, N. and Ito, T. (2021). Nucleosome assembly protein 1 (NAP-1) is a regulator of histone H1 acetylation. J Biochem. PubMed ID: 34551067
Summary:
Acetylation of histone H1 is generally considered to activate transcription, whereas deacetylation of H1 represses transcription. However, the precise mechanism of the acetylation is unknown. Using chromatography this study identified nucleosome assembly protein 1 (NAP-1) as having inhibitory activity against histone H1 acetylation by acetyltransferase p300. Native NAP-1 was found to interact with H1 in a Drosophila crude extract. It was also found to inhibit the deacetylation of histone H1 by histone deacetylase 1 (HDAC1). The core histones in nucleosomes were acetylated in a GAL4-VP16 transcriptional activator-dependent manner in vitro. This acetylation was strongly repressed by hypoacetylated H1 but to a lesser extent by hyperacetylated H1. Consistent with these findings, a micrococcal nuclease assay indicated that hypoacetylated H1, which represses activator-dependent acetylation, was incorporated into chromatin, whereas hyperacetylated H1 was not. To determine the contribution of NAP-1 to transcriptional regulation in vivo, NAP-1 knockdown (KD) was compared with coactivator CREB-binding protein (CBP) KD using RNA sequencing in Drosophila Schneider 2 cells. Most genes were downregulated rather than upregulated by NAP-1 KD, and those downregulated genes were also downregulated by CBP KD. These results suggest that NAP-1 plays a role in transcriptional regulation by fine-tuning the acetylation of histone H1.
Carlson, J., Price, L., Cook, I. and Deng, H. (2021). Drosophila Keap1 xenobiotic response factor regulates developmental transcription through binding to chromatin. Dev Biol. PubMed ID: 34662537
Summary:
The Keap1-Nrf2 complex is a central regulator that mediates transcriptional responses to xenobiotic stimuli and is highly related with multiple human diseases. The molecular mechanisms and biological functions of Keap1 and Nrf2 are not fully understood. The Drosophila Keap1 homolog (dKeap1) is conserved with mammalian Keap1 except that dKeap1 contains a 156 aa C-terminal tail (CTD). A dKeap1 truncation with the CTD removed (dKeap1-ΔCTD) shows abolished nuclear localization and chromatin-binding. Expression of dKeap1-ΔCTD in the dKeap1 null background significantly rescues this mutant to the adult stage, but the files showed partial lethality, sterility and defects in adipose tissue. In the rescued flies, expression levels of ecdysone-response genes, ecdysone-synthetic genes and adipogenesis genes were down-regulated in specific tissues, indicating that the chromatin-binding of dKeap1 mediates the activation of these developmental genes. As the same time, dKeap1-ΔCTD can still suppress the basal expression of detoxifying genes and mediate the activation of these genes in response to xenobiotic stimuli, suggesting that the chromatin-binding of dKeap1 is not required for the regulation of detoxifying genes. These results support a model in which dKeap1 on one hand functions as an inhibitor for the Nrf2-mediated transcription in the xenobiotic response pathway and on the other hand functions as a chromatin-binding transcription activator in the developmental pathway. This study reveals a novel mechanism whereby Keap1-Nrf2 xenobiotic response signaling regulates development using a mechanism independent of redox signaling.
Zhang, S., Qi, H., Huang, C., Yuan, L., Zhang, L., Wang, R., Tian, Y. and Sun, L. (2021). Interaction of Male Specific Lethal complex and genomic imbalance on global gene expression in Drosophila. Sci Rep 11(1): 19679. PubMed ID: 34608252
Summary:
The inverse dosage effect caused by chromosome number variations shows global consequences in genomic imbalance including sexual dimorphism and an X chromosome-specific response. To investigate the relationship of the MSL complex to genomic imbalance, MSL2 was over-expressed in autosomal and sex chromosomal aneuploids, and the different transcriptomes were analyzed. Some candidate genes involved in regulatory mechanisms have also been tested during embryogenesis using TSA-FISH. This study showed that the de novo MSL complex assembled on the X chromosomes in females further reduced the global expression level on the basis of 2/3 down-regulation caused by the inverse dosage effect in trisomy through epigenetic modulations rather than induced dosage compensation. Plus, the sexual dimorphism effect in unbalanced genomes was further examined due to the pre-existing of the MSL complex in males. All these results demonstrate the dynamic functions of the MSL complex on global gene expression in different aneuploid genomes.
Voigt, S. and Kost, L. (2021). Differences in temperature-sensitive expression of PcG-regulated genes among natural populations of Drosophila melanogaster. G3 (Bethesda) 11(9). PubMed ID: 34544136
Summary:
Environmental temperature can affect chromatin-based gene regulation, in particular in ectotherms such as insects. Genes regulated by the Polycomb group (PcG) vary in their transcriptional output in response to changes in temperature. Expression of PcG-regulated genes typically increases with decreasing temperatures. This study examined variations in temperature-sensitive expression of PcG target genes in natural populations from different climates of Drosophila melanogaster, and differences thereof across different fly stages and tissues. Temperature-induced expression plasticity was found to be stage- and sex-specific with differences in the specificity between the examined PcG target genes. Some tissues and stages, however, showed a higher number of PcG target genes with temperature-sensitive expression than others. Overall, higher levels of temperature-induced expression plasticity was found in African tropical flies from the ancestral species range than in flies from temperate Europe. Differences were also observed between temperate flies, however, with more reduction of expression plasticity in warm-temperate than in cold-temperate populations. Although in general, temperature-sensitive expression appeared to be detrimental in temperate climates, there were also cases in which plasticity was increased in temperate flies, as well as no changes in expression plasticity between flies from different climates.

Friday, November 5th - Disease Models

Blazquez-Bernal, A., Fernandez-Costa, J. M., Bargiela, A. and Artero, R. (2021). Inhibition of autophagy rescues muscle atrophy in a LGMDD2 Drosophila model. Faseb j 35(10): e21914. PubMed ID: 34547132
Summary:
Limb-girdle muscular dystrophy D2 (LGMDD2) is an ultrarare autosomal dominant myopathy caused by mutation of the normal stop codon of the TNPO3 nuclear importin. The mutant protein carries a 15 amino acid C-terminal extension associated with pathogenicity. This study reports the first animal model of the disease by expressing the human mutant TNPO3 gene in Drosophila musculature or motor neurons and concomitantly silencing the endogenous expression of the fly protein ortholog, Tnpo-SR. A similar genotype expressing wildtype TNPO3 served as a control. Phenotypes characterization revealed that mutant TNPO3 expression targeted at muscles or motor neurons caused LGMDD2-like phenotypes such as muscle degeneration and atrophy, and reduced locomotor ability. Notably, LGMDD2 mutation increase TNPO3 at the transcript and protein level in the Drosophila model. Upregulated muscle autophagy observed in LGMDD2 patients was also confirmed in the fly model, in which the anti-autophagic drug chloroquine was able to rescue histologic and functional phenotypes. Overall, this study provides a proof of concept of autophagy as a target to treat disease phenotypes, and a neurogenic component is proposed to explain mutant TNPO3 pathogenicity in diseased muscles.
Wen, D. T., Zheng, L., Lu, K. and Hou, W. Q. (2021). Activation of cardiac Nmnat/NAD+/SIR2 pathways mediates endurance exercise resistance to lipotoxic cardiomyopathy in aging Drosophila. J Exp Biol 224(18). PubMed ID: 34495320
Summary:
Endurance exercise is an important way to resist and treat high-fat diet (HFD)-induced lipotoxic cardiomyopathy, but the underlying molecular mechanisms are poorly understood. This study used Drosophila to identify whether cardiac Nmnat/NAD+/SIR2 pathway activation mediates endurance exercise-induced resistance to lipotoxic cardiomyopathy. The results showed that endurance exercise activated the cardiac Nmnat/NAD+/SIR2/FOXO pathway and the Nmnat/NAD+/SIR2/PGC-1α pathway, including up-regulating cardiac Nmnat, SIR2, FOXO and PGC-1α expression, superoxide dismutase (SOD) activity and NAD+ levels, and it prevented HFD-induced or cardiac Nmnat knockdown-induced cardiac lipid accumulation, malondialdehyde (MDA) content and fibrillation increase, and fractional shortening decrease. Cardiac Nmnat overexpression also activated heart Nmnat/NAD+/SIR2 pathways and resisted HFD-induced cardiac malfunction, but it could not protect against HFD-induced lifespan reduction and locomotor impairment. Exercise improved lifespan and mobility in cardiac Nmnat knockdown flies. Therefore, the current results confirm that cardiac Nmnat/NAD+/SIR2 pathways are important antagonists of HFD-induced lipotoxic cardiomyopathy. Cardiac Nmnat/NAD+/SIR2 pathway activation is an important underlying molecular mechanism by which endurance exercise and cardiac Nmnat overexpression give protection against lipotoxic cardiomyopathy in Drosophila.
Zhu, Y., Cai, Q., Zheng, X., Liu, L., Hua, Y., Du, B., Zhao, G., Yu, J., Zhuo, Z., Xie, Z. and Ji, S. (2021). Aspirin Positively Contributes to Drosophila Intestinal Homeostasis and Delays Aging through Targeting Imd. Aging Dis 12(7): 1821-1834. PubMed ID: 34631223
Summary:
The intestine, a high-turnover tissue, plays a critical role in regulating aging and health in both vertebrates and invertebrates. Maintaining the epithelial barrier function of the intestine by preserving innate immune homeostasis significantly delays aging and prevents mortality. In an effort to explore effective chemicals and materials that can improve intestinal integrity, a nonbiased screen was performed utilizing Drosophila as an animal model. Long-term uptake of aspirin markedly prevented age-onset gut leakage, the over-proliferation of intestinal stem cells, and the dysbiosis of commensal microbiota in fruit flies. Mechanistically, aspirin efficiently downregulated chronic activation of intestinal immune deficiency signaling during aging. Furthermore, in vivo and in vitro biochemical analyses indicated that aspirin is a negative modulator in control of the K63-linked ubiquitination of Imd. These findings uncover a novel regulatory mechanism by which aspirin positively modulates intestinal homeostasis, thus delaying aging, in Drosophila.
Zuko, A., Mallik, M., Thompson, R., Spaulding, E. L., Wienand, A. R., Been, M., Tadenev, A. L. D., van Bakel, N., Sijlmans, C., Santos, L. A., Bussmann, J., Catinozzi, M., Das, S., Kulshrestha, D., Burgess, R. W., Ignatova, Z. and Storkebaum, E. (2021). tRNA overexpression rescues peripheral neuropathy caused by mutations in tRNA synthetase. Science 373(6559): 1161-1166. PubMed ID: 34516840
Summary:
Heterozygous mutations in six transfer RNA (tRNA) synthetase genes cause Charcot-Marie-Tooth (CMT) peripheral neuropathy. CMT mutant tRNA synthetases inhibit protein synthesis by an unknown mechanism. This study found that CMT mutant glycyl-tRNA synthetases bound tRNAGly but failed to release it, resulting in tRNAGly sequestration. This sequestration potentially depleted the cellular tRNAGly pool, leading to insufficient glycyl-tRNAGly supply to the ribosome. Accordingly, this study found ribosome stalling at glycine codons and activation of the integrated stress response (ISR) in affected motor neurons. Moreover, transgenic overexpression of tRNAGly rescued protein synthesis, peripheral neuropathy, and ISR activation in Drosophila and mouse CMT disease type 2D (CMT2D) models. Conversely, inactivation of the ribosome rescue factor GTPBP2 exacerbated peripheral neuropathy. These findings suggest a molecular mechanism for CMT2D, and elevating tRNAGly levels may thus have therapeutic potential.
Yu, C. C., Chang, F. C., Hong, Y. H., Li, J. C., Chen, P. L., Chen, C. H., Chiu, T. W., Lu, T. T., Wang, Y. M. and Kao, C. F. (2021). Assessing the cognitive status of Drosophila by the value-based feeding decision. NPJ Aging Mech Dis 7(1): 24. PubMed ID: 34526491
Summary:
Decision-making is considered an important aspect of cognitive function. Impaired decision-making is a consequence of cognitive decline caused by various physiological conditions, such as aging and neurodegenerative diseases. This study exploited the value-based feeding decision (VBFD) assay, which is a simple sensory-motor task, to determine the cognitive status of Drosophila. The results indicated the deterioration of VBFD is notably correlated with aging and neurodegenerative disorders. Restriction of the mushroom body (MB) neuronal activity partly blunted the proper VBFD. Furthermore, using the Drosophila polyQ disease model, this study demonstrated the impaired VBFD is ameliorated by the dinitrosyl iron complex (DNIC-1), a novel and steady nitric oxide (NO)-releasing compound. Therefore it is proposed that the VBFD assay provides a robust assessment of Drosophila cognition and can be used to characterize additional neuroprotective interventions.
Zhu, J. Y., Huang, X., Fu, Y., Wang, Y., Zheng, P., Liu, Y. and Han, Z. (2021). Pharmacological or genetic inhibition of hypoxia signaling attenuates oncogenic RAS-induced cancer phenotypes. Dis Model Mech. PubMed ID: 34580712
Summary:
Oncogenic Ras mutations are highly prevalent in hematopoietic malignancies. However, it is difficult to directly target oncogenic RAS proteins for therapeutic intervention. This study has developed a Drosophila Acute Myeloid Leukemia (AML) model induced by human KRASG12V, which exhibits a dramatic increase in myeloid-like leukemia cells. Both genetic and drug screens were performed using this model. The genetic screen identified 24 candidate genes able to attenuate the oncogenic RAS-induced phenotype, including two key hypoxia pathway genes HIF1A and ARNT (HIF1B). The drug screen revealed echinomycin, an inhibitor of HIF1A, could effectively attenuate the leukemia phenotype caused by KRASG12V. Furthermore, this study showed that echinomycin treatment could effectively suppress oncogenic RAS-driven leukemia cell proliferation using both human leukemia cell lines and a mouse xenograft model. These data suggest that inhibiting the hypoxia pathway could be an effective treatment approach for oncogenic RAS-induced cancer phenotype, and that echinomycin is a promising targeted drug to attenuate oncogenic RAS-induced cancer phenotypes.

Thursday, November 4th - Adult neural development and function

Vrontou, E., Groschner, L. N., Szydlowski, S., Brain, R., Krebbers, A. and Miesenbock, G. (2021). Response competition between neurons and antineurons in the mushroom body. Curr Biol. PubMed ID: 34610272
Summary:
The mushroom bodies of Drosophila contain circuitry compatible with race models of perceptual choice. When flies discriminate odor intensity differences, opponent pools of αβ core Kenyon cells (on and off αβ(c) KCs) accumulate evidence for increases or decreases in odor concentration. These sensory neurons and "antineurons" connect to a layer of mushroom body output neurons (MBONs) which bias behavioral intent in opposite ways. All-to-all connectivity between the competing integrators and their MBON partners allows for correct and erroneous decisions; dopaminergic reinforcement sets choice probabilities via reciprocal changes to the efficacies of on and off KC synapses; and pooled inhibition between αβ(c) KCs can establish equivalence with the drift-diffusion formalism known to describe behavioral performance. The response competition network gives tangible form to many features envisioned in theoretical models of mammalian decision making, but it differs from these models in one respect: the principal variables-the fill levels of the integrators and the strength of inhibition between them-are represented by graded potentials rather than spikes. In pursuit of similar computational goals, a small brain may thus prioritize the large information capacity of analog signals over the robustness and temporal processing span of pulsatile codes.
Byers, N., Hahm, E. T. and Tsunoda, S. (2021). Slo2/K(Na) Channels in Drosophila Protect Against Spontaneous and Induced Seizure-like Behavior Associated with an Increased Persistent Na(+) Current. J Neurosci. PubMed ID: 34544836
Summary:
Na(+)-sensitivity is a unique feature of Na(+)-activated K(+) (K(Na)) channels, making them naturally suited to counter a sudden influx in Na(+) ions. As such, it has long been suggested that K(Na) channels may serve a protective function against excessive excitation associated with neuronal injury and disease. This study examined K(Na) channels encoded by the Drosophila Slo2 (dSlo2) gene in males and females. dSlo2/K(Na) channels are selectively expressed in cholinergic neurons in the adult brain, as well as in glutamatergic motor neurons, where dampening excitation may function to inhibit global hyperactivity and seizure-like behavior. Indeed, this study shows that effects of feeding Drosophila a cholinergic agonist are exacerbated by the loss of dSlo2/K(Na) channels. dSlo2/K(Na) channels encode a TTX-sensitive K(+) conductance, indicating that dSlo2/K(Na) channels can be activated by Na(+) carried by voltage-dependent Na(+) channels. The role of dSlo2/K(Na) channels was tested in established genetic seizure models in which the voltage-dependent persistent Na(+) current (I(Nap)) was elevated. The absence of dSlo2/K(Na) channels increased susceptibility to mechanically-induced seizure-like behavior. Finally, this study showed that loss of dSlo2/K(Na) channels in both genetic and pharmacologically-primed seizure models resulted in the appearance of spontaneous seizures. Together, these results support a model in which dSlo2/K(Na) channels, activated upon neuronal over-excitation, contribute to a protective threshold to suppress the induction of seizure-like activity.
Zeng, X., Komanome, Y., Kawasaki, T., Inada, K., Jonaitis, J., Pulver, S. R., Kazama, H. and Nose, A. (2021). An electrically coupled pioneer circuit enables motor development via proprioceptive feedback in Drosophila embryos. Curr Biol. PubMed ID: 34666002
Summary:
Precocious movements are widely seen in embryos of various animal species. Whether such movements via proprioceptive feedback play instructive roles in motor development or are a mere reflection of activities in immature motor circuits is a long-standing question. This study imaged the emerging motor activities in Drosophila embryos that lack proprioceptive feedback and showed that proprioceptive experience is essential for the development of locomotor central pattern generators (CPGs). Downstream of proprioceptive inputs, a pioneer premotor circuit was identified composed of two pairs of segmental interneurons, whose gap-junctional transmission requires proprioceptive experience and plays a crucial role in CPG formation. The circuit autonomously generates rhythmic plateau potentials via IP(3)-mediated Ca(2+) release from internal stores, which contribute to muscle contractions and hence produce proprioceptive feedback. These findings demonstrate the importance of self-generated movements in instructing motor development and identify the cells, circuit, and physiology at the core of this proprioceptive feedback.
Weiss, S., Clamon, L. C., Manoim, J. E., Ormerod, K. G., Parnas, M. and Littleton, J. T. (2021). Glial ER and GAP junction mediated Ca(2+) waves are crucial to maintain normal brain excitability. Glia. PubMed ID: 34528727
Summary:
Astrocytes play key roles in regulating multiple aspects of neuronal function from invertebrates to humans and display Ca(2+) fluctuations that are heterogeneously distributed throughout different cellular microdomains. Changes in Ca(2+) dynamics represent a key mechanism for how astrocytes modulate neuronal activity. An unresolved issue is the origin and contribution of specific glial Ca(2+) signaling components at distinct astrocytic domains to neuronal physiology and brain function. The Drosophila model system offers a simple nervous system that is highly amenable to cell-specific genetic manipulations to characterize the role of glial Ca(2+) signaling. This study has identified a role for ER store-operated Ca(2+) entry (SOCE) pathway in perineurial glia (PG), a glial population that contributes to the Drosophila blood-brain barrier. PG cells display diverse Ca(2+) activity that varies based on their locale within the brain. Ca(2+) signaling in PG cells does not require extracellular Ca(2+) and is blocked by inhibition of SOCE, Ryanodine receptors, or gap junctions. Disruption of these components triggers stimuli-induced seizure-like episodes. These findings indicate that Ca(2+) release from internal stores and its propagation between neighboring glial cells via gap junctions are essential for maintaining normal nervous system function.
You, S., Yu, A. M., Roberts, M. A., Joseph, I. J. and Jackson, F. R. (2021). Circadian regulation of the Drosophila astrocyte transcriptome. PLoS Genet 17(9): e1009790. PubMed ID: 34543266
Summary:
Recent studies have demonstrated that astrocytes cooperate with neurons of the brain to mediate circadian control of many rhythmic processes including locomotor activity and sleep. Transcriptional profiling studies have described the overall rhythmic landscape of the brain, but few have employed approaches that reveal heterogeneous, cell-type specific rhythms of the brain. Using cell-specific isolation of ribosome-bound RNAs in Drosophila, the first circadian "translatome" for astrocytes was generated. This analysis identified 293 "cycling genes" in astrocytes, most with mammalian orthologs. A subsequent behavioral genetic screen identified a number of genes whose expression is required in astrocytes for normal sleep behavior. In particular, this study showed that certain genes known to regulate fly innate immune responses are also required for normal sleep patterns.
Yamasaki, Y., Lim, Y. M., Minami, R. and Tsuda, L. (2021). A splicing variant of Charlatan, a Drosophila REST-like molecule, preferentially localizes to axons. Biochem Biophys Res Commun 578: 35-41. PubMed ID: 34536827
Summary:
Neuron-restrictive silencing factor (NRSF), also known as RE-1 silencing transcription factor (REST), has pivotal functions in many neuron-specific genes. Previous studies revealed that neuron-specific alternative splicing (AS) of REST produces divergent forms of REST variants and provides regulatory complexity in the nervous system. However, the biological significance of these variants in the regulation of neuronal activities remains to be clarified. This study revealed that Charlatan (Chn), a Drosophila REST-like molecule, is also regulated by neuron-specific AS. Neuron-specific AS produced six divergent variants of Chn proteins, one of which preferentially localized to axons. A small sequence of this variant was especially important for the axonal localization. These data suggest that some variants have roles beyond the transcriptional regulation of neuronal activities.

Wednesday, November 3rd - Signaling

Brown, B., Mitra, S., Roach, F. D., Vasudevan, D. and Ryoo, H. D. (2021). The transcription factor Xrp1 is required for PERK-mediated antioxidant gene induction in Drosophila. Elife 10. PubMed ID: 34605405
Summary:
PERK is an endoplasmic reticulum (ER) transmembrane sensor that phosphorylates eIF2α to initiate the Unfolded Protein Response (UPR). eIF2α phosphorylation promotes stress-responsive gene expression most notably through the transcription factor ATF4 that contains a regulatory 5' leader. Possible PERK effectors other than ATF4 remain poorly understood. This study reports that the bZIP transcription factor Xrp1 is required for ATF4-independent PERK signaling. Cell-type-specific gene expression profiling in Drosophila indicated that delta-family glutathione-S-transferases (gstD) are prominently induced by the UPR-activating transgene Rh1(G69D). Perk was necessary and sufficient for such gstD induction, but ATF4 was not required. Instead, Perk and other regulators of eIF2α phosphorylation regulated Xrp1 protein levels to induce gstDs. The Xrp1 5' leader has a conserved upstream Open Reading Frame (uORF) analogous to those that regulate ATF4 translation. The gstD-GFP reporter induction required putative Xrp1 binding sites. These results indicate that antioxidant genes are highly induced by a previously unrecognized UPR signaling axis consisting of PERK and Xrp1.
Yasumura, M., Hagiwara, A., Hida, Y. and Ohtsuka, T. (2021). Planar cell polarity protein Vangl2 and its interacting protein Ap2m1 regulate dendritic branching in cortical neurons. Genes Cells. PubMed ID: 34626136
Summary:
Van Gogh-like 2 (Vangl2) is a mammalian homolog of Drosophila core planar cell polarity (PCP) protein Vang/Strabismus, which organizes asymmetric cell axes for developmental proliferation, fate determination, and polarized movements in multiple tissues, including neurons. Although the PCP pathway has an essential role for dendrite and dendritic spine formation, the molecular mechanism remains to be clarified. To investigate the mechanism of Vangl2-related neuronal development, this study screened for proteins that interact with the Vangl2 cytosolic N-terminus from postnatal day 9 mouse brains using a yeast two-hybrid system. From 61 genes, adaptor-related protein complex 2, mu 1 subunit (Ap2m1: Drosophila homolog AP-2mu) was identified as the Vangl2 N-terminal binding protein. Intriguingly, however, the pull-down assay demonstrated that Vangl2 interacted with Ap2m1 not only at its N-terminus but also at the C-terminal Prickle binding domain. Furthermore, it was verified that the downregulation of Ap2m1 in the developing cortical neurons reduced the dendritic branching similar to what occurs in a knockdown of Vangl2. From these results, it is suggested that the membrane internalization regulated by the PCP pathway is required for the developmental morphological change in neurons.
Wei, Y., Du, J. and Zhao, Z. (2021). Integrative Role of 14-3-3epsilon in Sleep Regulation. Int J Mol Sci 22(18). PubMed ID: 34575915
Summary:
Sleep is a crucial factor for health and survival in all animals. This study found by proteomic analysis that some cancer related proteins were impacted by the circadian clock. The 14-3-3ε protein, expression of which is activated by the circadian transcription factor Clock, regulates adult sleep of Drosophila independent of circadian rhythm. Detailed analysis of the sleep regulatory mechanism shows that 14-3-3ε directly targets the Ultrabithorax (Ubx) gene to activate transcription of the pigment dispersing factor (PDF). The dopamine receptor (Dop1R1) and the octopamine receptor (Oamb), are also involved in the 14-3-3ε pathway, which in 14-3-3ε mutant flies causes increases in the dopR1 and OAMB, while downregulation of the DopR1 and Oamb can restore the sleep phenotype caused by the 14-3-3ε mutation. In conclusion, 14-3-3ε is necessary for sleep regulation in Drosophila.
Xu, Y., Erdjument-Bromage, H., Phoon, C. K. L., Neubert, T. A., Ren, M. and Schlame, M. (2021)s. Cardiolipin remodeling enables protein crowding in the inner mitochondrial membrane. EMBO J: e108428. PubMed ID: 34661298
Summary:
Mitochondrial cristae are extraordinarily crowded with proteins, which puts stress on the bilayer organization of lipids. This study tested the hypothesis that the high concentration of proteins drives the tafazzin-catalyzed remodeling of fatty acids in cardiolipin, thereby reducing bilayer stress in the membrane. Specifically, whether protein crowding induces cardiolipin remodeling and whether the lack of cardiolipin remodeling prevents the membrane from accumulating proteins were tested. In vitro, the incorporation of large amounts of proteins into liposomes altered the outcome of the remodeling reaction. In yeast, the concentration of proteins involved in oxidative phosphorylation (OXPHOS) correlated with the cardiolipin composition. Genetic ablation of either remodeling or biosynthesis of cardiolipin caused a substantial drop in the surface density of OXPHOS proteins in the inner membrane of the mouse heart and Drosophila flight muscle mitochondria. These data suggest that OXPHOS protein crowding induces cardiolipin remodelling and that remodeled cardiolipin supports the high concentration of these proteins in the inner mitochondrial membrane.

Tuesday, November 2nd - Gonads

Kumari, J. and Sinha, P. (2021). Developmental expression patterns of toolkit genes in male accessory gland of Drosophila parallels those of mammalian prostate. Biol Open 10(8). PubMed ID: 34342345
Summary:
Conservation of genetic toolkits in disparate phyla may help reveal commonalities in organ designs transcending their extreme anatomical disparities. A male accessory sexual organ in mammals, the prostate, for instance, is anatomically disparate from its analogous, phylogenetically distant counterpart - the male accessory gland (MAG) - in insects like Drosophila. It has not been ascertained if the anatomically disparate Drosophila MAG shares developmental parallels with those of the mammalian prostate. This study shows that the development of Drosophila mesoderm-derived MAG entails recruitment of similar genetic toolkits of tubular organs like that seen in endoderm-derived mammalian prostate. For instance, like mammalian prostate, Drosophila MAG morphogenesis is marked by recruitment of fibroblast growth factor receptor (FGFR) - a signalling pathway often seen recruited for tubulogenesis - starting early during its adepithelial genesis. A specialisation of the individual domains of the developing MAG tube, on the other hand, is marked by the expression of a posterior Hox gene transcription factor, Abd-B, while Hh-Dpp signalling marks its growth. Drosophila MAG, therefore, reveals the developmental design of a unitary bud-derived tube that appears to have been co-opted for the development of male accessory sexual organs across distant phylogeny and embryonic lineages.
Bubnell, J. E., Fernandez-Begne, P., Ulbing, C. K. S. and Aquadro, C. F. (2021). Diverse wMel variants of Wolbachia pipientis differentially rescue fertility and cytological defects of the bag of marbles partial loss of function mutation in Drosophila melanogaster. G3 (Bethesda). PubMed ID: 34580706
Summary:
In Drosophila melanogaster, the maternally inherited endosymbiont Wolbachia pipientis interacts with germline stem cell genes during oogenesis. One such gene, bag of marbles (bam) is the key switch for differentiation and also shows signals of adaptive evolution for protein diversification. These observations have led to a hypothesis that W. pipientis could be driving the adaptive evolution of bam for control of oogenesis. To test this hypothesis, the specificity of the genetic interaction between bam and W. pipientis must be understood. This study used CRISPR/Cas9 to engineer the original single amino acid bam hypomorphic mutation (bamL255F) and a new bam null disruption mutation into the w1118 isogenic background. The fertility was assessed of wildtype bam, bamL255F/bamnull hypomorphic, and bamL255F/bamL255F mutant females, each infected individually with 10 W. pipientis wMel variants representing three phylogenetic clades. Overall, it was found that all of the W. pipientis variants tested rescue bam hypomorphic fertility defects with wMelCS-like variants exhibiting the strongest rescue effects. In addition, these variants did not increase wildtype bam female fertility. Therefore, both bam and W. pipientis interact in genotype-specific ways to modulate female fertility, a critical fitness phenotype.
Sahu, A., Karmakar, S., Halder, S., Ghosh, G., Acharjee, S., Dasgupta, P., Ghosh, R., Deshpande, G. and Prasad, M. (2021). Germline soma communication mediated by gap junction proteins regulates epithelial morphogenesis. PLoS Genet 17(8): e1009685. PubMed ID: 34343194
Summary:
Gap junction (GJ) proteins, the primary constituents of GJ channels, are conserved determinants of patterning. Canonically, a GJ channel, made up of two hemi-channels contributed by the neighboring cells, facilitates transport of metabolites/ions. This study demonstrates the involvement of GJ proteins during cuboidal to squamous epithelial transition displayed by the anterior follicle cells (AFCs) from Drosophila ovaries. Somatically derived AFCs stretch and flatten when the adjacent germline cells start increasing in size. GJ proteins, Innexin2 (Inx2) and Innexin4 (Inx4), functioning in the AFCs and germline respectively, promote the shape transformation by modulating calcium levels in the AFCs. These observations suggest that alterations in calcium flux potentiate STAT activity to influence actomyosin-based cytoskeleton, possibly resulting in disassembly of adherens junctions. These data have uncovered sequential molecular events underlying the cuboidal to squamous shape transition and offer unique insight into how GJ proteins expressed in the neighboring cells contribute to morphogenetic processes.
Praggastis, S. A., Nam, H. J., Lam, G., Child Vi, M. B., Castillo, D. M. and Thummel, C. S. (2021). Regulation of male fertility and accessory gland gene expression by the Drosophila HR39 nuclear receptor. Dev Biol 479: 51-60. PubMed ID: 34331899
Summary:
Successful reproduction is dependent on the transfer of male seminal proteins to females upon mating. These proteins arise from secretory tissues in the male reproductive tract, including the prostate and seminal vesicles in mammals and the accessory gland in insects. Although detailed functional studies have provided important insights into the mechanisms by which accessory gland proteins support reproduction, much less is known about the molecular mechanisms that regulate their expression within this tissue. This study shows that the Drosophila HR39 nuclear receptor is required for the proper expression of most genes that encode male accessory gland proteins. Consistent with this role, HR39 mutant males are infertile. In addition, tissue-specific RNAi and genetic rescue experiments indicate that HR39 acts within the accessory glands to regulate gene expression and male fertility. These results provide new directions for characterizing the mammalian orthologs of HR39, the SF-1 and LRH-1 nuclear receptors, both of which are required for glandular secretions and reproduction. In addition, these studies provide a molecular mechanism to explain how the accessory glands can maintain the abundant levels of seminal fluid production required to support fertility.
Yang, Y., Kong, R., Goh, F. G., Somers, W. G., Hime, G. R., Li, Z. and Cai, Y. (2021).. dRTEL1 is essential for the maintenance of Drosophila male germline stem cells. PLoS Genet 17(10): e1009834. PubMed ID: 34644293
Summary:
Stem cells have the potential to maintain undifferentiated state and differentiate into specialized cell types. Despite numerous progress has been achieved in understanding stem cell self-renewal and differentiation, many fundamental questions remain unanswered. In this study, dRTEL1, the Drosophila homolog of Regulator of Telomere Elongation Helicase 1 (CG4078), was identified as a novel regulator of male germline stem cells (GSCs). Genome-wide transcriptome analysis and ChIP-Seq results suggest that dRTEL1 affects a set of candidate genes required for GSC maintenance, likely independent of its role in DNA repair. Furthermore, dRTEL1 prevents DNA damage-induced checkpoint activation in GSCs. Finally, dRTEL1 functions to sustain Stat92E protein levels, the key player in GSC maintenance. Together, these findings reveal an intrinsic role of the DNA helicase dRTEL1 in maintaining male GSC and provide insight into the function of dRTEL1.
Muller, K., Herrera, K., Talyn, B. and Melchiorre, E. (2021). Toxicological Effects of Roundup(®) on Drosophila melanogaster Reproduction. Toxics 9(7). PubMed ID: 34357904
Summary:
Herbicide use has increased dramatically since 2001, particularly Roundup. Effective in agricultural practice, Roundup adversely affects non-target organisms, including reproductive and endocrine systems. This study exposed fruit flies, Drosophila melanogaster, to either Roundup Ready to Use, containing pelargonic acid and glyphosate, or Roundup Super Concentrate, that includes glyphosate and POEA, at sublethal concentrations. Both Roundup formulations reduced ovary volume with fewer mature oocytes, most adversely at the highest concentration tested. Flies exposed within 2 h of eclosion were affected more than at 4 h, suggesting a critical period of increased ovarian sensitivity. These results support multi-species evidence that glyphosate-based herbicides interfere with normal development of the reproductive systems of non-target organisms.

Monday, November 1st - Methods

Zheng, L., Liu, Z., Yang, Y. and Shen, H. B. (2021). Accurate inference of gene regulatory interactions from spatial gene expression with deep contrastive learning. Bioinformatics. PubMed ID: 34664632
Summary:
Reverse engineering of gene regulatory networks (GRNs) has long been an attractive research topic in system biology. Computational prediction of gene regulatory interactions has remained a challenging problem due to the complexity of gene expression and scarce information resources. The high-throughput spatial gene expression data, like in situ hybridization images that exhibit temporal and spatial expression patterns, has provided abundant and reliable information for the inference of GRNs. However, computational tools for analyzing the spatial gene expression data are highly underdeveloped. This study developed a new method for identifying gene regulatory interactions from gene expression images, called ConGRI. The method is featured by a contrastive learning scheme and deep Siamese CNN architecture, which automatically learns high-level feature embeddings for the expression images and then feeds the embeddings to an artificial neural network to determine whether or not the interaction exists. The method was applied to a Drosophila embryogenesis dataset and GRNs of eye development and mesoderm development were identified. Experimental results show that ConGRI outperforms previous traditional and deep learning methods by a large margin, which achieves accuracies of 76.7% and 68.7% for the GRNs of early eye development and mesoderm development, respectively. It also reveals some master regulators for Drosophila eye development.
Meiler, A., Marchiano, F., Haering, M., Weitkunat, M., Schnorrer, F. and Habermann, B. H. (2021). AnnoMiner is a new web-tool to integrate epigenetics, transcription factor occupancy and transcriptomics data to predict transcriptional regulators. Sci Rep 11(1): 15463. PubMed ID: 34326396
Summary:
Gene expression regulation requires precise transcriptional programs, led by transcription factors in combination with epigenetic events. Recent advances in epigenomic and transcriptomic techniques provided insight into different gene regulation mechanisms. However, to date it remains challenging to understand how combinations of transcription factors together with epigenetic events control cell-type specific gene expression. This study has developed the AnnoMiner web-server, an innovative and flexible tool to annotate and integrate epigenetic, and transcription factor occupancy data. First, AnnoMiner annotates user-provided peaks with gene features. Second, AnnoMiner can integrate genome binding data from two different transcriptional regulators together with gene features. Third, AnnoMiner offers to explore the transcriptional deregulation of genes nearby, or within a specified genomic region surrounding a user-provided peak. AnnoMiner's fourth function performs transcription factor or histone modification enrichment analysis for user-provided gene lists by utilizing hundreds of public, high-quality datasets from ENCODE for the model organisms human, mouse, Drosophila and C. elegans. Thus, AnnoMiner can predict transcriptional regulators for a studied process without the strict need for chromatin data from the same process. AnnoMiner is compared to existing tools and experimentally validated several transcriptional regulators predicted by AnnoMiner to indeed contribute to muscle morphogenesis in Drosophila.
Kogler, A. C., Kherdjemil, Y., Bender, K., Rabinowitz, A., Marco-Ferreres, R. and Furlong, E. E. M. (2021). Extremely rapid and reversible optogenetic perturbation of nuclear proteins in living embryos. Dev Cell 56(16): 2348-2363. PubMed ID: 34363757
Summary:
Many developmental regulators have complex and context-specific roles in different tissues and stages, making the dissection of their function extremely challenging. As regulatory processes often occur within minutes, perturbation methods that match these dynamics are needed. This paper presents the improved light-inducible nuclear export system (iLEXY), an optogenetic loss-of-function approach that triggers translocation of proteins from the nucleus to the cytoplasm. By introducing a series of mutations, LEXY's efficiency and generated variants was substantially increased with different recovery times. iLEXY enables rapid (t(1/2) < 30 s), efficient, and reversible nuclear protein depletion in embryos, and is generalizable to proteins of diverse sizes and functions. Applying iLEXY to the Drosophila master regulator Twist, loss-of-function mutants were phenocopied, the Twist-sensitive embryonic stages were precisely mapped, and the effects of timed Twist depletions were investigated. The results demonstrate the power of iLEXY to dissect the function of pleiotropic factors during embryogenesis with unprecedented temporal precision.
Vierock, J., Rodriguez-Rozada, S., Dieter, A., Pieper, F., Sims, R., Tenedini, F., Bergs, A. C. F., Bendifallah, I., Zhou, F., Zeitzschel, N., Ahlbeck, J., Augustin, S., Sauter, K., Papagiakoumou, E., Gottschalk, A., Soba, P., Emiliani, V., Engel, A. K., Hegemann, P. and Wiegert, J. S. (2021). BiPOLES is an optogenetic tool developed for bidirectional dual-color control of neurons. Nat Commun 12(1): 4527. PubMed ID: 34312384
Summary:
Optogenetic manipulation of neuronal activity through excitatory and inhibitory opsins has become an indispensable experimental strategy in neuroscience research. For many applications bidirectional control of neuronal activity allowing both excitation and inhibition of the same neurons in a single experiment is desired. This requires low spectral overlap between the excitatory and inhibitory opsin, matched photocurrent amplitudes and a fixed expression ratio. Moreover, independent activation of two distinct neuronal populations with different optogenetic actuators is still challenging due to blue-light sensitivity of all opsins. This study reports BiPOLES, an optogenetic tool for potent neuronal excitation and inhibition with light of two different wavelengths. BiPOLES enables sensitive, reliable dual-color neuronal spiking and silencing with single- or two-photon excitation, optical tuning of the membrane voltage, and independent optogenetic control of two neuronal populations using a second, blue-light sensitive opsin. The utility of BiPOLES is demonstrated in worms, flies, mice and ferrets.
Asaoka, M., Sakamaki, Y., Fukumoto, T., Nishimura, K., Tomaru, M., Takano-Shimizu, T., Tanaka, D. and Kobayashi, S. (2021). Offspring production from cryopreserved primordial germ cells in Drosophila. Commun Biol 4(1): 1159. PubMed ID: 34621004
Summary:
There is an urgent need to cryopreserve Drosophila stocks that have been maintained as living cultures for a long time. Long-term culture increases the risk of accidental loss and of unwanted genetic alteration. This paper reports that cryopreserved primordial germ cells (PGCs) can produce F1 progeny when transplanted into hosts. The cryopreserved donor PGCs could form germline stem cells in host gonads and contributed to continuous offspring production. Furthermore, the ability to produce offspring did not appear to vary with either differences between donor strains or cryopreservation duration. Therefore, it is proposed that the cryopreservation method is feasible for long-term storage of various Drosophila strains. These results underscore the potential usefulness of this cryopreservation method for backing up living stocks to avoid either accidental loss or genetic alteration.
Bylino, O. V., Ibragimov, A. N., Pravednikova, A. E. and Shidlovskii, Y. V. (2021). Investigation of the Basic Steps in the Chromosome Conformation Capture Procedure. Front Genet 12: 733937. PubMed ID: 34616432
Summary:
A constellation of chromosome conformation capture methods (С-methods) are an important tool for biochemical analysis of the spatial interactions between DNA regions that are separated in the primary sequence. All these methods are based on the long sequence of basic steps of treating cells, nuclei, chromatin, and finally DNA, thus representing a significant technical challenge. This paper presents an in-depth study of the basic steps in the chromatin conformation capture procedure (3С), which was performed using Drosophila Schneider 2 cells as a model. The steps of cell lysis, nuclei washing, nucleoplasm extraction, chromatin treatment with SDS/Triton X-100, restriction enzyme digestion, chromatin ligation, reversion of cross-links, DNA extraction, treatment of a 3C library with RNases, and purification of the 3C library were investigated. Several options were studied, and optimal conditions were found. This work contributes to the understanding of the 3C basic steps and provides a useful guide to the 3C procedure.

Friday, October 29th- Disease Models

Meiler, A., Marchiano, F., Haering, M., Weitkunat, M., Schnorrer, F. and Habermann, B. H. (2021). AnnoMiner is a new web-tool to integrate epigenetics, transcription factor occupancy and transcriptomics data to predict transcriptional regulators. Sci Rep 11(1): 15463. PubMed ID: 34326396
Summary:
Gene expression regulation requires precise transcriptional programs, led by transcription factors in combination with epigenetic events. Recent advances in epigenomic and transcriptomic techniques provided insight into different gene regulation mechanisms. However, to date it remains challenging to understand how combinations of transcription factors together with epigenetic events control cell-type specific gene expression. This study has developed the AnnoMiner web-server, an innovative and flexible tool to annotate and integrate epigenetic, and transcription factor occupancy data. First, AnnoMiner annotates user-provided peaks with gene features. Second, AnnoMiner can integrate genome binding data from two different transcriptional regulators together with gene features. Third, AnnoMiner offers to explore the transcriptional deregulation of genes nearby, or within a specified genomic region surrounding a user-provided peak. AnnoMiner's fourth function performs transcription factor or histone modification enrichment analysis for user-provided gene lists by utilizing hundreds of public, high-quality datasets from ENCODE for the model organisms human, mouse, Drosophila and C. elegans. Thus, AnnoMiner can predict transcriptional regulators for a studied process without the strict need for chromatin data from the same process. AnnoMiner is compared to existing tools and experimentally validated several transcriptional regulators predicted by AnnoMiner to indeed contribute to muscle morphogenesis in Drosophila.
Zheng, L., Liu, Z., Yang, Y. and Shen, H. B. (2021). Accurate inference of gene regulatory interactions from spatial gene expression with deep contrastive learning. Bioinformatics. PubMed ID: 34664632
Summary:
Reverse engineering of gene regulatory networks (GRNs) has long been an attractive research topic in system biology. Computational prediction of gene regulatory interactions has remained a challenging problem due to the complexity of gene expression and scarce information resources. The high-throughput spatial gene expression data, like in situ hybridization images that exhibit temporal and spatial expression patterns, has provided abundant and reliable information for the inference of GRNs. However, computational tools for analyzing the spatial gene expression data are highly underdeveloped. This study developed a new method for identifying gene regulatory interactions from gene expression images, called ConGRI. The method is featured by a contrastive learning scheme and deep Siamese CNN architecture, which automatically learns high-level feature embeddings for the expression images and then feeds the embeddings to an artificial neural network to determine whether or not the interaction exists. The method was applied to a Drosophila embryogenesis dataset and GRNs of eye development and mesoderm development were identified. Experimental results show that ConGRI outperforms previous traditional and deep learning methods by a large margin, which achieves accuracies of 76.7% and 68.7% for the GRNs of early eye development and mesoderm development, respectively. It also reveals some master regulators for Drosophila eye development.
Vierock, J., Rodriguez-Rozada, S., Dieter, A., Pieper, F., Sims, R., Tenedini, F., Bergs, A. C. F., Bendifallah, I., Zhou, F., Zeitzschel, N., Ahlbeck, J., Augustin, S., Sauter, K., Papagiakoumou, E., Gottschalk, A., Soba, P., Emiliani, V., Engel, A. K., Hegemann, P. and Wiegert, J. S. (2021). BiPOLES is an optogenetic tool developed for bidirectional dual-color control of neurons. Nat Commun 12(1): 4527. PubMed ID: 34312384
Summary:
Optogenetic manipulation of neuronal activity through excitatory and inhibitory opsins has become an indispensable experimental strategy in neuroscience research. For many applications bidirectional control of neuronal activity allowing both excitation and inhibition of the same neurons in a single experiment is desired. This requires low spectral overlap between the excitatory and inhibitory opsin, matched photocurrent amplitudes and a fixed expression ratio. Moreover, independent activation of two distinct neuronal populations with different optogenetic actuators is still challenging due to blue-light sensitivity of all opsins. This study reports BiPOLES, an optogenetic tool for potent neuronal excitation and inhibition with light of two different wavelengths. BiPOLES enables sensitive, reliable dual-color neuronal spiking and silencing with single- or two-photon excitation, optical tuning of the membrane voltage, and independent optogenetic control of two neuronal populations using a second, blue-light sensitive opsin. The utility of BiPOLES is demonstrated in worms, flies, mice and ferrets.
Kogler, A. C., Kherdjemil, Y., Bender, K., Rabinowitz, A., Marco-Ferreres, R. and Furlong, E. E. M. (2021). Extremely rapid and reversible optogenetic perturbation of nuclear proteins in living embryos. Dev Cell 56(16): 2348-2363. PubMed ID: 34363757
Summary:
Many developmental regulators have complex and context-specific roles in different tissues and stages, making the dissection of their function extremely challenging. As regulatory processes often occur within minutes, perturbation methods that match these dynamics are needed. This paper presents the improved light-inducible nuclear export system (iLEXY), an optogenetic loss-of-function approach that triggers translocation of proteins from the nucleus to the cytoplasm. By introducing a series of mutations, LEXY's efficiency and generated variants was substantially increased with different recovery times. iLEXY enables rapid (t(1/2) < 30 s), efficient, and reversible nuclear protein depletion in embryos, and is generalizable to proteins of diverse sizes and functions. Applying iLEXY to the Drosophila master regulator Twist, loss-of-function mutants were phenocopied, the Twist-sensitive embryonic stages were precisely mapped, and the effects of timed Twist depletions were investigated. The results demonstrate the power of iLEXY to dissect the function of pleiotropic factors during embryogenesis with unprecedented temporal precision.
Asaoka, M., Sakamaki, Y., Fukumoto, T., Nishimura, K., Tomaru, M., Takano-Shimizu, T., Tanaka, D. and Kobayashi, S. (2021). Offspring production from cryopreserved primordial germ cells in Drosophila. Commun Biol 4(1): 1159. PubMed ID: 34621004
Summary:
There is an urgent need to cryopreserve Drosophila stocks that have been maintained as living cultures for a long time. Long-term culture increases the risk of accidental loss and of unwanted genetic alteration. This paper reports that cryopreserved primordial germ cells (PGCs) can produce F1 progeny when transplanted into hosts. The cryopreserved donor PGCs could form germline stem cells in host gonads and contributed to continuous offspring production. Furthermore, the ability to produce offspring did not appear to vary with either differences between donor strains or cryopreservation duration. Therefore, it is proposed that the cryopreservation method is feasible for long-term storage of various Drosophila strains. These results underscore the potential usefulness of this cryopreservation method for backing up living stocks to avoid either accidental loss or genetic alteration.
Bylino, O. V., Ibragimov, A. N., Pravednikova, A. E. and Shidlovskii, Y. V. (2021). Investigation of the Basic Steps in the Chromosome Conformation Capture Procedure. Front Genet 12: 733937. PubMed ID: 34616432
Summary:
A constellation of chromosome conformation capture methods (С-methods) are an important tool for biochemical analysis of the spatial interactions between DNA regions that are separated in the primary sequence. All these methods are based on the long sequence of basic steps of treating cells, nuclei, chromatin, and finally DNA, thus representing a significant technical challenge. This paper presents an in-depth study of the basic steps in the chromatin conformation capture procedure (3С), which was performed using Drosophila Schneider 2 cells as a model. The steps of cell lysis, nuclei washing, nucleoplasm extraction, chromatin treatment with SDS/Triton X-100, restriction enzyme digestion, chromatin ligation, reversion of cross-links, DNA extraction, treatment of a 3C library with RNases, and purification of the 3C library were investigated. Several options were studied, and optimal conditions were found. This work contributes to the understanding of the 3C basic steps and provides a useful guide to the 3C procedure.
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